Lauren McBryde Gray THE OF University ofNorth Carolina ...

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF THESIMULTANEOUS UV-VIS DETECTION OF FOSINOPRIL SODIUM,

BENAZEPRIL HYDROCHLORIDE, HYDROCHLOROTHIAZIDE AND ALLKNOWN RELATED COMPOUNDS WHEN IN THE FINISHED PRODUCT

DOSAGE FORM USING HPLC AND UPLC

Lauren McBryde Gray

A Thesis Submitted to theUniversity of North Carolina Wilmington in Partial Fulfillment

of the Requirements for the Degree ofMaster of Science

Department of Chemistry and Biochemistry

University of North Carolina Wilmington

2011

Approved By

Advisory Committee

Nadeem Zia

_____ _____

Jeremy Moraan

John TyrellChair

Accepted By

Dean, Graduate School

TABLE OF CONTENTSACKNOWLEDGMENTS.v

DEDICATION vi

LIST OF TABLES vii

LIST OF FIGURES xvi

INTRODUCTION 1

History and Overview of Liquid Chromatography 1

Current Methods Available in Literature 10

EXPERIMENTAL 17

Materials for Assay CU and Related Compounds 17

Equipment for Assay CU and Related Compounds 17

Components of Interest for Related Compounds 19

Analysis of Pharmaceutical Formulations for Assay CU and Related Compounds 20

Materials for Dissolution Testing 25

Equipment for Dissolution Testing 25

Analysis of Pharmaceutical Formulations for Dissolution 26

RESULTS AND DISSCUSSION 27

Determination of Molecular PKa Values 27

Effect of Mobile Phase pH 27

Selection of Extraction Diluent 28

Selection of a Gradient vs. Isocratic Separation Technique 30

Gradient Optimization 30

Discussion of Method Validation 32

Results of Method Validation Experiments for Assay Content Uniformity 36

Results of Method Validation Experiments for Related Compounds 75

Results of Method Validation Experiments for Dissolution 205

Discussion of the UPLC Dissolution Method and Trouble-Shooting 217

CONCLUSION 221

REFERENCES 224

AB STRACT

Liquid chromatography has been a key tool in the arsenal of an analytical chemist.

Through the years the advancements of high performance liquid chromatography and new

technology have allowed the pharmaceutical industry to grow by leaps and bounds. Being able

to produce and successfully release generic drugs into the market to satisfy the customer need is

an important aspect of a successful generic pharmaceutical company. Drug availability can

always be traced back to an efficient and successful quality GMP (good manufacturing practices)

laboratory. As the times change and technology advances, these companies need to recognize

and apply new knowledge to improve laboratory efficiency. Currently, Sandoz Inc at Wilson,

NC has several important generic drug products in the market that are used to battle

hypertension. These molecules are angiotension converting enzyme (ACE) inhibitors and may

be paired with a diuretic to combat ensuing water retention in a combination product. However,

when these products are in these combined formulations each active pharmaceutical ingredient

(API) must be analyzed individually in the laboratory for potency and purity. The product itself

is also tested for rate of dissolution and uniformity of the dosage units. Therefore, the use of a

fast, efficient chromatographic method for the simultaneous detection of all individual aspects of

each API can be very profitable for a generic company. The purpose of this report is to outline

the development and validation of a method that can be used to accurately quantitate the API and

all related compounds simultaneously in four different Sandoz products.

The four products that will be impacted by the new method for assay content uniformity and

related compounds are as listed:

1. Fosinopril Sodium Tablets, 10 mg, 20 mg, and 40 mg

2. Fosinopril SodiumlHydrochlorothiazide Tablets, 10 mg/l2.5 mg and 20 mgIl2.5 mg

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3. Benazepril HC1 Tablets, 5 mg, 10 mg, 20 mg, and 40 mg

4. Benazepril HCllHydrochlorothiazide Tablets, 5 mg/6.25 mg, 10 mg/12.5 mg, 20 mg/12.5

mg and 20 mg/25 mg

Each of the above listed products also may contain a number of known related

compounds that have to be separated chromatographically for accurate quantitation and

detection. The new method that has been developed will simultaneously detect each of the four

products listed for assay, content uniformity, blend uniformity and amount of related

compounds.

A new method for the simultaneous determination of the rate of dissolution for Fosinopril

Sodium and Hydrochlorothiazide in the finished product dosage form was also developed and

validated as per this project. The two products listed below may use this new dissolution

method.

1. Fosinopril Sodium Tablets, 10 mg, 20 mg, and 40 mg

2. Fosinopril SodiumlHydrochlorothiazide Tablets, 10 mg/12.5 mg and 20 mg/l2.5 mg

ACKNOWLEDGMENTS

First and foremost, I would like to acknowledge the hard work of Jason Miller (JSM) and

Mohammad Jashim Uddin (JU). It was because of their hard work, long hours, and excellent

laboratory expertise that the completion of this project was made possible. If Jason and Jashim

had not been such selfless and hardworking individuals this project would never have been

completed during the projected timeline. I owe these men my deepest thanks and gratitude.

I would like to acknowledge my manager John Bredin for his time, patience and valuable

guidance during this thesis project. His extensive technical knowledge allowed for the

completion of this project whenever any obstacles were encountered.

I would like to acknowledge the Director of Quality Control at Sandoz, Inc in Wilson,

NC, Richard Uveges for giving me the time to complete this project even though it kept taking

much longer than expected. His guidance was a valuable asset during this process.

I want to extend my gratitude to my committee members Dr. Nadeem Zia, Dr. John

Tyrell, and Dr. Jeremy Morgan for their guidance and help during this endeavor and allowing me

to have this incredible opportunity.

DEDICATION

I would like to dedicate this thesis project to my husband Ashley Gray whose constant

support and understanding allowed me to focus on the completion of my degree, and to my

unborn daughter who was unknowingly along for the ride.

LIST OF TABLES

Table 1: Solubility Properties of Fosinopril Sodium in Various Solvents 6Table 2: Solubility Properties of Fosinopril Sodium in Various Buffer pHs 6Table 3: Saglik Chromatographic Conditions 10Table 4: Sandoz Monograph vs. USP Monograph Solutions for Fosinopril Na Tablets 13Table 5: Sandoz Monograph vs. USP Monograph Solutions for Fosinopril Na in Fosinopril Na/HCTZ Tablets 13Table 6: Sandoz Monograph vs. USP Monograph Solutions for Benazepril HC1 Tablets 13Table 7: Sandoz Monograph vs. USP Monograph Solutions for Benazepril HC1 in BenazeprilHC1/HCTZ Tablets 13Table 8: Sandoz Monograph vs. USP Monograph Solutions for HCTZ in Fosinopril Na/HCTZTablets 14Table 9: Sandoz Monograph vs. USP Monograph Solutions for HCTZ in Benazepril HCI HCTZTablets 14Table 10: Sandoz Monograph vs. USP Monograph for Detection Wavelength for Fosinopril NaTablets 14Table 11: Sandoz Monograph vs. USP Monograph for Detection Wavelength for BenazeprilHC1 Tablets 14Table 12: Sandoz Monograph vs. USP Monograph for Detection Wavelength for Fosinopril Nain Fosinopril Na/HCTZ Tablets 14Table 13: Sandoz Monograph vs. USP Monograph for Detection Wavelength for BenazeprilHCI in Benazepril HC1/HCTZ Tablets 15Table 14: Sandoz Monograph vs. USP Monograph for Detection Wavelength for HCTZ inFosinopril Na/HCTZ Tablets 15Table 15: Sandoz Monograph vs. USP Monograph for Detection Wavelength for HCTZ inBenazepril HCI/HCTZ Tablets 15Table 16: Sandoz Monograph vs. USP Monograph for Chromatographic Columns for FosinoprilNa Tablets 15Table 17: Sandoz Monograph vs. USP Monograph for Chromatographic Columns forBenazepril HC1 Tablets 15Table 18: Sandoz Monograph vs. USP Monograph for Chromatographic Columns for FosinoprilNa in Fosinopril NaIHCTZ Tablets 16Table 19: Sandoz Monograph vs. USP Monograph for Chromatographic Columns for HCTZ inFosinopril NaIHCTZ Tablets 16Table 20: Sandoz Monograph vs. USP Monograph for Chromatographic Columns forBenazepril HC1 in Benazepril HCI/HCTZ Tablets 16Table 21: Sandoz Monograph vs. USP Monograph for Chromatographic Columns for HCTZ inBenazepril HC1/HCTZ Tablets 16Table 22: HPLC Linear Gradient for Assay/CU and Related Compounds 18Table 23: UPLC Linear Gradient for Assay/CU and Related Compounds 18Table 24: Related Compounds with Scientific and Common Name 19Table 25: HPLC Gradient for Dissolution 25Table 27: pI(a Values for Each API of Interest 27Table 28: Optimization of Sample Diluent 29Table 29: Common Degradation Pathways of Various Functional Groups 33

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Table 30: Minimum Analytical Ranges for Method Validation 34Table 31: Assay Linearity Results for UPLC 36Analyst: JSM 36Table 32: Assay Linearity Results for HPLC 36Table 33: Assay Accuracy Results for Fosinopril Sodium Tablets for UPLC 37Table 34: Assay Accuracy Results for Fosinopril SodiumJHCTZ Tablets (Fosinopril Sodium)for UPLC 37Table 35: Assay Accuracy Results for Fosinopril SodiumJHCTZ Tablets (HCTZ) for UPLC .. 38Table 36: Assay Accuracy Results for Benazepril HC1 Tablets for UPLC 38Table 37: Assay Accuracy Results for Benazepril HCI HCTZ Tablets (Benazepril HC1) UPLC38Table 38: Assay Accuracy Results for Benazepril HCI HCTZ Tablets (HCTZ) for UPLC 39Table 39: Assay Accuracy Results for Fosinopril Sodium Tablets for HPLC 39Table 40: Assay Accuracy Results for Fosinopril SodiumJHCTZ Tablets (Fosinopril Sodium)forHPLC 39Table 41: Assay Accuracy Results for Fosinopril SodiumJHCTZ Tablets (HCTZ) for HPLC .. 40Table 42: Assay Accuracy Results for Benazepril HC1 Tablets for HPLC 40Table 43: Assay Accuracy Results for Benazepril HC1 HCTZ Tablets (Benazepril HC1) HPLC4OTable 44: Assay Accuracy Results for Benazepril HC1/HCTZ Tablets (HCTZ) for HPLC 41Table 45: Assay Precision Results for Fosinopril Sodium 10 mg Tablets on UPLC 42Table 46: Assay Precision Results for Fosinopril Sodium 10 mg Tablets on HPLC 42Table 47: Assay Precision Results for Fosinopril SodiumJHCTZ 10 mg/12.5 mg Tablets onUPLC 42Table 48: Assay Precision Results for Fosinopril SodiumJHCTZ 10 mg/12.5 mg Tablets onHPLC 43Table 49: Assay Precision Results for Benazepril HCI 5 mg Tablets on UPLC 43Table 50: Assay Precision Results for Benazepril HC1 5 mg Tablets on HPLC 43Table 51: Assay Precision Results for Benazepril HC1 HCTZ 5 mg/6.25 mg Tablets on UPLC 44Table 52: Assay Precision Results for Benazepril HC1IHCTZ 5 mg/6.25 mg Tablets on HPLC 44Table 53: Content Uniformity Results for UPLC 45Table 54: Content Uniformity Results for HPLC 45Table 55: Forced Degradation Conditions for Fosinopril Sodium Tablets and Fosinopril 50SodiumIHCTZ Tablets 50Table 56: Forced Degradation Conditions for Benazepril HC1 Tablets and BenazeprilHC1 HCTZ Tablets 50Table 57: Forced Degradation Results for Fosinopril Sodium Tablets on UPLC 52Table 58: Forced Degradation Results for Fosinopril SodiumJHCTZ Tablets (Fosinopril) onUPLC 52Table 59: Forced Degradation Results for Fosinopril SodiumfHCTZ Tablets (HCTZ) on UPLC

53Table 60: Forced Degradation Results for Benazepril HC1 Tablets on UPLC 53Table 61: Forced Degradation Results for Benazepril HCI HCTZ Tablets (Benazepril) on UPLC

54Table 62: Forced Degradation Results for Benazepril HC1 HCTZ Tablets (HCTZ) on UPLC .. 54Table 63: Forced Degradation Results for Fosinopril Sodium Tablets (HPLC) 55Table 64: Forced Degradation Results for Fosinopril SodiumJHCTZ Tablets (Fosinopril) onHPLC 55

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Table 65: Forced Degradation Results for Fosinopril SodiumJHCTZ Tablets (HCTZ) on HPLC56

Table 66: Forced Degradation Results for Benazepril HC1 Tablets on HPLC 56Table 67: Forced Degradation Results for Benazepril HC1 HCTZ Tablets (Benazepril) on HPLC

57Table 68: Forced Degradation Results for Benazepril HC1 HCTZ Tablets (HCTZ) on HPLC.. 57Table 69: Assay Filter Validation Using the External Standard 58Table 70: Assay Filter Validation for Fosinopril Sodium 10 mg Tablets 58Table 71: Assay Filter Validation for Fosinopril SodiumJHCTZ 10 mg/12.5 mg Tablets 59Table 72: Assay Filter Validation for Benazepril HC1 5 mg Tablets 59Table 73: Assay Filter Validation for Benazepril HCIJHCTZ 5 mgI6.25 mg Tablets 59Table 74: Assay Extraction Validation for Fosinopril Sodium 10 mg Tablets 60Table 75: Assay Extraction Validation for Fosinopril SodiumJHCTZ 10 mg/12.5 mg Tablets.. 60Table 76: Assay Extraction Validation for Benazepril HC1 5 mg Tablets 60Table 77: Assay Extraction Validation for Benazepril HC1 HCTZ 5 mg/6.25 mg Tablets 61Table78 61Table 79: Robustness Parameters for UPLC 62Table 80: Robustness Results for Fosinopril Sodium Tablets on UPLC 62Table 81: Robustness Results for Fosinopril SodiumJHCTZ Tablets on UPLC 62Table 82: Robustness Results for Benazepril HC1 Tablets on UPLC 63Table 83: Robustness Results for Benazepril HC1/HCTZ Tablets on UPLC 63Table 84: Robustness Results for System Suitability Criterion for Fosinopril 63Table 85: Robustness Results for System Suitability Criterion for Benazepril 64Table 86: Robustness Results for System Suitability Criterion for HCTZ 64Table 87: Robustness Parameters for HPLC 64Table 88: Robustness Results for Fosinopril Sodium Tablets on HPLC 65Table 89: Robustness Results for Fosinopril Sodium!HCTZ Tablets on HPLC 65Table 90: Robustness Results for Benazepril HCI Tablets on HPLC 66Table 91: Robustness Results for Benazepril HC1 HCTZ Tablets on HPLC 66Table 92: Robustness Results for System Suitability Criterion for Fosinopril 67Table 93: Robustness Results for System Suitability Criterion for Benazepril 67Table 94: Robustness Results for System Suitability Criterion for HCTZ 68Table 95: Solution Stability of the Fosinopril (Alone) Working Standard 69Table 96: Solution Stability of the FosinopriuIHCTZ Working Standard (Fosinopril) 69Table 97: Solution Stability of the Fosinopril/HCTZ Working Standard (HCTZ) 69Table 98: Solution Stability of the Benazepril (Alone) Working Standard 69Table 99: Solution Stability of the Benazepril HCTZ Standard (Benazepril) 69Table 100: Solution Stability of the Benazepril HCTZ Standard (HCTZ) 70Table 101: Solution Stability of the FosinoprillBenazepril/HCTZ Working Standard (Fosinopril)

70Table 102: Solution Stability of the Fosinopril/Benazepril/HCTZ Working Standard(Benazepril) 70Table 103: Solution Stability of the Fosinopriu/Benazepril/HCTZ Working Standard (HCTZ). 70Table 104: Solution Stability of the Fosinopril Sodium 10 mg Tablets 70Table 105: Solution Stability of the Fosinopril SodiumJHCTZ lOmg/12.5mg Tablets(Fosinopril) 71

Table 106: Solution Stability of the Fosinopril SodiumJHCTZ lOmgIl2.5mg Tablets (HCTZ) 71Table 107: Solution Stability of the Benazepril HC1 5 mg Tablets 71Table 108: Solution Stability of the Benazepril HCI/HCTZ 5mg/6.25 mg (Benazepril) 71Table 109: Solution Stability of the Benazepril HC1 HCTZ 5mg/6.25 mg (HCTZ) 71Table 110: Assay Equivalency Results for Fosinopril Sodium 10 mg Tablets on UPLC 72Table 111: Assay Equivalency Results for Fosinopril Sodium 10 mg Tablets on HPLC 72Table 112: Assay Equivalency Results for Fosinopril SodiumIHCTZ 10 mg/12.5 mg Tablets onUPLC 73Table 113: Assay Equivalency Results for Fosinopril SodiumIHCTZ 10 mg/12.5 mg Tablets onHPLC 73Table 114: Assay Equivalency Results for Benazepril HC1 5 mg Tablets on UPLC 73Table 115: Assay Equivalency Results for Benazepril HC1 5 mg Tablets on HPLC 74Table 116: Assay Equivalency Results for Benazepril HC1 HCTZ 5 mg/6.25 mg Tablets onUPLC 74Table 117: Assay Equivalency Results for Benazepril HC1/HCTZ 5 mg/6.25 mg Tablets onHPLC 74Table 118: Linearity Results for UPLC 76Table 119: Linearity Results for HPLC 76Table 120: Limit of Quantitation for Fosinopril Sodium Tablets 77Table 121: Limit of Quantitation for Fosinopril Sodium!HCTZ Tablets 77Table 122: Limit of Quantitation for Benazepril HC1 Tablets 77Table 123: Limit of Quantitation for Bena.zepril HC1IHCTZ Tablets 78Table 124: Acceptance Criteria for Related Compound Accuracy 78Table 125: Accuracy Results for Related Compound A in Fosinopril Sodium Tablets 78Table 126: Accuracy Results for Related Compound B in Fosinopril Sodium Tablets 78Table 127: Accuracy Results for Related Compound C in Fosinopril Sodium Tablets 79Table 128: Accuracy Results for Related Compound D in Fosinopril Sodium Tablets 79Table 129: Accuracy Results for Related Compound A in Fosinopril SodiumJHCTZ Tablets.. 79Table 130: Accuracy Results for Related Compound B in Fosinopril SodiumJHCTZ Tablets... 79Table 131: Accuracy Results for Related Compound C in Fosinopril SodiumIHCTZ Tablets... 79Table 132: Accuracy Results for Related Compound D in Fosinopril SodiumJHCTZ Tablets.. 80Table 133: Accuracy Results for 4-Amino in Fosinopril SodiumIHCTZ Tablets 80Table 134: Accuracy Results for CTZ in Fosinopril SodiumJHCTZ Tablets 80Table 135: Accuracy Results for Dimer in Fosinopril SodiumIHCTZ Tablets 80Table 136: Accuracy Results for S-Amine in Benazepril HC1 Tablets 81Table 137: Accuracy Results for Benazeprilat in Benazepril HC1 Tablets 81Table 138: Accuracy Results for R,S-Isomer in Benazepril HC1 Tablets 81Table 139: Accuracy Results for S-Amine in Benazepril HC1 HCTZ Tablets 82Table 140: Accuracy Results for Benazeprilat in Benazepril HC1 HCTZ Tablets 82Table 141: Accuracy Results for R,S-Isomer in Benazepril HC1 HCTZ Tablets 82Table 142: Accuracy Results for 4-Amino in Benazepril HC1 HCTZ Tablets (For InformationOnly) 82Table 143: Accuracy Results for CTZ in Benazepril HC1/HCTZ Tablets (For Information Only)

82Table 144: Acceptance Criteria for RC Repeatability 83Table 145: Acceptance Criteria for RC Intermediate Precision 83

.98Table 186: Forced Degradation Results for Benazepril HCI Tablets on UPLC 98Table 187: Forced Degradation Results for Benazepril HC1 HCTZ Tablets (Benazepril) onUPLC 98

Table 146: RC Precision Results for RC-A in Fosinopril Sodium Tablets on UPLC 84Table 147: RC Precision Results for RC-A in Fosinopril Sodium Tablets on HPLC 84Table 148: RC Precision Results for RC-B in Fosinopril Sodium Tablets on UPLC 84Table 149: RC Precision Results for RC-B in Fosinopril Sodium Tablets on HPLC 85Table 150: RC Precision Results for RC-C in Fosinopril Sodium Tablets on UPLC 85Table 151: RC Precision Results for RC-C in Fosinopril Sodium Tablets on HPLC 85Table 152: RC Precision Results for RC-D in Fosinopril Sodium Tablets on UPLC 86Table 153: RC Precision Results for RC-D in Fosinopril Sodium Tablets on HPLC 86Table 154: RC Precision Results for RC-A in Fosinopril SodiumfHCTZ Tablets on UPLC 86Table 155: RC Precision Results for RC-A in Fosinopril SodiumlHCTZ Tablets on HPLC 87Table 156: RC Precision Results for RC-B in Fosinopril SodiumIHCTZ Tablets on UPLC 87Table 157: RC Precision Results for RC-B in Fosinopril SodiumIHCTZ Tablets on HPLC 87Table 158: RC Precision Results for RC-C in Fosinopril SodiumIHCTZ Tablets on UPLC 88Table 159: RC Precision Results for RC-C in Fosinopril SodiumJHCTZ Tablets on HPLC 88Table 160: RC Precision Results for RC-D in Fosinopril SodiumIHCTZ Tablets on UPLC 88Table 161: RC Precision Results for RC-D in Fosinopril SodiumJHCTZ Tablets on HPLC 89Table 162: RC Precision Results for 4-Amino in Fosinopril SodiumJHCTZ Tablets on UPLC. 89Table 163: RC Precision Results for 4-Amino in Fosinopril SodiumJHCTZ Tablets on HPLC. 89Table 164: RC Precision Results for CTZ in Fosinopril Sodiunv’HCTZ Tablets on UPLC 90Table 165: RC Precision Results for CTZ in Fosinopril SodiumIHCTZ Tablets on HPLC 90Table 166: RC Precision Results for Dimer in Fosinopril SodiumIHCTZ Tablets on UPLC 90Table 167: RC Precision Results for Dimer in Fosinopril Sodium!HCTZ Tablets on HPLC 91Table 168: RC Precision Results for S-Amine in Benazepril HC1 Tablets on UPLC 91Table 169: RC Precision Results for S-Amine in Benazepril HC1 Tablets on HPLC 91Table 170: RC Precision Results for Benazeprilat in Benazepril HC1 Tablets on UPLC 92Table 171: RC Precision Results for Benazeprilat in Benazepril HC1 Tablets on HPLC 92Table 172: RC Precision Results for R,S-Isomer in Benazepril HC1 Tablets on UPLC 92Table 173: RC Precision Results for R,S-Isomer in Benazepril HC1 Tablets on HPLC 93Table 174: RC Precision Results for S-Amine in Benazepril HCI/HCTZ Tablets on UPLC 93Table 175: RC Precision Results for S-Amine in Benazepril HCI HCTZ Tablets on HPLC 93Table 176: RC Precision Results for Benazeprilat in Benazepril HCI HCTZ Tablets on UPLC 94Table 177: RC Precision Results for Benazeprilat in Benazepril HC1 HCTZ Tablets on HPLC 94Table 178: RC Precision Results for R,S-Isomer in Benazepril HC1/HCTZ Tablets on UPLC.. 94Table 179: RC Precision Results for R,S-Isomer in Benazepril HC1/HCTZ Tablets on HPLC.. 95Table 180: Forced Degradation Results for Fosinopril Sodium Raw Material on UPLC 96Table 181: Forced Degradation Results for Benazepril HCI Raw Material on UPLC 96Table 182: Forced Degradation Results for HCTZ Raw Material on UPLC 97Table 183: Forced Degradation Results for Fosinopril Sodium Tablets on UPLC 97Table 184: Forced Degradation Results for Fosinopril SodiumIHCTZ Tablets (Fosinopril) onUPLC 97Table 185: Forced Degradation Results for Fosinopril SodiumJHCTZ Tablets (HCTZ) on UPLC

Table 188: Forced Degradation Results for Benazepril HC1 HCTZ Tablets (HCTZ) on UPLC(For Information Only) 99Table 189: Forced Degradation Results for Fosinopril Sodium Raw Material on HPLC 139Table 190: Forced Degradation Results for Benazepril HC1 Raw Material on HPLC 139Table 191: Forced Degradation Results for HCTZ Raw Material on HPLC 140Table 192: Forced Degradation Results for Fosinopril Sodium Tablets on HPLC 140Table 193: Forced Degradation Results for Fosinopril SodiumJHCTZ Tablets (Fosinopril) onHPLC 140Table 194: Forced Degradation Results for Fosinopril SodiumJHCTZ Tablets (HCTZ) on HPLC

141Table 195: Forced Degradation Results for Benazepril HC1 Tablets on HPLC 141Table 196: Forced Degradation Results for Benazepril HC1 HCTZ Tablets (Benazepril) onHPLC 141Table 197: Forced Degradation Results for Benazepril HCI/HCTZ Tablets (HCTZ) on HPLC(For Information Only) 142Table 199: Filter Study Results for Fosinopril Sodium 10 mg Tablets 187Table 200: Filter Study Results for Fosinopril SodiumIHCTZ 10 mg/12.5 mg Tablets 187(Fosinopril) 187Table 201: Filter Study Results for Fosinopril SodiumJHCTZ 10 mg/12.5 mg Tablets 187(HCTZ) 187Table 202: Filter Study Results for Benazepril HC1 5 mg Tablets 188Table 203: Filter Study Results for Benazepril HC1 HCTZ 5 mg/6.25 mg Tablets (Benazepril)

188Table 204: Filter Study Results for Benazepril HCI HCTZ 5 mg/6.25 mg Tablets (HCTZ) .... 188Table 205: Acceptance Criteria for RC Robustness 189Table 206: System Suitability Criteria 189Table 207: Resolution of Fosinopril Sodium Resolution Solution on UPLC 190Table 208: Resolution of Fosinopril Sodium Resolution Solution on HPLC 191Table 209: Resolution of Fosinopril Sodiumi’HCTZ Resolution Solution on UPLC 191Table 210: Resolution of Fosinopril SodiumIHCTZ Resolution Solution on HPLC 192Table 211: Resolution of Benazepril HC1 Resolution Solution on UPLC 192Table 212: Resolution of Benazepril HC1 Resolution Solution on HPLC 193Table 213: Resolution of Benazepril HC1 HCTZ Resolution Solution on UPLC 193Table 214: Resolution of Benazepril HC1 HCTZ Resolution Solution on HPLC 194Table 215: Robustness 00 Change for Fosinopril Sodium Tablets on UPLC 195Table 216: Robustness Results for Fosinopril Sodiumi’HCTZ Tablets on UPLC 195Table 227: Robustness Results for Benazepril HCI Tablets on UPLC 195Table 218: Robustness Results for Benazepril HC1/HCTZ Tablets on UPLC 196Table 219: Robustness Results for Fosinopril Sodium Tablets on HPLC 196Table 220: Robustness Results for Fosinopril SodiumIHCTZ Tablets on HPLC 197Table 221: Robustness Results for Benazepril HCI Tablets on HPLC 197Table 222: Robustness Results for Benazepril HC1/HCTZ Tablets on HPLC 198Table 223: Solution Stability Acceptance Criteria for Related Compounds Samples 198Table 224: Solution Stability of Fosinopril Sodium 10 mg Tablets (Clear Flasks RoomTemperature) 198

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Table 225: Solution Stability of Fosinopril Sodium 10 mg Tablets (Amber Flasks RoomTemperature) 199Table 226: Solution Stability of Fosinopril Sodium 10 mg Tablets (Clear Flasks Refrigeration)

199Table 227: Solution Stability of Fosinopril Sodium 10 mg Tablets (Amber FlasksRefrigeration) 199Table 228: Solution Stability of Fosinopril SodiumlHCTZ 10 mg/12.5 mg Tablets (Clear Flasks

Room Temperature - Fosinopril) 199Table 229: Solution Stability of Fosinopnl SodiumJHCTZ 10 mg/12.5 mg Tablets (AmberFlasks Room Temperature - Fosinopril) 199Table 230: Solution Stability of Fosinopril SodiumJHCTZ 10 mg/12.5 mg Tablets (Clear Flasks

Refrigeration - Fosinopril) 200Table 231: Solution Stability of Fosinopril SodiumIHCTZ 10 mg/12.5 mg Tablets (AmberFlasks Refrigeration - Fosinopril) 200Table 232: Solution Stability of Fosinopril SodiumIHCTZ 10 mg/12.5 mg Tablets (Clear Flasks

Room Temperature - HCTZ) 200Table 233: Solution Stability of Fosinopril SodiumJHCTZ 10 mg/12.5 mg Tablets (AmberFlasks Room Temperature - HCTZ) 200Table 234: Solution Stability of Fosinopril Sodium/HCTZ 10 mg/12.5 mg Tablets (Clear Flasks

Refrigeration - HCTZ) 200Table 235: Solution Stability of Fosinopril SodiumJHCTZ 10 mg/12.5 mg Tablets (AmberFlasks Refrigeration - HCTZ) 201Table 236: Solution Stability of Benazepril HC1 5 mg Tablets (Clear Flasks RoomTemperature) 201Table 237: Solution Stability of Benazepnl HCI 5 mg Tablets (Amber Flasks RoomTemperature) 201Table 238: Solution Stability of Benazepril HC1 5 mg Tablets (Clear Flasks Refrigeration) 201Table 239: Solution Stability of Benazepril HCI/HCTZ 5 mg/6.25 mg Tablets (Clear FlasksRoom Temperature - Benazepril) 201Table 240: Solution Stability of Benazepril HC1 HCTZ 5 mgI6.25 mg Tablets (Amber FlasksRoom Temperature - Benazepril) 202Table 241: Solution Stability of Benazepril HC1 HCTZ 5 mg/6.25 mg Tablets (Clear FlasksRefrigeration - Benazepril) 202Table 242: Solution Stability of Benazepril HCI!HCTZ 5 mg/6.25 mg Tablets (Amber FlasksRefrigeration - Benazepril) 202Table 243: Solution Stability of Benazepril HC1 HCTZ 5 mg/6.25 mg Tablets (Clear FlasksRoom Temperature - HCTZ) 202Table 244: Solution Stability of Benazepril HC1 HCTZ 5 mg/6.25 mg Tablets (Amber FlasksRoom Temperature - HCTZ) 202Table 245: Solution Stability of Benazepril HC1 HCTZ 5 mg/6.25 mg Tablets (Clear FlasksRefrigeration - HCTZ) 203Table 246: Solution Stability of Benazepril HC1 HCTZ 5 mg/6.25 mg Tablets (Amber FlasksRefrigeration - HCTZ) 203Table 247: Method Equivalency for Fosinopril Sodium 10 mg Tablets Related Compounds onUPLC 203

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Table 248: Method Equivalency for Fosinopril Sodium 10 mg Tablets Related Compounds onHPLC 204Table 249: Method Equivalency for Fosinopril SodiumfHCTZ 10 mg/12.5mg Tablets RelatedCompounds on UPLC (Fosinopril) 204Table 250: Method Equivalency for Fosinopril SodiumJHCTZ 10 mg/12.5mg Tablets RelatedCompounds on HPLC (Fosinopril) 204Table 251: Method Equivalency for Fosinopril SodiumJHCTZ 10 mg/12.5mg Tablets RelatedCompounds on UPLC (HCTZ) 204Table 252: Method Equivalency for Fosinopril SodiumJHCTZ 10 mg/12.5mg Tablets RelatedCompounds on HPLC (HCTZ) 204Table 253: Method Equivalency for Benazepril 5 mg Tablets Related Compounds on UPLC 205Table 254: Method Equivalency for Benazepril 5 mg Tablets Related Compounds on HPLC 205Table 255: Method Equivalency for Benazepril HC1IHCTZ 5 mg/6.25mg Tablets RelatedCompounds on UPLC (Benazepril) 205Table 256: Method Equivalency for Benazepril HC1IHCTZ 5 mg/6.25mg Tablets RelatedCompounds on HPLC (Benazepril) 205Table 257: Dissolution Linearity Results for HPLC 206Table 258: Dissolution Accuracy Results for Fosinopril Sodium Tablets for HPLC 207Table 259: Dissolution Accuracy Results for Fosinopril SodiumJHCTZ Tablets for HPLC .... 207(Fosinopril) 207Table 260: Dissolution Accuracy Results for Fosinopril SodiumJHCTZ Tablets for HPLC .... 208(HCTZ) 208Table 261: Dissolution Precision Results for Fosinopril Sodium 10 mg Tablets on HPLC 209Table 262: Dissolution Precision Results for Fosinopril SodiumJHCTZ 10 mgl2.5 mg Tabletson HPLC (Fosinopril) 209Table 263: Dissolution Precision Results for Fosinopril SodiumJHCTZ 10 mgl2.5 mg Tabletson HPLC (HCTZ) 209Table 264: Dissolution Results for the Degassing Procedure for Fosinopril Sodium 10 mg.... 211Tablets 211Table 265: Dissolution Results for the Degassing Procedure for Fosinopril SodiumJHCTZ 10mg 12.5 mg Tablets (Fosinopril) 212Table 266: Dissolution Results for the Degassing Procedure for Fosinopril SodiumIHCTZ 10mg 12.5 mg Tablets (HCTZ) 212Table 267: Dissolution Results for the Sampling Procedure for Fosinopril Sodium 10 mg 212Tablets 212Table 268: Dissolution Results for the Sampling Procedure for Fosinopril Sodium/HCTZ 10mg12.5 mgTablets (Fosinopril) 213

Table 269: Dissolution Results for the Sampling Procedure for Fosinopril SodiumIHCTZ 10 mg12.5 mg Tablets (HCTZ) 213

Table 270: Dissolution Chromatographic Robustness Parameters 213Table 271: Dissolution Robustness Results for Fosinopril Sodium 10mg Tablets 214Table 272: Dissolution Robustness Results for Fosinopril Sodium!HCTZ lOmg/12.Smg Tablets

214Table 273: Dissolution Solution Stability for the Fosinopril Sodium Standard 215Table 274: Dissolution Solution Stability for the Fosinopril SodiumIHCTZ Standard 215Table 275: Dissolution Solution Stability for Fosinopril Sodium 10 mg Tablets 215

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Table 276: Dissolution Solution Stability for the Fosinopril SodiumJHCTZ lOmg/12.5 mgTablets 215Table 277: Method Equivalency for Fosinopril Sodium 10mg Tablets 216Table 278: Method Equivalency for Fosinopril SodiumJHCTZ lOmg/12.5mg Tablets 216Table 279: Method Equivalency for Fosinopril SodiumJHCTZ 10mg/i 2.5mg Tablets 216

LIST OF FIGURES

Figure 1: The Van Deemter Equation 3Figure 2: Fosinopril Sodium (Active Pharmaceutical Ingredient # 1) and Fosinoprilat (RC-A) . 4Figure 3: Benazepril Hydrochloride (Active Pharmaceutical Ingredient 2) and Benazeprilat.. 8Figure 4: Hydrochlorothiazide (Active Pharmaceutical Ingredient # 3) 9Figure 5: Chromatogram of the Reference Standard (UPLC) 21Figure 6: Chromatogram of the Fosinopril Sodium ID Solution (UPLC) 21Figure 7: Chromatogram of the Fosinopril SodiuniJHCTZ ID Solution (UPLC) 21Figure 8: Chromatogram of the Benazepril HC1 ID Solution (UPLC) 22Figure 9: Chromatogram of the Benazepril HCI HCTZ ID Solution (UPLC) 22Figure 10: Chromatogram of the Ink ID Solution (UPLC) 22Figure 11: Chromatogram of the Reference Standard (UPLC) 23Figure 12: Chromatogram of the Fosinopril Sodium ID Solution (HPLC) 23Figure 13: Chromatogram of the Fosinopril SodiumJHCTZ ID Solution (HPLC) 23Figure 14 Chromatogram of the Benazepril HCI ID Solution (HPLC) 24Figure 15 Chromatogram of the Benazepril HC1 HCTZ ID Solution (HPLC) 24Figure 16 Chromatogram of the Ink ID Solution (HPLC) 24Figure 17 Standard Preparation Using HPLC Method for Dissolution 26Figure 18 Equation for Theoretical Plates 35Figure 19 Equation for Resolution 36Figure 20 Chromatogram of the Blank (Diluent) on UPLC 46Figure 21 Chromatogram of the Fosinopril Sodium Tablets Placebo for Assay (UPLC) 46Figure 22 Chromatogram of the Fosinopril SodiumJHCTZ Tablets Placebo for Assay (UPLC) 47Figure 23 Chromatogram of the Benazepril HC1 Tablets Placebo for Assay (UPLC) 47Figure 24 Chromatogram of the Benazepril HC1/HCTZ Tablets Placebo for Assay (UPLC) ... 47Figure 25 Chromatogram of the Blank (Diluent) on HPLC 48Figure 26 Chromatogram of the Fosinopril Sodium Tablets Placebo for Assay (HPLC) 48Figure 27 Chromatogram of the Fosinopril SodiuniJHCTZ Tablets Placebo for Assay (HPLC)48Figure 28 Chromatogram of the Benazepril HC1 Tablets Placebo for Assay (HPLC) 49Figure 29 Chromatogram of the Benazepril HC1IHCTZ Tablets Placebo for Assay (HPLC) ... 49Figure 30: Chromatogram of the Blank (Diluent) - UPLC 99Figure 31: Chromatogram of the Fosinopril Sodium Raw Material (Control) - UPLC 100Figure 32: Chromatogram of the Benazepril HC1 Raw Material (Control) - UPLC 100Figure 33: Chromatogram of the HCTZ Raw Material (Control) - UPLC 100Figure 34: Chromatogram of the Fosinopril Sodium Tablets Placebo (Control) - UPLC 101Figure 35: Chromatogram of the Fosinopril Sodium Tablets Blend (Control) - UPLC 101Figure 36: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Placebo (Control) - UPLC

101Figure 37: Chromatogram of the Fosinopril SodiumfHCTZ Tablets Blend (Control) - UPLC 102Figure 38: Chromatogram of the Benazepril HC1 Tablets Placebo (Control) - UPLC 102Figure 39: Chromatogram of the Benazepril HC1 Tablets Blend (Control) - UPLC 102Figure 40: Chromatogram of the Benazepril HC1 HCTZ Tablets Placebo (Control) - UPLC. 103Figure 41: Chromatogram of the Benazepril HCI/HCTZ Tablets Blend (Control) - UPLC .... 103Figure 42: Chromatogram of the Fosinopril Sodium Raw Material Sample Solution After UVExposure for 5 Days - UPLC 104

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Figure 43: Chromatogram of the Benazepril HC1 Raw Material Sample Solution After UVExposure for 5 Days - UPLC 104Figure 44: Chromatogram of the HCTZ Raw Material Sample Solution After UV Exposure for5Days-UPLC 104Figure 45: Chromatogram of the Fosinopril Sodium Tablets Placebo Sample Solution After UVExposure for 5 Days - UPLC 105Figure 46: Chromatogram of the Fosinopril Sodium Tablets Blend Sample Solution After UVExposure for 5 Days - UPLC 105Figure 47: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Placebo Sample SolutionAfter UV Exposure for 5 Days - UPLC 105Figure 48: Chromatogram of the Fosinopril SodiumlHCTZ Tablets Blend Sample SolutionAfter UV Exposure for 5 Days - UPLC 106Figure 48: Chromatogram of the Benazepril HC1 Tablets Placebo Sample Solution After UVExposure for 5 Days UPLC 106Figure 49: Chromatogram of the Benazepril HC1 Tablets Blend Sample Solution After UVExposure for 5 Days - UPLC 106Figure 50: Chromatogram of the Benazepril HCIIHCTZ Tablets Placebo Sample Solution AfterUV Exposure for 5 Days - UPLC 107Figure 51: Chromatogram of the Benazepril HCI HCTZ Tablets Blend Sample Solution AfterUV Exposure for 5 Days - UPLC 107Figure 52: Chromatogram of the Fosinopril Sodium Raw Material Sample Solution After UVExposure for 3 Hours - UPLC 108Figure 53: Chromatogram of the Benazepril HC1 Raw Material Sample Solution After UVExposure for 3 Hours - UPLC 108Figure 54: Chromatogram of the HCTZ Raw Material Sample Solution After UV Exposure for3Hours-UPLC 108Figure 55: Chromatogram of the Fosinopril Sodium Tablets Placebo Sample Solution After UVExposure for 3 Hours - UPLC 109Figure 56: Chromatogram of the Fosinopril Sodium Tablets Blend Sample Solution After UVExposure for 3 Hours - UPLC 109Figure 57: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Placebo Sample SolutionAfter UV Exposure for 3 Hours - UPLC 110Figure 58: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Blend Sample SolutionAfter UV Exposure for 3 Hours - UPLC 110Figure 59: Chromatogram of the Benazepril HC1 Tablets Placebo Sample Solution After UVExposure for 3 Hours - UPLC 111Figure 60: Chromatogram of the Benazepril HC1 Tablets Blend Sample Solution After UVExposure for 3 Hours - UPLC 111Figure 61: Chromatogram of the Benazepril HC1/HCTZ Tablets Placebo Sample Solution AfterUV Exposure for 3 Hours - UPLC 111Figure 62: Chromatogram of the Benazepril HCI HCTZ Tablets Blend Sample Solution AfterUV Exposure for 3 Hours - UPLC 112Figure 63: Chromatogram of the Fosinopril Sodium Raw Material Dry Powders After UVExposure for 5 Days UPLC 112Figure 64: Chromatogram of the Benazepril HCI Raw Material Dry Powders After UVExposure for 5 Days UPLC 112

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Figure 65: Chromatogram of the HCTZ Raw Material Dry Powders After UV Exposure for 5Days—UPLC 113Figure 66: Chromatogram of the Fosinopril Sodium Tablets Placebo Dry Powders After UVExposure for 5 Days UPLC 113Figure 67: Chromatogram of the Fosinopril Sodium Tablets Blend Dry Powders After UVExposure for 5 Days UPLC 113Figure 68: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Placebo Dry Powders After

114Figure 69: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Blend Dry Powders AfterUV Exposure for 5 Days — UPLC 114Figure 70: Chromatogram of the Benazepril HC1 Tablets Placebo Dry Powders After UVExposure for 5 Days UPLC 114Figure 71: Chromatogram of the Benazepril HC1 Tablets Blend Dry Powders After UVExposure for 5 Days UPLC 115Figure 72: Chromatogram of the Benazepril HC1/HCTZ Tablets Placebo Dry Powders AfterUV Exposure for 5 Days — UPLC 115Figure 73: Chromatogram of the Benazepril HC1/HCTZ Tablets Blend Dry Powders After UVExposure for 5 Days UPLC 115Figure 74: Chromatogram of the Fosinopril Sodium Raw Material Powder Exposed toHumidity for 5 Days at Room Temperature - UPLC 116Figure 75: Chromatogram of the Benazepril HC1 Raw Material Powder Exposed to Humidityfor 5 Days at Room Temperature - UPLC 116Figure 76: Chromatogram of the HCTZ Raw Material Sample Powder Exposed to Humidity for5 Days at Room Temperature - UPLC 116Figure 77: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder Exposed toHumidity for 5 Days at Room Temperature - UPLC 117Figure 78: Chromatogram of the Fosinopril Sodium Tablets Blend Powder Exposed toHumidity for 5 Days at Room Temperature UPLC 117Figure 79: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Placebo Powder Exposed toHumidity for 5 Days at Room Temperature - UPLC 117Figure 80: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Blend Powder Exposed toHumidity for 5 Days at Room Temperature - UPLC 118Figure 81: Chromatogram of the Benazepril HC1 Tablets Placebo Powder Exposed to Humidityfor 5 Days at Room Temperature - UPLC 118Figure 82: Chromatogram of the Benazepril HC1 Tablets Blend Powder Exposed to Humidityfor 5 Days at Room Temperature - UPLC 118Figure 83: Chromatogram of the Benazepril HC1 HCTZ Tablets Placebo Powder Exposed toHumidity for 5 Days at Room Temperature - UPLC 119Figure 84: Chromatogram of the Benazepril HC1 HCTZ Tablets Blend Powder Exposed toHumidity for 5 Days at Room Temperature - UPLC 119Figure 85: Chromatogram of the Fosinopril Sodium Raw Material Powder After ThermalDegradation at 105°C for 5 Days- UPLC 119Figure 86: Chromatogram of the Benazepril HC1 Raw Material Powder After ThermalDegradation at 105°C for 2 Days- UPLC 120Figure 87: Chromatogram of the HCTZ Raw Material Sample Powder After ThermalDegradation at 105°C for 5 Days- UPLC 120

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Figure 88: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder After ThermalDegradation at 105°C for 5 Days- UPLC 120Figure 89: Chromatogram of the Fosinopril Sodium Tablets Blend Powder After ThermalDegradation at 105°C for 5 Days- UPLC 121Figure 90: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Placebo Powder AfterThermal Degradation at 105°C for 5 Days- UPLC 121Figure 91: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Blend Powder AfterThermal Degradation at 105°C for 5 Days- UPLC 122Figure 92: Chromatogram of the Benazepril HC1 Tablets Placebo Powder After ThermalDegradation at 105°C for 5 Days- UPLC 122Figure 93: Chromatogram of the Benazepril HC1 Tablets Blend Powder After ThermalDegradation at 105°C for 2 Days- UPLC 122Figure 94: Chromatogram of the Benazepril HC1 HCTZ Tablets Placebo Powder After ThermalDegradation at 105°C for 2 Days- UPLC 123Figure 95: Chromatogram of the Benazepril HCI/HCTZ Tablets Blend Powder After ThermalDegradation at 105°C for 2 Days- UPLC 123Figure 96: Chromatogram of the Fosinopril Sodium Raw Material Powder After MoistureSaturated Headspace at 90°C for 30 minutes - UPLC 123Figure 97: Chromatogram of the Benazepril HC1 Raw Material Powder After MoistureSaturated Headspace at 90°C for 30 minutes - UPLC 124Figure 98: Chromatogram of the HCTZ Raw Material Sample Powder After Moisture SaturatedHeadspace at 90°C for 30 minutes - UPLC 124Figure 99: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder After MoistureSaturated Headspace at 90°C for 30 minutes - UPLC 124Figure 100: Chromatogram of the Fosinopril Sodium Tablets Blend Powder After MoistureSaturated Headspace at 90°C for 30 minutes - UPLC 125Figure 101: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Placebo Powder AfterMoisture Saturated Headspace at 90°C for 30 minutes - UPLC 125Figure 102: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Blend Powder AfterMoisture Saturated Headspace at 90°C for 30 minutes - UPLC 125Figure 103: Chromatogram of the Benazepril HCI Tablets Placebo Powder After MoistureSaturated Headspace at 90°C for 30 minutes - UPLC 126Figure 104: Chromatogram of the Benazepril HC1 Tablets Blend Powder After MoistureSaturated Headspace at 90°C for 30 minutes - UPLC 126Figure 105: Chromatogram of the Benazepril HC1JHCTZ Tablets Placebo Powder AfterMoisture Saturated Headspace at 90°C for 30 minutes - UPLC 126Figure 106: Chromatogram of the Benazepril HCI/HCTZ Tablets Blend Powder After MoistureSaturated Headspace at 90°C for 30 minutes - UPLC 127Figure 107: Chromatogram of the Fosinopril Sodium Raw Material Powder After AcidHydrolysis - UPLC 127Figure 108: Chromatogram of the Benazepril HCI Raw Material Powder After Acid Hydrolysis-UPLC 127Figure 109: Chromatogram of the HCTZ Raw Material Sample Powder After Acid Hydrolysis -

UPLC 128Figure 110: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder After AcidHydrolysis - UPLC 128

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Figure 111: Chromatogram of the Fosinopril Sodium Tablets Blend After Acid Hydrolysis -

UPLC 128Figure 112: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Placebo Powder After AcidHydrolysis - UPLC 129Figure 113: Chromatogram of the Fosinopril Sodium!HCTZ Tablets Blend Powder After AcidHydrolysis - UPLC 129Figure 114: Chromatogram of the Benazepril HCI Tablets Placebo Powder After AcidHydrolysis UPLC 129Figure 115: Chromatogram of the Benazepril HC1 Tablets Blend Powder After Acid Hydrolysis-UPLC 130Figure 116: Chromatogram of the Benazepril HC1 HCTZ Tablets Placebo Powder After AcidHydrolysis - UPLC 130Figure 117: Chromatogram of the Benazepril HC1/HCTZ Tablets Blend Powder After AcidHydrolysis UPLC 131Figure 118: Chromatogram of the Fosinopril Sodium Raw Material Powder After BasicHydrolysis - UPLC 131Figure 119: Chromatogram of the Benazepril HC1 Raw Material Powder After BasicHydrolysis - UPLC 131Figure 120: Chromatogram of the HCTZ Raw Material Sample Powder After Basic Hydrolysis-UPLC 132Figure 121: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder After BasicHydrolysis - UPLC 132Figure 122: Chromatogram of the Fosinopril Sodium Tablets Blend Powder After BasicHydrolysis - UPLC 133Figure 123: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Placebo Powder AfterBasic Hydrolysis - UPLC 133Figure 124: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Blend Powder After BasicHydrolysis - UPLC 133Figure 125: Chromatogram of the Benazepril HCI Tablets Placebo Powder After BasicHydrolysis - UPLC 134Figure 126: Chromatogram of the Benazepril HC1 Tablets Blend Powder After BasicHydrolysis - UPLC 134Figure 127: Chromatogram of the Benazepril HCI HCTZ Tablets Placebo Powder After BasicHydrolysis - UPLC 135Figure 128: Chromatogram of the Benazepril HC1 HCTZ Tablets Blend Powder After BasicHydrolysis - UPLC 135Figure 129: Chromatogram of the Fosinopril Sodium Raw Material Powder After Oxidation -

UPLC 135Figure 130: Chromatogram of the Benazepril HC1 Raw Material Powder After Oxidation -

UPLC 136Figure 131: Chromatogram of the HCTZ Raw Material Sample Powder After Oxidation -

UPLC 136Figure 132: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder After Oxidation -

UPLC 136Figure 133: Chromatogram of the Fosinopril Sodium Tablets Blend Powder After Oxidation -

UPLC 137

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Figure 134: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Placebo Powder AfterOxidation - UPLC 137Figure 135: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Blend Powder AfterOxidation - UPLC 137Figure 136: Chromatogram of the Benazepril HC1 Tablets Placebo Powder After Oxidation -

UPLC 138Figure 137: Chromatogram of the Benazepril HC1 Tablets Blend Powder After Oxidation -

UPLC 138Figure 138: Chromatogram of the Benazepril HC1 HCTZ Tablets Placebo Powder AfterOxidation - UPLC 138Figure 139: Chromatogram of the Benazepril HC1 HCTZ Tablets Blend Powder AfterOxidation - UPLC 139Figure 140: Chromatogram of the Blank (Diluent) HPLC 143Figure 141: Chromatogram of the Fosinopril Sodium Raw Material (Control) - HPLC 143Figure 142: Chromatogram of the Benazepril HC1 Raw Material (Control) - HPLC 143Figure 143: Chromatogram of the HCTZ Raw Material (Control) - HPLC 144Figure 144: Chromatogram of the Fosinopril Sodium Tablets Placebo Control - HPLC 144Figure 145: Chromatogram of the Fosinopril Sodium Tablets Blend Control - HPLC 144Figure 146: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Placebo Control - HPLC

145Figure 147: Chromatogram of the Fosinopril Sodium!HCTZ Tablets Blend Control - HPLC 145Figure 148: Chromatogram of the Benazepril HC1 Tablets Placebo Control - HPLC 145Figure 149: Chromatogram of the Benazepril HCI Tablets Blend Control - HPLC 146Figure 150: Chromatogram of the Benazepril HC1/HCTZ Tablets Placebo Control - HPLC. 146Figure 151: Chromatogram of the Benazepril HC1 HCTZ Tablets Blend Control - HPLC.... 146Figure 152: Chromatogram of Fosinopril Sodium Raw Material Sample Solution After UVLight Exposure for 5 Days - HPLC 147Figure 153: Chromatogram of HCTZ Raw Material Sample Solution After UV Light Exposurefor5 Days HPLC 147Figure 154: Chromatogram of HCTZ Raw Material Sample Solution After UV Light for 3Hours-HPLC 148Figure 155: Chromatogram of Benazepril HC1 Raw Material Sample Solution After UV Lightfor5 Days- HPLC 148Figure 156: Chromatogram of Fosinopril Sodium Tablets Placebo Sample Solution After UVLight Exposure for 5 Days HPLC 148Figure 157: Chromatogram of Fosinopril Sodium Tablets Blend Sample Solution After UVLight Exposure for 5 Days - HPLC 149Figure 158: Chromatogram of Fosinopril SodiumJHCTZ Tablets Placebo Sample Solution AfterUV Light Exposure for 5 Days - HPLC 149Figure 159: Chromatogram of Fosinopril SodiumJHCTZ Tablets Blend Sample Solution AfterUV Light Exposure for 5 Days - HPLC 149Figure 160: Chromatogram of Fosinopril SodiumJHCTZ Tablets Placebo Sample Solution AfterUV Light Exposure for 3 Hours - HPLC 150Figure 161: Chromatogram of Fosinopril SodiumJHCTZ Tablets Blend Sample Solution AfterUV Light Exposure for 3 Hours - HPLC 150

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Figure 162: Chromatogram of Benazepril HC1 Tablets Placebo Sample Solution After UV LightExposure for 5 Days - HPLC 151Figure 163: Chromatogram of Benazepril HC1 Tablets Blend Sample Solution After UV LightExposure for 5 Days - HPLC 151Figure 164: Chromatogram of Benazepril HCI HCTZ Tablets Placebo Sample Solution AfterUV Light Exposure for 5 Days - HPLC 151Figure 165: Chromatogram of Benazepril HCI HCTZ Tablets Blend Sample Solution After UVLight Exposure for 5 Days - HPLC 152Figure 166: Chromatogram of Benazepril HC1 HCTZ Tablets Placebo Sample Solution AfterUV Light Exposure for 3 Hours - HPLC 152Figure 167: Chromatogram of Benazepril HCI HCTZ Tablets Blend Sample Solution After UVLight Exposure for 3 Hours - HPLC 152Figure 168: Chromatogram of Fosinopril Sodium Raw Material Dry Powder After StorageUnder UV Light for 5 Days HPLC 153Figure 169: Chromatogram of HCTZ Raw Material Dry Powder After Storage Under UV Lightfor5 Days HPLC 153Figure 170: Chromatogram of Benazepril HC1 Raw Material Dry Powder After Storage UnderUV Light for 5 Days HPLC 154Figure 171: Chromatogram of Fosinopril Sodium Tablets Placebo Dry Powder After StorageUnder UV Light for 5 Days HPLC 154Figure 172: Chromatogram of Fosinopril Sodium Tablets Blend Dry Powder After StorageUnder UV Light for 5 Days HPLC 154Figure 173: Chromatogram of Fosinopril SodiumJHCTZ Tablets Placebo Dry Powder AfterStorage Under UV Light for 5 Days HPLC 155Figure 174: Chromatogram of Fosinopril SodiumIHCTZ Tablets Blend Dry Powder AfterStorage Under UV Light for 5 Days HPLC 155Figure 175: Chromatogram of Benazepril HC1 Tablets Placebo Dry Powder After StorageUnder UV Light for 5 Days - HPLC 155Figure 176: Chromatogram of Benazepril HCI Tablets Blend Dry Powder After Storage UnderUV Light for 5 Days - HPLC 156Figure 177: Chromatogram of Benazepril HC1IHCTZ Tablets Placebo After Storage Under UVLightfor5 Days HPLC 156Figure 178: Chromatogram of Benazepril HCI HCTZ Tablets Blend Dry Powder After StorageUnder UV Light for 5 Days - HPLC 156Figure 179: Chromatogram of the Fosinopril Sodium Raw Material Powder Exposed toHumidity for 5 Days at Room Temperature HPLC 157Figure 180: Chromatogram of the HCTZ Raw Material Sample Powder Exposed to Humidityfor 5 Days at Room Temperature - HPLC 157Figure 181: Chromatogram of Benazepril HC1 Raw Material After Storage Under Humidity for5 Days HPLC 158Figure 182: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder Exposed toHumidity for 5 Days at Room Temperature - HPLC 158Figure 183: Chromatogram of the Fosinopril Sodium Tablets Blend Powder Exposed toHumidity for 5 Days at Room Temperature - HPLC 158Figure 184: Chromatogram of the Fosinopril Sodium!HCTZ Tablets Placebo Powder Exposedto Humidity for 5 Days at Room Temperature - HPLC 159

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Figure 185: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Blend Powder Exposed toHumidity for 5 Days at Room Temperature - HPLC 159Figure 186: Chromatogram of Benazepril HC1 Tablets Placebo After Storage Under Humidityfor5Days-HPLC 159Figure 187: Chromatogram of Benazepril HC1 Tablets Blend After Storage Under Humidity for5Days-HPLC 160Figure 188: Chromatogram of Benazepril HCI HCTZ Tablets Placebo After Storage UnderHumidity for 5 Days - HPLC 160Figure 189: Chromatogram of Benazepril HC1 HCTZ Tablets Blend After Storage UnderHumidity for 5 Days - HPLC 160Figure 190: Chromatogram of the Fosinopril Sodium Raw Material Powder After ThermalDegradation at 105°C for 5 Days- HPLC 161Figure 191: Chromatogram of the Benazepril HC1 Raw Material Powder After ThermalDegradation at 105°C for 2 Days- HPLC 161Figure 192: Chromatogram of the HCTZ Raw Material Sample Powder After ThermalDegradation at 105°C for 5 Days- HPLC 161Figure 193: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder After ThermalDegradation at 105°C for 5 Days- HPLC 162Figure 194: Chromatogram of the Fosinopril Sodium Tablets Blend Powder After ThermalDegradation at 105°C for 5 Days- HPLC 162Figure 195: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Placebo Powder AfterThermal Degradation at 105°C for 5 Days- HPLC 162Figure 196: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Blend Powder AfterThermal Degradation at 105°C for 5 Days- HPLC 163Figure 197: Chromatogram of the Benazepril HC1 Tablets Placebo Powder After ThermalDegradation at 105°C for 2 Days- HPLC 163Figure 198: Chromatogram of the Benazepril HC1 Tablets Blend Powder After ThermalDegradation at 105°C for 2 Days- HPLC 163Figure 199: Chromatogram of the Benazepril HCI HCTZ Tablets Placebo Powder AfterThermal Degradation at 105°C for 2 Days- HPLC 164Figure 200: Chromatogram of the Benazepril HC1 HCTZ Tablets Blend Powder After ThermalDegradation at 105°C for 2 Days- HPLC 164Figure 201: Chromatogram of the Fosinopril Sodium Raw Material Powder After MoistureSaturated Headspace at 90°C for 48 Hours - HPLC 165Figure 202: Chromatogram of the Benazepril HC1 Raw Material Powder After MoistureSaturated Headspace at 90°C for 48 Hours - HPLC 165Figure 203: Chromatogram of the HCTZ Raw Material Sample Powder After MoistureSaturated Headspace at 90°C for 48 Hours - HPLC 166Figure 204: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder After MoistureSaturated Headspace at 90°C for 48 Hours - HPLC 166Figure 205: Chromatogram of the Fosinopril Sodium Tablets Blend Powder After MoistureSaturated Headspace at 90°C for 48 Hours - HPLC 167Figure 206: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Placebo Powder AfterMoisture Saturated Headspace at 90 C for 48 Hours - HPLC 167Figure 207: Chromatogram of the Fosinopril Sodiumi’HCTZ Tablets Blend Powder AfterMoisture Saturated Headspace at 90°C for 48 Hours - HPLC 168

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Figure 208: Chromatogram of the Benazepril HCI Tablets Placebo Powder After MoistureSaturated Headspace at 90°C for 48 Hours - HPLC 168Figure 209: Chromatogram of the Benazepril HC1 Tablets Blend Powder After MoistureSaturated Headspace at 90°C for 48 Hours - HPLC 169Figure 210: Chromatogram of the Benazepril HC1/HCTZ Tablets Placebo Powder AfterMoisture Saturated Headspace at 90°C for 48 Hours - HPLC 169Figure 211: Chromatogram of the Benazepril HC1/HCTZ Tablets Blend Powder After MoistureSaturated Headspace at 90°C for 48 Hours - HPLC 170Figure 212: Chromatogram of the Fosinopril Sodium Raw Material Powder After AcidHydrolysis - HPLC 170Figure 213: Chromatogram of the Benazepril HC1 Raw Material Powder After Acid Hydrolysis-HPLC 171Figure 214: Chromatogram of the HCTZ Raw Material Sample Powder After Acid Hydrolysis

HPLC 171Figure 215: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder After AcidHydrolysis - HPLC 172Figure 216: Chromatogram of the Fosinopril Sodium Tablets Blend After Acid HydrolysisHPLC 172Figure 217: Chromatogram of the Fosinopril Sodiuni/HCTZ Tablets Placebo Powder AfterAcid Hydrolysis - HPLC 173Figure 218: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Blend Powder After AcidHydrolysis - HPLC 173Figure 219: Chromatogram of the Benazepril HCI Tablets Placebo Powder After AcidHydrolysis - HPLC 174Figure 220: Chromatogram of the Benazepril HC1 Tablets Blend Powder After Acid Hydrolysis-HPLC 174Figure 221: Chromatogram of the Benazepril HC1/HCTZ Tablets Placebo Powder After AcidHydrolysis HPLC 175Figure 222: Chromatogram of the Benazepril HC1 HCTZ Tablets Blend Powder After AcidHydrolysis - HPLC 175Figure 223: Chromatogram of the Fosinopril Sodium Raw Material Powder After BasicHydrolysis - HPLC 175Figure 224: Chromatogram of the Benazepril HC1 Raw Material Powder After Basic Hydrolysis-HPLC 176Figure 225: Chromatogram of the HCTZ Raw Material Sample Powder After Basic Hydrolysis

HPLC 176Figure 226: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder After BasicHydrolysis - HPLC 177Figure 227: Chromatogram of the Fosinopril Sodium Tablets Blend Powder After BasicHydrolysis - HPLC 177Figure 228: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Placebo Powder AfterBasic Hydrolysis HPLC 178Figure 229: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Blend Powder After BasicHydrolysis - HPLC 178Figure 230: Chromatogram of the Benazepril HCI Tablets Placebo Powder After BasicHydrolysis - HPLC 179

xxiv

Figure 231: Chromatogram of the Benazepril HC1 Tablets Blend Powder After BasicHydrolysis - HPLC 179Figure 232: Chromatogram of the Benazepril HCI HCTZ Tablets Placebo Powder After BasicHydrolysis - HPLC 180Figure 233: Chromatogram of the Benazepril HC1 HCTZ Tablets Blend Powder After BasicHydrolysis - HPLC 180Figure 234: Chromatogram of the Fosinopril Sodium Raw Material Powder After OxidationHPLC 181Figure 235: Chromatogram of the Benazepril HC1 Raw Material Powder After OxidationHPLC 181Figure 236: Chromatogram of the HCTZ Raw Material Sample Powder After OxidationHPLC 182Figure 237: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder After OxidationHPLC 182Figure 238: Chromatogram of the Fosinopril Sodium Tablets Blend Powder After OxidationHPLC 183Figure 239: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Placebo Powder AfterOxidation - HPLC 183Figure 240: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Blend Powder AfterOxidation - HPLC 184Figure 241: Chromatogram of the Benazepril HCI Tablets Placebo Powder After - HPLC .... 184Figure 242: Chromatogram of the Benazepril HC1 Tablets Blend Powder After OxidationHPLC 185Figure 243: Chromatogram of the Benazepril HC1/HCTZ Tablets Placebo Powder AfterOxidation - HPLC 185Figure 244: Chromatogram of the Benazepril HC1 HCTZ Tablets Blend Powder AfterOxidation - HPLC 186Figure 245: Chromatogram of the Blank (Dissolution Medium) 210Figure 246: Chromatogram of the Blank (Prepared per Standard preparation with methanol).210Figure 247: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder 210Figure 248: Chromatogram of the Fosinopril Sodium!HCTZ Tablets Placebo Powder 211

xxv

INTRODUCTION

History and Overview of Liquid Chromatography

The analysis of each product was executed using a high performance liquid

chromatographic system (HPLC) and an ultra performance liquid chromatographic system

(UPLC). Chromatography was first discovered by Mikhail Tswett in 1903 in an effort to

separate different plant pigments.” Through his studies, Tswett separated the different pigments

using precipitated calcium carbonate packed inside a glass tube. He then passed a variety of

solutions through the tube (all of different polarity), which would separate each of the pigments

according to their different affinities for the solutions. This new method of separation was then

used for quantitative purposes and thus the science of analytical chromatography was born.

However, very little work regarding Tswett’s first attempts at chromatography were revisited

until 1931 by Richard Khun. It was during this time that interest in using in this new technique

for the analysis of not only plants pigments, but vitamins and steroids was increasing.1’Khun’ s

interest in this new science spread outward to other laboratories, including one run by Paul

Karrer in Zurich and Lazlo Zechmeister in Hungary.1’ Many of these new pioneers went on to

win Noble Prizes for their ground-breaking work in the advancement of the chromatographic

sciences. Over the years this technique has become more and more specific as different column

packing materials are constantly being introduced to the market’ and the technique is now a

staple for the pharmaceutical industry.

The primary objective of chromatography is simply “the science of separation.” In the

1 960s the first High Performance Liquid Chromatographic (HPLC) system was developed. This

system could utilize smaller column packings and higher pressures. The first objective of any

chromatographer is to achieve the desired separation as fast as possible.’ Chromatography at

the molecular level uses a mobile phase and a stationary phase to elute different molecules

depending on their affinity of one of the two phases. Being as “like attracts like” a polar

molecule will likely want to stay in a more polar phase, while a non-polar molecule will likely

want to stay in a more non-polar phase. The stationary phase in liquid chromatography is that of

the column, which may be packed with various materials that may be polar or non-polar.

The two most common forms of liquid chromatography may be called either normal

phase or reverse phase chromatography. During normal phase chromatography, the stationary

phase in the packed column is the polar phase, and the mobile phase is the non-polar phase. The

most common form of chromatography is that of reverse phase chromatography in which the

mobile phase is poiar and the stationary phase is the non-polar phase. Therefore, if the column

consists of a non-polar bonded silica gel, the polar phase (or mobile phase) will be the more

polar of the two. The affinity of the molecule for the mobile phase versus the stationary non

poiar column packing will be determined by intermolecular interactions of the molecule in

question. When analyzing multiple compounds, a chromatographer must capitalize on the

different intermolecular interactions of molecules in order to achieve separation between them as

they elute through the column.

The elution of the mobile phase through the packed column can be that of an isocratic or

gradient separation. Isocratic separations utilize one mobile phase that is pushed through the

column at a constant flow without a change in composition. Gradient elution will utilize two or

more different mobile phase solutions, which can be introduced to the column over time at

various concentrations. These gradients usually proceed from a higher percentage aqueous phase

to a more organic containing phase. Gradients can be instrumental in the separation of different

analytes that contain a variety of hydrophobicities.

The mathematical theory behind HPLC is directed by the Van Deemter equation (Figure

1) which is a description of the relationship between terms a, b and c. The a term relates to the

particle size (dr) in the column’s packing material which affects the eddy diffusion of matenal

through the column and the packing constant (?).2 The b term is the relationship between the

longitudinal and linear velocity (u) of the mobile phase in relation to the stationary phase, y an

obstruction factor, and Drn the diffusion coefficient for the mobile phase.2 The c term gives the

mass transfer and is calculated based on the linear velocity, the square of the particle size, the

diffusion coefficient, andf(lc) which is a function of the retention factor (k).2 The sum of these

variables will yield H which is the height equivalent to a theoretical plate. This theoretical plate

in itself is simply the bed length divided by the plate count (N).” Therefore, the smaller the

equivalent of theoretical plates (HETP), the more theoretical plates (N) there are in the column.

Figure 1: The Van Deemter Equation

27D d2 bH =22d + “ +f(k) ° =a+—+cu

u uOnce each variable is plotted, the A term gives a horizontal line, the B term gives a

downward sloping curve as the linear velocity increases, and the C term gives a positive linear

relationship between plate count and particle size. This equation demonstrates that if the particle

size used in a column is reduced by half, the H value would be reduced by half thereby

increasing the column efficiency.1°It is this concept that makes using smaller particles in the

column packing material so beneficial. However, the reduction of particle size causes a marked

increase in system back pressure. Conventional HPLC analysis can occur with back pressures up

to about 400 bar, or 6,000 psi.

A new, improved chromatographic application, Ultra Performance Chromatography

(UPLC) has been developed. As shown by the previously discussed Van Deemter equation, the

efficiency of a chromatographic separation will increase proportionally to a decrease in the

3

particle size.10 This means that a smaller particle size inside the colunm will reduce the height

equivalent of theoretical plates (HETP), which in turn reduces the band broadening seen in the

peaks. As the HETP is further reduced, the Van Deemter curve will begin to flatten, meaning

that as the flow rate is increased there will be fewer negative effects on the column efficiency.’°

As the particle size is decreased, the back pressure on the columns is subsequently

increased.’0 Ultra Performance Liquid Chromatography (UPLC) can take advantage of the sub

2.0 micron particle size and still allows for pressures up to 1030 bar, or 15,000 psi.2 The new

UPLC also utilizes much smaller system volumes so that speed, sensitivity, and resolution during

the sample runs are maintained.’0 One of the first particles developed by Waters to work with

this new technology was that of the bridged ethylsiloxane silica hybrid (BEH) with a size of 1.7

jim. This new packing allowed for much higher pressures with a wide pH range from 1 to 12.10

A form of this particle that was utilized during the development of the method discussed in this

report.

Figure 2: Fosinopril Sodium (Active Pharmaceutical Ingredient # 1) and Fosinoprilat (RC-A)

Fosinopril Sodium is an angiotension converting enzyme (ACE) inhibitor (Figure 2).

Fosinopril Sodium is used in the treatment of hypertension and congestive heart failure.’5 This

compound is administered orally, and may be in a product alone or paired with a diuretic (i.e.

hydrochlorothiazide).’5When in the body, Fosinopril Sodium is converted to the active

4

Fosinopril Sodium

0

0 0

Fosinoprilat

metabolite, Fosinoprilat.3 There are three different classes for ACE inhibitor molecules. The

first is the thiol-containing ACE inhibitors, the second (and largest) is that of the dicarboxyl

containing, and the last and smallest (consisting of Fosinopril alone) is that of the phosphorus-

containing inhibitors.’5 The Fosinopril molecule contains a carboxylic acid group (pKa 4.38)

and is fully ionized at physiological pH.’4 However, the active Fosinoprilat (Figure 2) will

contain a more acidic function of a phosphinic acid group, which is even more acidic than the

carboxylic acid group in water (pKa 3.1). During the metabolism of Fosinopril, the

phosphinate is hydrolyzed to a phosphinic acid. The ACE enzyme active site is where this

phosphinic acid in the Fosinoprilat becomes coordinated to the zinc atom in the enzyme of

interest. 14

For the purposes of separation chemistry, the chemical properties of the molecule of

interest play a significant role in the approach toward the development of the method that will be

used to analyze it in the laboratory. The difference in pKa values between Fosinopril Sodium and

Fosinoprilat in the literature indicated a difference in polarity, which would result in a diverse

affinity to stationary phase.5 The pICa value of Fosinopril Sodium is 4.48 0.11 and for the

corresponding phosphinic acid of Fosinoprilat is 3.356 ± 0.042. The technical package from the

API manufacturer, Hetero Drugs, listed the solubility of Fosinopril Sodium in various pH

solutions. This technical package listed the following results for the solubility studies in Table 1

and 2:

Table 1: Solubilit Pro • erties of Fosino • ru Sodium in Various SolventsName of Solubiity Specification as per Solubifity SolubiitySolvent USP (°C:mglmL) Status

0.1 g of substance is soluble in 3 28°C: 10 SparinglyWaterto 10 mL of solvent 37°C: 10 Soluble

0.1 g of substance is soluble in 0.1 28°C: 100Methanol Freely Solubleto 1 mL is solvent 37°C: 100Methylene 0.01 g of substance is soluble in 1 28°C: 1

Slightly SolubleChloride to 10 mL of solvent 37°C: 10.01 g of substance is soluble in

28°C: 0.1Dimethylgreater than to equal to 100 mL of Insoluble

37°C: 0.1Formamidesolvent

0.01 g of substance is soluble in28°C: 0.1greater than to equal to 100 mL of3 7°C: 0.1 Insoluble

solventAcetone

Hetero Drugs Technical Package

Table 2: Solubility Pro • erties of Fosino • ru Sodium in Various Buffer • HsSolubifity

Solubility SolubilityBuffer pH Solvent Media specifications as per(°C:mg/mL) StatusUSP

3.727 g of KC1 isdissolved in a mixture of

0.01 g of substance is28°C: 0.1soluble in 1 to 100 mL65 mL of 0.2M HC1 and 37°C: 0.1of solvent Insoluble935 mLofHO

10.21 g of potassiumbiphthalate is dissolved 0.01 g of substance is

28°C: 0.1in a mixture of 0.5 mL of soluble in 1 to 100 mL37°C: 0.10.2M of HCI and 999.5 of solvent

mL ofH206.805 g ofKH2PO4are

dissolved in a mixture of0.1 g of substance is

28°C: 10 Sparinglysoluble in 3 to 10 mL145.5 mL of 0.2M NaOH 37°C: 10 Solubleof solventand 854.5 mL ofH203.092g of boric acid and

3.727g of KC1 are 0.1 g of substance is28°C: 10 Sparinglydissolved in a mixture of soluble in 3 to 10 mL37°C: 10 Soluble19.5 mL of 0.2M NaOH of solvent

and 980.5 mL ofH20Hetero Drugs Technical Package

A study by Sunanda A. Ranadive entitled “Relative Lipophilicities and Structural-

Pharmacological Considerations of Various Angiotensin-Converting (ACE) Inhibitors” defined

the lipophilicity of Fosinopril Sodium by determining their octanol-water distribution

6

2.0 Buffer

4.0 Buffer

7.0 Buffer

8.0 Buffer

Insoluble

coefficient.’2 This study utilized various aqueous pHs, which were used to determine not only

the lipophilicity of the ACE inhibitory products, but how this lipophilicity may change with

different pH values. The octanol-water distribution coefficients were determined in pHs ranging

from I to 712 During this study, a known amount of the API of interest was dissolved in

aqueous prior to introduction to the octanol.’2 Many of the compounds of interest were dissolved

in acidic aqueous phase; however, Fosinopril sodium was an exception as the solubility was

already known to be very low at acidic pH. For the lower pH studies, Fosinopril Sodium had to

be dissolved in the octanol first prior to the study.’2 All samples were then mixed and

centrifuged. The separated aqueous phase was then analyzed using HPLC for the amount of API

present.’2 Out of the 11 ACE inhibitory products tested, Fosinopril Sodium was shown to be the

most lipophilic of all.’2 At pH 1, there was no Fosinopril Sodium detected in the aqueous phase

at all. However, the solubility appeared to increase through each pH unit, until the highest tested

pH of 7 demonstrated the highest solubility.’2

During the testing of the two products containing Fosinopril Sodium (Fosinopril Sodium

Tablets and Fosinopril SodiumJHCTZ Tablets) there have been many investigations regarding

possible extraction problems (due to various results) of the assay procedure as well as failing

°0RSD values for replicate injections of the dissolution standard preparation. The dissolution

standard would show a°0RSD of greater than 2.0% for replicate standard injections and for the

assay a 0 difference of greater than 3.00o between replicate assay preparations would cause

failing results. Currently the assay content uniformity (CU) procedure for Fosinopril Sodium

uses a diluent consisting ofO.20oby weight phosphoric acid to acetonitrile in the ratio of 40 to 60

by volume. The dissolution media and standard diluent is water. The low pH of the assay CU

diluent may attribute to the inconsistent extraction results for the product because Fosinopril

Sodium is protonated and, therefore, insoluble at low pH. However, the high organic percentage

in this diluent may have helped to mitigate the solubility problems resulting in sporadic method

failings. However, the previous study outlined above demonstrates that Fosinopril Sodium

should be slightly soluble at neutral pH. Therefore, the discrepancy for the standard preparation

in water still exists.

A study demonstrated that Fosinopril Sodium will form micelles during a self-association

reaction when present in water.’3 This study determined that the higher the concentration of

Fosinopril Sodium in the solution the more pronounced this effect became. At a concentration of

1.5 mg/mL of Fosinopril Sodium, the product would form a typical micelle self-association in

water. Due to the large side chain in Fosinopril Sodium, this product will in turn form micelles

that are present in higher concentrations than expected. At a concentration of 1.81 mg/mL, the

measured size of the micelles was found to be 149 nm. In current Sandoz methods, the

Fosinopril Sodium is not present at this concentration (0.022 mg/mL of Fosinopril Sodium in the

dissolution and 0.2 mg/mL in the assay CU at most). However, this study does not outline the

behavior of the Fosinopril Sodium at lower concentrations as those used the Sandoz analytical

laboratories due to sensitivity issues.

Figure 3: Benazepril Hydrochloride (Active Pharmaceutical Ingredient #2) and Benazeprilat

HCIHC1

Like Fosinopril Sodium, Benazepril Hydrochloride (HC1) is an ACE inhibitor taken

orally for the treatment of hypertension and heart failure (Figure 3))5 Benazepril HC1 can be

8

Benazeprilat

present alone or in a combination product containing the diuretic After

oral administration, Benazepril HC1 will be metabolized in the body to the active form,

Benazeprilat. While Fosinopril Sodium belongs to the class of phosphorus-containing ACE

inhibitors, Benazepril HC1 is in the largest class, that of the carboxylic acid containing ACE

inhibitors.’4 The active Benazeprilat molecule contains two carboxylic acid equivalents after the

hydrolysis of the ester group on the Benazepril molecule.14 At physiological pH (pH 7.4) both

carboxylic acid groups exist as completely ionized, indicating that Benazepril will behave as an

acid.’4 Like Fosinoprilat the Benazeprilat coordinates to the active-site zinc atom in the

angiotension enzyme; however, Benazeprilat will coordinate through the second carboxyl group

near the N-terminus of the molecule.’4

Unlike Fosinopril, Benazepril contains low lipophilicity, with the Benazeprilat metabolite

being even lower.’4 Because Benazepril has much lower lipophilicity than Fosinopril, solubility

problems in aqueous solutions should not pose as much of a problem as with the Fosinopril.

Figure 4: Hydrochiorothiazide (Active Pharmaceutical Ingredient # 3)

00 00\\//

Hydrochlorothiazide (HCTZ) is a thiazide diuretic and used to treat hypertension (Figure

4)•1 6 HCTZ is commonly prescribed to treat edema that can be associated with congestive heart

failure as well as kidney and liver djsorders.’6 Hydrochiorothiazide is characterized as being

slightly soluble in water and sparingly soluble in methanol.’7 HCTZ has over time been shown

to be unstable in aqueous solution; however, the use of an acidic mobile phase can be helpful in

ensuring the stability of the HCTZ molecule during analysis.’6

9

Current Methods Available in Literature

Currently there are several methods available through different publications for the

simultaneous detection of Fosinopril Sodium, Benazepril Hydrochloride, and

Hydrochlorothiazide alone and in combination products. The first study to be discussed was

published in 2001 by Sibel A. Ozkan et al. and utilizes UV detection at 245 nm.3 A reversed

phase HPLC method was developed using a Bondapak C18 10 m 300 mm x 3.9 mm column

and a mobile phase consisting of methanol and water (40:60) with pH adjusted to 4.0 with

phosphoric acid. A sample chromatogram was provided in the publication. However, the

resolution between the two compounds of interest appears to be poor along with the peak shape.

The Fosinopril peak also demonstrates poor peak response. This method was informational in

that the Fosinopril Sodium was shown to be soluble in a diluent consisting of 60° o water.

Another publication was found for the detection of both Fosinopril and HCTZ by Serap

Saglik, et al. in 2001. This method utilizes a gradient HPLC run with a C18 column (250 mm x

4.6 mm, 10 tim) and a mobile phase proportioning between aqueous 10 mM phosphoric acid and

acetonitrile.4 The gradient parameters are described in the Table 3. The samples were analyzed

at 215 nm using UV detection.

Table 3: Saglik Chromatographic ConditionsMobile Phase A (%) Mobile Phase BTime

(mm)(Aqueous 10mM (%)

Flow Rate

H3P04) (Acetonitrile)0.0—4.0 60 40 1.0

4.0 — 6.0 20 1.0 until 5.0 mm then changeto 2.0 mL/min

The method specifies that a 10 minute equilibration time was also added to the run to

ensure that the system had returned to initial conditions before the next sample was injected.

This would give a total run time of at least 16 to possibly 20 minutes.4 However, it was noted

10

that the samples were prepared in 10000 methanol, which could cause some evaporation

problems during sample analysis. Sample analysis using such a high percentage of organic can

also cause peak fronting. Though this problem can sometimes be mitigated through a reduction

in the injection volume, this may be difficult for samples needed for related compound analysis.

A more recent publication in 2008 regarding the analysis of Fosinopril Sodium by Jancic

Biljana and coworkers utilizes a more modem C18 3.5 jim XBridge Column 3.0 mm x 100 mm.5

The analysis was run with UV detection at 220 nm. The run was isocratic using a mobile phase

composition of 50 50 acetonitrile and 25mM potassium phosphate buffer pH 3.0. Though this

method is shown to be adequate for Fosinopril detection, there is no indication of where the

HCTZ peak would elute.5 It was during my developmental activities that this ratio of 50 50

acetonitrile and phosphate buffer was shown to be unsatisfactory, as the HCTZ peak eluted in the

solvent front.

Another method was found that outlines the simultaneous detection of Fosinopril

Sodium, Benazepril HCI, and HCTZ (along with another ACE inhibitor). After consideration of

the advantages that such a method would have, this thesis project was expanded to include that

of Benazepril HC1 as well. This method by L. Manna uses an isocratic reversed phase

chromatography with a mobile phase consisting of 20 mM sodium heptanesulphonate pH 2.5 and

methanol in the ratio of 32 to 68 respectively.’5 The flow rate was set at 1.0 mL mm at 220 nm.

The HCTZ was shown to elute first in the provided chromatogram, followed by Benazepril, and

ending with Fosinopril much later. Now not only will this new method need to incorporate the

assay, content uniformity, and dissolution procedure, but will be used to detect all related

compounds. Assay, content uniformity, and dissolution methods are relatively easy to develop

as you are only interested in your API peaks, but related compounds methods are considerably

much more difficult.

The current USP 33 also provided several monographs for the detection of Fosinopril

Sodium Raw Material, Fosinopril Sodium Tablets, Benazepril HC1 Raw Material, Benazepril

HC1 Tablets, Hydrochiorothiazide Raw Material, Hydrochiorothiazide Tablets, and Fosinopril

SodiumlHCTZ Tablets. The Fosinopril Sodium Raw Material method provided in the USP is the

current method in use at Sandoz, Inc at Wilson, NC for the release of raw materials. As

Fosinopril Sodium contains many different stereoisomers, which are formed during the synthetic

process, the USP monograph employs different related compound tests for the detection of them

all. As these stereoisomers are considered to be process related impurities and are controlled at

such low levels through the raw material method, the need for their separation in the finished

product testing monograph is not required. The separation of these stereoisomers from the active

Fosinopril Sodium also requires different forms of chromatography including ion-exchange and

use of chiral columns. The same situation also applied for the testing of Benazepril HC1 Raw

Material (i.e. many stereoisomers), and therefore, these stereoisomers will be controlled through

the testing of the raw material. The same impurities listed for the testing of the HCTZ raw

material are the same as that given in the USP monograph for HCTZ Tablets. Though there is no

simultaneous method for the determination of Benazepril HC1 HCTZ Tablets, there is one

provided for the determination of Fosinopril SodiumIHCTZ Tablets. Each of the finished

product methods from Sandoz and the USP are outlined in Tables 4-21.

Table 4: Sandoz Monograph vs. USP Monograph Solutions for Fosinopril Na TabletsSandoz HPLC USP HPLCSandoz Diluent USP DiluentFosinopril Na Tablets Mobile Phase Mobile Phase

Assay CU(40:60) (40:60) (78:22) (80:20)

0.2°oH3P04:ACN 0.2%H3P04:ACN MeOH: 0.2°cH3P04 0.2M Urea:ACN

Related Compounds(40:60)

Water Same as Assay Same as Assay0.2°oH3P04:ACN

Dissolution(40:60)

Water (64:3 6)Water0.2°oH3P04:ACN ACN:0.2°o H3P04

Table 5: Sandoz Monograph vs. USP Monograph Solutions for Fosinopril Na in Fosinopril NaIHCTZ Tablets

Fosinopril NaIHCTZ Sandoz HPLC USP HPLCSandoz Diluent USP DiluentTablets Mobile Phase Mobile Phase(40:60) (40:60) (A) 0.O1MH2KPO4

(2:1) 0.OO1N HC1:Assay CU 0.2°0H3P04 0.2° o H3P04 pH 2.0ACNpH 2.0:ACN pH 2.00:ACN (B) ACN - gradient

Related(40:60)

0.2%H3PO4pH Water Same as Assay Same as AssayCompounds2.0:ACN(40:60) (45:35:20)0.O1M

Dissolution 0.2% H3P04pH Water KH2PO4pH Water2.0:ACN 3.0:MeOH:ACN

Table 6: Sandoz Monograph vs. USP Monograph Solutions for Benazepril HC1 Tablets.

Benazepril HCI Sandoz HPLCSandoz Diluent

USP HPLC MobileUSP DiluentTablets Mobile Phase Phase

(36:64) (36:64)(65 :35)0.05M (65 :35)0.05M Tetrabutylammonium TetrabutylammoniumAssay/CU

KH2PO4pH 2.5:ACN KH2PO4pH 2.5:ACN Bromide BromideSolution:MeOH Solution:MeOH

RelatedSame as Assay Same as Assay Same as Assay Same as AssayCompounds

Dissolution Same as Assay Water Same as Assay Water

Table 7: Sandoz Monograph vs. USP Monograph Solutions for Benazepril HC1 in BenazeprilHC1IHCTZ Tablets.

Benazepril HCI Sandoz HPLCSandoz Diluent

USP HPLC MobileUSP Diluent/HCTZ Tablets Mobile Phase Phase

(74:26)0.05M (74:26)0.05MAssay/CUKH2PO4pH 2.5:ACN KH2PO4pH 2.5:ACN

Related NA NASame as Assay Same as AssayCompoundsDissolution Same as Assay 0. iN HC1

Table 8: Sandoz Monograph vs. USP Monograph Solutions for HCTZ in Fosinopril NaJHCTZ

Tablets

FosinoprilSandoz HPLC USP HPLC MobileNaIHCTZ Sandoz Diluent USP DiluentMobile Phase PhaseTablets(90:l0)0.05M (70:30)0.05M (A) 0.O1MKH2PO4pH (2:1)0.OO1NHC1:Assay/CU

KH2PO4pH 2.5:ACN KH2PO4pH 2.5:ACN 2.0 (B) ACN - gradient ACNRelated

Same as Assay Same as Assay Same as Assay Same as AssayCompoundsUV-Vis (45:35:20)0.O1MDissolution Water WaterSpectrophotometer KH2PO4:MeOH:ACN

Table 9: Sandoz Monograph vs. USP Monograph Solutions for HCTZ in Benazepril HC1/HCTZTablets

BenazeprilSandoz HPLC USP HPLC MobileHC1IHCTZ Sandoz Diluent USP DiluentMobile Phase PhaseTablets(74:26)0.05M (74:26)0.05MAssay/CU

KH2PO4pH 2.5:ACN KH2PO4pH 2.5:ACNRelated (90:10) 0.05M NA NASame as AssayCompounds KH2PO4pH 2.5:ACN

Dissolution Same as Assay 0.1 N HC1

Table 10: Sandoz Monograph vs. USP Monograph for Detection Wavelength for Fosinopril NaTablets.

Sandoz UVFosinopril Na Tablets USP WavelengthWavelengthAssayCU 2lOnm 215nm

Related Compounds 2lOnm 215nmDissolution 210 tim 215 nm

Table 11: Sandoz Monograph vs. USP Monograph for Detection Wavelength for Benazepril

HC1 Tablets.

Sandoz UVBenazepril HC1 Tablets USP WavelengthWavelengthAssayCU 2lOmn 240nm

Related Compounds 210 tim 240 urnDissolution 210 tim 240 nm

Table 12: Sandoz Monograph vs. USP Monograph for Detection Wavelength for Fosinopril Nain Fosinopril Na/HCTZ Tablets.

________________ __________________

SandozUVFosinoprilfHCTZ TabletsWavelength [ USP Wavelength

Assay CU 210 tim 206 timRelated Compounds 210 tim 206 urn

Dissolution 210 nm 206 tim

14

Table 13: Sandoz Monograph vs. USP Monograph for Detection Wavelength for Benazepril

HC1 in Benazepril HC1IHCTZ Tablets.

Benazepril HCI IHCTZ Sandoz U’USP WavelengthTablets Wavelength

AssayCU 2lOnmRelated Compounds 210 nm NA

Dissolution 210 nm

Table 14: Sandoz Monograph vs. USP Monograph for Detection Wavelength for HCTZ in

Fosinopril NaJHCTZ Tablets.

I

SandozUVFosinopril/HCTZUSP WavelengthTablets Wavelength

Assay CU 1 270 nm 206 nmRelated Compounds 1 270 nm 206 nm

Dissolution j UV-Vis Method 206 nm

Table 15: Sandoz Monograph vs. USP Monograph for Detection Wavelength for HCTZ in

Benazepril HC1/HCTZ Tablets.

_______________ _______________

Benazepril/HCTZSandoz UV Wavelength USP WavelengthTablets

Assay CU 210Related Compounds 210 NA

Dissolution 210

Table 16: Sandoz Monograph vs. USP Monograph for Chromatographic Columns for

Fosinopril Na Tablets.Fosinopril Na Tablets Sandoz Column USP Column

(Apex ODS)Phenomenex Luna Phenyl HexylLl PacgAssay/CU

5im 4.6 x 250mm4.6 x 300 mm

Related Compounds Same as Assay Same as AssayLi PackingDissolution Same as Assay

4.6 x_150mm 5-.tm


Benazepnl HC1 Tablets.

Benazepril HC1 Tablets Sandoz Column USP Column(Microbondapak Cl 8)

Li PackingWaters Symmetry C8 5-iim 2.6 x 300 mmAssay/CU

4.6mm x 250 mm connected to aLI Packing

3.9 x_300 mmWaters Symmetry C8 5-tim

Same as AssayRelated Compounds4.6mm x 250 mm

Waters Symmetry C8 5-gmDissolution4.6mm x 250 mm Same as Assay


Fosinopril Na in Fosinopril NaJHCTZ Tablets.

Fosinopril Na! HCTZ Tablets ] Sandoz Column USP Column

Phenomenex Luna Phenyl Hexyl (Bondclone Ci 8)Assay CU5jim 4.6 x 250mm Li Packing

Phenomenex Luna Phenyi Hexyl (Bondcione Cl 8)Related Compounds5jim 4.6 x 250mm Li Packing

Phenomenex Luna Phenyl Hexyl (Zorbax SB-CN)DissolutionSjim 4.6 x 250mm LiO Packing

Table 19: Sandoz Monograph vs. USP Monograph for Chromatographic Columns for HCTZ in

Fosinopril NaJHCTZ Tablets.

Fosinopril NaJ HCTZ Tablets Sandoz Column USP ColumnWaters Symmetry C8 Sjim 4.6 x 150 (Bondclone Ci8)Assay CU

mm Li PackingWaters Symmetry C8 Slim 4.6 x 150 (Bondclone Ci8)Related Compounds

mm Li Packing

(Zorbax SB-CN)Dissolution N A (UV-Vis Technique)Li0 Packing

Table 20: Sandoz Monograph vs. USP Monograph for Chromatographic Columns forBenazepril HC1 in Benazepril HC1 HCTZ Tablets.

Benazepril HCI/ HCTZ Tablets J Sandoz Column USP ColumnWaters Symmetry C8 5-jimAssay CU

4.6mm x 250 mmWaters Symmetry C8 5-jim

NARelated Compounds4.6n x 250 mm

Waters Symmetry C8 5-jimDissolution4.6mm x 250_mm

Table 21: Sandoz Monograph vs. USP Monograph for Chromatographic Columns for HCTZ in

Benazepril HCI/HCTZ Tablets.

Benazepril HCIJ HCTZ Tablets Sandoz Column USP Column

Assay CUWaters Symmetry C8 S-jim

4.6mm x_250_mmWaters Symmetry C8 S-jim

NARelated Compounds4.6nmi x 250 mm

Waters Symmetry C8 S-jimDissolution

4.6mm x_250_mm

16

EXPERIMENTAL

Materials for Assay CU and Related Compounds

Fosinopril Sodium (Hetero Drugs), Benazepril Hydrochloride (Farmahispania) and

Hydrochlorothiazide (Cambrex) were supplied by the aforementioned manufacturers. High

purity carbonyl free UV grade acetonitrile was purchased from Burdick and Jackson. Methanol,

concentrated hydrochloric acid (12N), and iN hydrochloric acid were purchased from Fisher

Scientific. The mobile phase consisted of a gradient elution partitioning from mobile phase A

prepared at (95:5 by volume) water pH adjusted to 2.1 0.05 using concentrated hydrochloric

acid and carbonyl free UV grade acetonitrile and mobile phase B consisting of 10000 of the

carbonyl free UV grade acetomtrile.

Equipment for Assay CU and Related Compounds

All analysis were run using a Waters High Performance Liquid Chromatographic (HPLC)

System 2998 and a Waters Acquity Ultra Performance Liquid Chromatographic (UPLC) System,

both equipped with a Photo Diode Array Detector capable of extracting UV spectra at 206-nm.

All data was collected using Waters Millennium Empower software. Excel was used for all data

calculations. The HPLC column was that of a Waters Atlantis T3 4.6mm x 150mm 3-gm

column maintained at 40°C. The UPLC column was a Waters Acquity BEH Phenyl 2.1mm x

100mm 1 .7-.tm column maintained at 40°C. The following gradient elution was used for

HPLC analysis with a flow rate of 1.2 mL mm and 25 jiL injections. The needle wash consisted

of (50:50 by volume) water and methanol. The column was washed in (50:50 by volume) water

and acetonitrile and stored in 1000o acetonitnie.

Table 22: HPLC Linear Gradient for Assay/CU and Related Compounds

Time %A %B0.0 93 7

5.50 93 735.00 15 8535.50 15 8536.00 93 740.00 93 7

The gradient elution used for UPLC analysis used a flow rate of 0.5 mL mm and 4.5 jiL

injections. The injection mode of partial loop with needle overfill was used for all injections.

The weak wash and seal wash was mobile phase A. The strong wash consisted of 10000

acetonitrile. The column was stored in 100% acetonitrile when not in use.

Table 23: UPLC Linear Gradient for Assay/CU and Related Compounds

Time % A % B Curve0.00 100 02.70 100 0 65.50 60 40 68.50 45 55 610.00 25 75 610.50 25 75 610.75 100 0 613.00 100 0 6

18

4-Amino

Components of Interest for Related Compounds

Table 24: Related Corn .ounds with Scientific and Common Name.Validation

Scientific Name Common Name API ProductIDUSP

4-Aznino-6-chloro- 1,3- BenzothiadiazideHCTZ (degradation)beazenedisulfonamide Related Compound I

ARS2H- 1 ,2,4-benzothiadiazine-7- USP Chiorthiazide HCTZ (process related and

sulfonamide,6-chloro- 1,1-dioxide RS degradation)[6-chloro-N-[(6-chloro-7-

sulfamoyl-2,3-dihydro-4H-1 ,2,4-benzothiadiazine-4-yl 1,1 -

dioxide)methyl]3 ,4-dthydro-2H-I ,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide]

Trans-4-cyclohexyl- 1-[[hydroxy- usp FosinoprilRelated Compound Fosinopril Sodium (degradation)phenylbutyl)Phosphinyl]acetyl]-L

AProline[(2-methyl-l-(1-

oxopropoxy)propoxy)(4- HeterodrugsFosinopril Sodium (process related)phenylbusyl)phosphinyl]acetic Impurity B

acid[1 [S*(R*),2a,413]_4phenyl_ 1 -[[[2- Heterodrugs Related

methyl-I-(1- Compound Coxopropoxy)propoxy](4- OR Fosinopril Sodium (degradation)

phenylbutyl)phosphinyl]acetyl]-L- USP Fosinoprilproline Related Compound E

[11 [[S*(R*),2a,4p1 2x,413]-4-cyclohexyl- 1 -[4-cyclohexyl- 1 -[[[2-

Heterodrugs Relatedmethyl- 1-( 1-Compound D Fosinopril Sodium (process related)oxopropoxy)propoxy](4-

phenylbutyl)phosphinyl]acetyl)-Lproline

(3 S)-3-amino-2,3,4,5-tetrahydro-2-Fannhispaniaoxo- 1 H- 1 -benzazepine- 1-acetic Benazepril HC1 (process related)S-Amineacid hydrochloride

(3S,l ‘S)-3-[(1’-Carboxy-3’-phenylproply)amino]-2,3,4,5-

USP BenazeprilBenazepril HC1 (degradation)Benazeprilat Tetrahydro-2-oxo- 1H- I

- Related Compound Cbenazepine-1-acetic acidHydrochloride

((3 S)-3 [[(1 R)- 1 -(Ethoxycarbonyl)3-phenylpropyl]amino]-2,3,4,5-

USP BenazeprilBenazepril HCI (process related)Tetrahydro-2-oxo- 1 H-i-

Related Compound Bbenazazepine-1 -acetic acid,monohydrochloride

Dimer

RC-A

HCTZ Dimer HCTZ (process related)

RC-B

RC-C

RC-D

S-Amine

R,S-Isomer

19

Analysis of Pharmaceutical Formulations for Assay CU and Related Compounds

All samples were assayed for potency by the random selection of 25 tablets from the

desired lot. The tablets were ground to a powder using a mortar and pestle. The equivalent to 20

mg of the present ACE inhibitor in the matrix was weighed into a 500-mL volumetric flask. All

samples were subsequently extracted using a diluent of (50:50 by volume) methanol:0.OO1N HC1

solution to obtain a resulting concentration of 40 ig/mL of the ACE inhibitor product and either

50 jig/mL or 25 .tg/mL of hydrochlorothiazide depending on the dosage strength. Each

preparation was subsequently filtered using a 0.45-.tm filter, discarding the first 5 mL of filtrate.

All samples were assayed for the uniformity of dosage units by the random selection of

10 individual tablets from the desired lot. The tablets were then placed, one each, into individual

volumetric size flasks to yield a target concentration of the ACE inhibitor of 40 .tg/mL. All

samples were subsequently extracted as per the assay for potency procedure using a diluent of

(50:50 by volume) Methanol:0.OO1N HC1 solution to obtain a resulting concentration of 40

.tg/mL of the ACE inhibitor product and either 50 .tg/mL or 25 .g/mL of hydrochlorothiazide

depending on the dosage strength. Each preparation was subsequently filtered using a 0.45-.tm

filter, discarding the first 5 mL of filtrate.

All samples were assayed for chromatographic purity by the random selection of 25

tablets from the desired lot. The tablets were ground to a powder using a mortar and pestle. The

equivalent to 100 mg of the present ACE inhibitor in the matrix was weighed into a 1 00-mL

volumetric flask. All samples were subsequently extracted using a diluent of (50:50 by volume)

Methanol:0.OO1N HC1 solution to obtain a resulting concentration of 1000 jig/mL of the ACE

inhibitor product and either 1250 .tg/mL or 625 ig/mL of hydrochlorothiazide depending on the

20

dosage strength. Each preparation was subsequently centrifuged and or filtered using a O.45-tm

filter, discarding the first 5 mL of filtrate.

Figure 5: Chromatogram of the Reference Standard (UPLC)

0.60

0.40U)C.,

iC.)

Figure 6: Chromatogram of the Fosinopril Sodium ID Solution (UPLC)0.05’

D

Mnutes

c’l

1L ‘-‘.0...

0.00 6.00 7.00

A’

Minutes13.00

-0.02’0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Figure 7: Chromatogram of the Fosinopril SodiumJHCTZ ID Solution (UPLC)0.05’

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00viflutes

21

Figure 8: Chromatogram of the Benazepril HCL ID Solution (UPLC)0.05’

D

Mnutes

Figure 9: Chromatogram of the Benazepril HC1 HCTZ ID Solution (UPLC)0.05’

D

D

-0.02’0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

-0.02’0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Mnutes

Figure 10: Chromatogram of the Ink ID Solution (UPLC)0.20

22

D

C

(I)0

U-

Figure 11: Chromatogram of the Reference Standard (UPLC)0.8’

0.7’

0.6.

0.5’

0.3’

0.2’

0.1’ 10.0 LA AVA

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00Mnutes

Figure 12: Chromatogram of the Fosinopril Sodium ID Solution (HPLC)0.05’

D

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00r’.inutes

Figure 13: Chromatogram of the Fosinopril Sodium/HCTZ ID Solution (HPLC)0.05’

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00Mnutes

23

U.UJU

L* .-0.010

-nn9n0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Mnutes

Figure 15 Chromatogram of the Benazepril HC1IHCTZ ID Solution (HPLC)

Figure 16 Chromatogram of the Ink ID Solution (HPLC)0.05’

0.04.

0.03’

0.02’D

0.01’

0.00i

-0.01’

Figure 14 Chromatogram of the Benazepril HC1 ID Solution (HPLC)

D

D ii:Li!0.000

-0.010

-vu_v I •I’’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 3400 36.00 38.00 48.00

Mnutes

-0.02.0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

inutes

24

Materials for Dissolution Testing

Fosinopril Sodium (Hetero Drugs) and Hydrochiorothiazide (Cambrex) were supplied by

the aforementioned manufacturers. High purity carbonyl free UV grade acetonitrile was

purchased from Burdick and Jackson. Methanol, concentrated hydrochloric acid (12N), and iN

hydrochloric acid were purchased from Fisher Scientific. The mobile phase consisted of a

gradient elution partitioning from mobile phase A prepared at (95:5 by volume) water pH

adjusted to 2.1 0.05 using concentrated hydrochloric acid and carbonyl free UV grade

acetonitrile and mobile phase B consisting of 10000 of the carbonyl free UV grade acetonitrile.

Equipment for Dissolution Testing

Vankel Dissolution Baths equipped with rotating paddles (USP apparatus II) were used

for all dissolution tests. All data analysis were run using a Waters High Performance Liquid

Chromatographic (HPLC) System 2998 equipped with a UV-Vis detector and/or a Photo Diode

Array Detector capable of extracting UV spectra at 206-nm were used. All data was collected

using Waters Millennium Empower software. The Empower Software or Excel was used for all

data calculations. The HPLC column was that of a Waters Atlantis T3 4.6mm x 150mm 3-j.tm

column maintained at 40°C. The following gradient elution was used for HPLC analysis with a

flow rate of 1.2 mL mm and 50 jiL injections. The needle wash consisted of (50:50 by volume)

water and methanol. The column was washed in (50:50 by volume) water and acetonitrile and

stored in 1000o acetonitrile.

Table 25: HPLC Gradient for Dissolution

25

Analysis of Pharmaceutical Formulations for Dissolution

All samples were assessed for rate of dissolution using dissolution apparatus 2 (paddles)

rotating at 50 rpm. Six tablets were tested for each dissolution determination using USP Water

as the dissolution medium. The six samples were prepared by taking the weight of each

individual tablet and performing the dissolution in 900 mL of degassed dissolution medium

allowed to thermally equilibrate to 37°C ± 0.5°C. Samples were collected after 30 minutes and

centrifuged for 10 minutes at 2000 rpm before analysis. Standards were prepared by weighing

22 mg of Fosinopril Sodium and 14 mg of Hydrochlorothiazide into 200-mL volumetric flasks.

Using a pipet 6.0 mL of methanol was added to each flask to dissolve the components.

Standards were diluted to volume using dissolution medium. A subsequent dilution was made

using dilution medium to obtain a final concentration of 22 p.g/mL of Fosinopril Sodium and 14

.ig/mL of Hydrochlorothiazide.

Figure 17 Standard Preparation Using HPLC Method for Dissolution

0.25

U,

C..)

0.150

D

—LL

0.10C’J

0

E

0.00

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00Minutes

_____

Channel 2487Channel 1; Processed Channel: ; Result Id: 31018; Processing Method:Fos HTZ_D iss MD_LC42_LMG

26

RESULTS AND DISSCUSSION

Determination of Molecular PKa Values

The first step that was initiated during the development of this combination method was

to determine the PKa of each analyte of interest through a literature search. The importance of

the PKa of the molecule allows one to determine the pH of the mobile phase used during the

chromatographic method. The pH of the mobile phase will thus determine whether the analyte is

in the ionized form or non-ionized form. If the pH of the mobile phase is near the PKa of the

molecule, complications can arise as the molecule would be present in the solution in both forms,

in equilibrium between the ionized vs. non-ionized. This will cause poor peak shape due to the

different forms of the molecule eluting at different rates resulting in broadening of the peak. The

reported PKaS for protonated Fosinopril Sodium, Benazepril Hydrochloride, and

Hydrochlorothiazide are as follows.

Table 27: •Ka Values for Each API of InterestComponent

Fosinopril Sodium’4Benazepril HC114

Hydrochlorthiazide’9 7.9 and 9.2

Effect of Mobile Phase pH

As a rule during the developmental process, one should control the pH of the mobile

phase preparation so that it is ± 2 pH units from the pKa of the analyte. Because the ACE

inhibitor compounds of interest (Fosinopril Sodium and Benazepril HC1) have PKa5 less than 7

when in their protonated forms, the molecules are more acidic when in lower pH solutions.

Therefore, the use of an acidic mobile phase with a low pH (2.5 or lower) was used to ensure that

all compounds of interest were present completely protonated. This allowed for better peak

27

shapes, which is reflective in lower peak tailing factors. The solubility properties of the

compounds of interest were also determined, as discussed previously in this thesis.

Selection of Extraction Diluent

Hydrochiorothiazide and Benazepril HC1 dissolved easily in all diluents that were

attempted. This included various mixtures of water, acetonitrile, methanol, and 0.2° o phosphoric

acid solution. However, it was much more difficult to determine the correct balance between

aqueous and organic phase for Fosinopril Sodium. The current in-house method for Fosinopril

Sodium and Fosinopril SodiumJHCTZ uses a diluent of (40:60) 0.2° a phosphoric acid solution to

acetonitrile. This high organic content allowed for the solvation of the Fosinopril and was

attempted first. The current method for the accurate detection of Fosinopril Sodium uses a

Phenyl Hexyl Column, or L9 column, with mobile phase being the same as the previously

mentioned diluent. The peak shapes are consistently acceptable with tailing not more than 2.0

and plate counts not less than 3000. However, the HCTZ molecule is very polar and therefore

highly attracted to the very polar mobile phase. The HCTZ would elute out of the column in the

solvent front of the injection and could not be accurately quantified. The peak shape for the

HCTZ was subsequently very poor with noticeable fronting due to the high organic content in

the diluent. This is not a problem for the later eluting Fosinopril peak, but it was problematic for

the HCTZ. The first developmental challenge was to determine a diluent that could be used to

consistently dissolve both components. The best diluent for a method is always the mobile phase

itself. When one uses the same mobile phase for the sample diluent, there will be minimal

chromatographic effects (i.e. spurious peaks) between the mobile phase and the sample injections

as they are already the same. The use of a sample diluent that is very different from that of the

mobile phase can cause various difficulties during chromatography. Fosinopril Sodium was

28

weighed into various solutions as follows in Table 28 and the solubility of the product was

monitored.

Table 28: 0 .timization of Sam.le DiluentDiluent

Soluble Peak Shape(Water:ACN)HCTZ: Yes HCTZ: Acceptable(100:0)

Fosinopril: No Fosinopril: NAHCTZ: Yes HCTZ: Acceptable(90:10)

Fosinopril: No Fosinopril: NAHCTZ: Yes HCTZ: Acceptable(80:20)

Fosinopnl: No Fosinopril: NAHCTZ: Yes HCTZ: Acceptable(70:30)

Fosinopril: Yes Fosinopril: Acceptable•HCTZ: Yes HCTZ: Peak Fronting(65:35)

Fosinopril: Yes Fosinopril: Acceptable

In order to maintain peak shape, while allowing for the highest organic content the

diluent of (70:30) water:ACN was first attempted. Though this diluent was shown to have

acceptable chromatography, it was consistently borderline with the ability to completely dissolve

Fosinopril Sodium. When the Fosinopril Sodium would not dissolve (denoted as a NA in the

table above), micelles were observed floating in suspension throughout the volumetric flask.

These micelles were observed at any concentration over 1 mg/mL and in any solution

preparation with less than 30° o acetonitrile. The Fosinopril Sodium would also not dissolve in

acidic pH adjusted water below pH of 2.5. This was true even if 30°0 of acetonitrile was present

in solution. The Fosinopril Sodium appeared to only dissolve when present in a diluent of water

with not less than 30° a acetonitrile. The Fosinopril Sodium would also not dissolve when

present in a phosphate buffer solution even at only 5 mM. Due to this very sensitive nature of

the Fosinopril Sodium and the known high solubility in methanol, the decision was made to

change the organic diluent from acetonitrile to methanol. By preparing a solution containing

29

5000 methanol and 500o water the Fosinopril Sodium would dissolve easily and rapidly without

the sacrifice of the peak shapes that are observed with the high acetonitrile content.

Selection of a Gradient vs. Isocratic Separation Technique

By reviewing each of the current data and analytical method validation reports for each

analyte, a method development approach could be established. As the HCTZ was currently

being detected with a very high aqueous mobile phase ratio and Fosinopril Sodium was lower

(only 40° o aqueous), the HCTZ is said to be more polar. The HCTZ appears to elute first at

(90:10) aqueous to acetonitrile having a higher affinity for the more polar mobile phase than the

non-polar stationary phase that exists on the silica column. The Fosinopril Sodium tends to

retain longer on an analytical column and will not preferentially elute with the mobile phase until

a much higher organic content is passed through the column. During isocratic chromatography,

the longitudinal diffusion will increase the bandwidth of the separating molecules over time.23

Due to the polarity differences between the two molecules, an isocratic elution that would allow

for reasonable retention of the HCTZ molecule would result in such a late eluting Fosinopril

peak that the broadening may make it undetectable from the baseline (if it even elutes from the

column at all). Therefore, a gradient separation was employed for this method.

Gradient Optimization

The first gradient separation for HCTZ and Fosinopril Sodium was achieved using the

HPLC method provided in the current USP monograph for Fosinopnl SodiumJHCTZ Tablets.

This gradient functioned by progressing through an initial starting condition of (88:12 v v) 0.2°c

phosphoric acid solution at pH 2.0 to acetonitrile to (10:90 v v) 0.2° o phosphoric acid solution

pH 2.0 to acetonitrile. However, during the gradient many unknown spurious peaks were

observed around the same time as the Fosinopril Sodium and the related compounds peaks,

30

which warranted further investigation. The first possible cause for these “ghost peaks” could be

that of mobile phase contamination and the band compression mechanism.23 One of the benefits

of gradient elutions is the band compression that occurs as the elutropic strength of the mobile

phase increases.23 By increasing the elutropic strength of the mobile phase as the gradient

progresses, the bands can become more focused on the head of the column, which improve peak

shapes for later eluting peaks.

As the amount of acetonitrile in the gradient was increased, the baseline noise was shown

to increase along with many spurious peaks. Though all solutions were prepared using the

common ACS grade materials, each proved to be unsuitable for a gradient analysis at such a low

wavelength (206 nm). The first culprit considered was that of the phosphoric acid that was used

to pH the mobile phase solution. As phosphoric acid is a weaker acid than one such as

hydrochloric acid, more is required to accurately adjust pH in an aqueous solution. Therefore, to

minimize the effect possible contaminants may have on the gradient separation the acid was

changed to use concentrated hydrochloric acid (12N).

The water that is currently supplied to the laboratory is standard USP de-ionized water.

After changing the mobile phase from using regular laboratory USP de-ionized water to high

purity water purchased from Burdick and Jackson with pH adjustment using hydrochloric acid,

many of the anomalous peaks were reduced. However, bacterial growth can cause ghost peaks

in chromatography. Based on the recommendation stated by Williams the aqueous mobile phase

preparation was mixed in the ratio of 9500 aqueous with 5° 0 acetonitrile to limit this bacterial

growth and extend the expiration period of the mobile phase.23

Organic impurities exiting the organic phase of the gradient separation may cause late

eluting ghost peaks.23 At the beginning of the gradient, which utilizes a low elutropic strength,

the organic impurities from the organic phase will retain on the column until the gradient reaches

about 9000 organic. To prevent these effects, ultra pure carbonyl free UV grade acetonitrile

purchased from Burdick and Jackson was used for the gradient elution.

Discussion of Method Validation

After the successful development of an analytical method, there soon follows the

analytical method validation phase of method qualification. The ICH (International Conference

on Harmonization) lists precision, accuracy, limit of detection, limit of quantitation, specificity,

linearity, range, robustness, and system suitability as the key parameters that must be defined

during analytical method validation. During the execution of the method validation a

representative placebo mixture was prepared for each finished product formulation. The placebo

for each product was prepared at the highest variance for every excipient in each formulation.

Therefore, the excipients used in the placebo preparation for each product would be present in

the matrix at the highest possible variance, and representative of all formulations for the

respective product.

The first factor for discussion is specificity. Specificity refers to “the ability to assess

unequivocally the analyte in the presence of components which may be expected to be

present.”25 Specificity is assessed through analysis of the placebo matrix for finished product

dosage forms, the diluent, impurities, and degradation products. The specificity of an analytical

method is determined through a series of studies. If the degradation profile of an API or drug

product is well established, the spiking of each impurity at appropriate levels and demonstrating

adequate separation (resolution not less than 1.0) is usually strong evidence of method

specificity.24 Subsequent recovery studies of these spiked samples are used to demonstrate the

sample matrix is unbiased on the report results.

32

Forced degradation studies may be used to further strengthen the specificity of an

analytical method. During these studies samples of placebo, API and finished product are

subjected to various stressed conditions to determine the degradative pathways of the product.

These conditions may consist of thermal, oxidative, light, ultraviolet, humidity, acid hydrolysis,

and basic hydrolysis. The target degradation for each condition is not more than 1500

degradation of the starting material.30 Common degradation routes for functional groups are

outlined in the Table 29 below.29

Table 29: Common Degradation Pathways of Various Functional GroupsFunctional Group Degradation Route - Degradants

Acetals Ketones/Aldehydes/AlcoholsEster/Lactones Acids/Alcohols

. HydrolysisAmides/Lactones Aimnes/AcidsAlkenes Alcohols

2° and 3° Amines N-Oxide, hydroxylamineThiols Disulfide

Thioethers Sulfoxide, sulfoneAlkenes Epoxide

Allylic Alcohols Oxidation (radical, light, metal, A, J3, unsaturated ketonesAldehyde peroxide mediated) AcidsAlcohol Ketones, acids

Oxazoles/Imidazoles Various productsDienes (able to aromatized) Aromatic rings

BenzyllAllylic Groups Benzylic/allylic alcoholsTable Provided by Anne-Francoise Aubry et al. “Chapter 7: Development of Stability Indicating Methods29”

Linearity is the next aspect of method validation, which is “the ability of an analytical

procedure to yield test results which are directly proportional to the concentration of the analyte

in the sample.”25 Linearity demonstrates that various concentration levels of an analyte will

yield a linear response across the range of the method, with a correlation coefficient of about 1,

when injected. The assay linearity should cover a minimum of five concentrations bracketing

the target concentration and cover the specified range of the method (see Table 30). For a related

compounds method this linearity must cover from the limit of quantitation (LOQ) up to 1200o of

the specification limit of each related compound.3°Dissolution should cover 1O°o of the lowest

detected analyte concentration up to at least 120° 0 of the target analyte concentration.

33

The Limit of Quantitation (LOQ) and limit of detection (LOD) must be determined for

chromatographic methods that determine very low levels of impurity degradation products and

residual solvents.24 The LOQ is the lowest level an analyte of interest can be quantitated with

suitable accuracy and precision, while the LOD is the lowest level that the concentration can be

detected.. While the LOD level may differ between chromatographic systems due to detector

age and system noise, the LOQ must be attained regardless of the system that is used. Therefore,

the determination of the signal-to-noise ratio (not less than 10:1 is acceptable) is determined

during the validation of the analytical method.

Accuracy is a measure of how close an experimental value is to the true value and may be

assessed by three samples at three concentrations each covering the specified range. The results

are calculated as a 0 recovery and must be within certain pre-set acceptance criteria (refer to

Attachment 3, SOP QC-117). The accuracy for each test should be assessed in the specific

sample matrix (i.e. placebo) to determine if the matrix is un-biased on the analytical results.

Table 30: Minimum Analytical Ranges for Method ValidationTest Range

Assay 80% - 120%Content Uniformity 70% - 130%

Impurities LOQ to 120% of the specification10% of the lowest detected analyte

Dissolution concentration up to 120% of the targetvalue

The next area of method validation is that of method precision and is defined as “the

measure of how close the data values agree to each other for a number of measurements under

the same analytical conditions.”25 Method precision may be further dissected into repeatability,

reproducibility, and intermediate precision. Method precision demonstrates that one analyst can

effectively execute a given method, with results demonstrating an acceptable standard deviation

34

from one another according to pre-set acceptance criteria (refer to Attachment 3 SOP QC- 117 for

these acceptance criteria). Reproducibility can be demonstrated as inter-laboratory precision, in

which two separate labs can achieve similar results. Intermediate precision shows that a separate

chemist on a different day using all new solutions and equipment can achieve similar results

when compared to the initial repeatability testing.

Robustness aids to establish the reliability of an analytical method when exposed to

deliberate variations from the set parameters.24 The robustness of the mobile phase preparation,

buffer pH, extraction procedures, filtration, columns, systems, and pre-set chromatographic

parameters are all exposed to these deliberate changes which later help to establish the

permissible variations of the analytical method during routine laboratory use. These robustness

results also aid in establishing the system suitability criteria for the final analytical method.

The establishment of system suitability is an important part of determining that a

chromatographic system is adequate for the intended test. During development and method

validation robustness experiments various adjustments to the chromatographic parameters may

be made to establish the tolerances of the analytical method. The first factor to be assessed

during system suitability is the tailing factor (T), which is a measure of the peak symmetry. As

peak tailing is increased the precision of the method decreases as integration of the peak become

less reliable. The number of theoretical plates (N) is used to demonstrate the efficiency of the

chromatographic system. The number of theoretical plates is determined by the following

equation where t is the retention time of the peak and W is the peak width.

Figure 18 Equation for Theoretical Plates

N=161

An important factor in the establishment of system suitability is the resolving power of

the chromatographic system (R). When separating two components one needs to be able to en

35

sure that they components are separated enough for accurate analytical quantitation. The resolu

tion of two peaks may be determined by the following equation where t is the retention times of

the two components and W are the widths of the two peaks.

Figure 19 Equation for Resolution

R2(t2 — t1)

w2+

Another aspect of robustness testing is the establishment of the stability of the sample

solutions. During solution stability testing all solutions will be stored under various conditions

(i.e. room temperature, under refrigeration, amber vs. clear glassware, etc) to determine the

optimum storage conditions for samples during time of possible use.

Results of Method Validation Experiments for Assay Content Uniformity

The linearity for assay was assessed across the range of the analytical method. Because

the external standards used in the analytical method are the same for both the assay content

uniformity and the related compounds analysis the linearity was performed for the main

components across the range of 2000 o of the target sample concentration down to the limit of

quantitation (0.05° o). The acceptance criteria for the linearity is not less than 0.999 for the

correlation coefficient and the °oy-intercept must be less than 2° o of the target sample

concentration. As shown in the table below all results met the preset acceptance criteria.

Table 31: Assa Linearit Results for UPLCComponent Correlation Coefficient (R) %Y-Intercept

Fosinopril Sodium 0.999982796Benazepril Hydrochloride 0.999995467

Hydrochlorothiazide 0.999996050Aia1yst JSM

Table 32: Assa Linearit Results for HPLCComponent Correlation Coefficient (R) %Y-Intercept

Fosinopril Sodium 0.999991241Benazepril Hydrochloride 0.999997088

Hydrochlorothiazide 0.999995615

36

The accuracy for assay was assessed across the range of 50% of the analyte concentration

(20 WmL) up to 200°c of the analyte concentration (80 ig/mL) for ACE inhibitors. The

accuracy study was executed for HCTZ from 50% of the lowest analyte concentration up to

200°c of the highest analyte concentration. The study was executed by the preparation of three

representative samples at three concentrations. Each sample was spiked with known amounts of

raw material in placebo matrix and prepared according to the analytical method for assay. The

acceptance criteria for the assay accuracy study are 97° o - 103° o for each individual preparation

and 98° o - 102° o for the mean recovery at each level. As shown in the following table all aspects

of the accuracy study met all preset acceptance criteria.

Table 33: Assay Accuracy Results for Fosinopril Sodium Tablets for UPLC%Level Individual Results Mean Level Result

100%50% 100% 100%

99%99%

100% 99% 99%100%99%

200% 100% 100%100%

Analyst: JU

Table 34: Assay Accuracy Results for Fosinopril Sodium/HCTZ Tablets (Fosinopril Sodium)for UPLC

%Level Individual Results Mean Level Result100%

50% 100% 100%100%100%

100% 100% 100%100%100%

200% 100% 100%100%

Analyst: JSM

Table 35: Assay Accuracy Results for Fosinopril Sodium!HCTZ Tablets (HCTZ) for UPLC%Level (Based on 5Ojtg/InL) Individual Results Mean Level Result

100%25% 100% 100%

100%100%

100% 100% 100%100%100%

200% 101% 100%100%

Analyst: JU

Table 36: Assay Accuracy Results for Benazepril HC1 Tablets for UPLC%Level Individual Results Mean Level Result

100%50% 100% 100%

100%100%

100% 100% 100%100%100%

200% 100% 100%100%

Analyst: JU

Table 37: Assay Accuracy Results for Benazepril HC1/HCTZ Tablets (Benazepril HC1) UPLC%Level Individual Results Mean Level Result

100%50% 100% 100%

100%101%

100% 100% 100%100%100%

200% 100% 100%100%

Analyst: JU

38

Table 38: Assay Accuracy Results for Benazepril HC1/HCTZ Tablets (HCTZ) for UPLC%Level (Based on 5Otg/inL) Individual Results Mean Level Result

100%25% 100% 100%

99%99%

100% 99% 99%99%99%

200% 99% 99%99%

Analyst: JU

Table 39: Assay Accuracy Results for Fosinopril Sodium Tablets for HPLC%Level Individual Results Mean Level Result

100%50% 100% 100%

100%100%

100% 100% 100%100%100%

200% 100% 100%100%

Analyst: JU

Table 40: Assay Accuracy Results for Fosinopril SodiumJHCTZ Tablets (Fosinopril Sodium)for HPLC

%Level Individual Results Mean Level Result100%

50% 100% 100%100%100%

100% 100% 100%100%100%

200% 100% 100%100%

Analyst: JSM

39

Table 41: Assay Accuracy Results for Fosinopril SodiumfHCTZ Tablets (HCTZ) for HPLC%Level (Based on 5Oig/inL) Individual Results Mean Level Result

100%25% 100% 100%

100%100%

100% 100% 100%100%99%

200% 99% 99%99%

Analyst: JU

Table 42: Assay Accuracy Results for Benazepril HCI Tablets for HPLC%Level Individual Results Mean Level Result

100%50% 100% 100%

100%100%

100% 100% 100%100%100%

200% 99% 100%101%

Analyst: JU

Table 43: Assay Accuracy Results for Benazepril HC1/HCTZ Tablets (Benazepril HC1) HPLC%Level Individual Results Mean Level Result

100%50% 100% 100%

100%100%

100% 100% 100%100%100%

200% 100% 100%100%

Analyst: JU

40

Table 44: Assay Accuracy Results for Benazepril HCI/HCTZ Tablets (HCTZ) for HPLC%Level (Based on 5Optg/mL) Individual Results Mean Level Result

99%25% 98% 99%

99%99%

100% 99% 99%99%99%

200% 99% 99%99%

Analyst: JU

The precision studies for the assay method were executed for both repeatability and

intermediate precision. As this test method will be in use at Sandoz, Inc in Wilson, NC only,

reproducibility (inter-laboratory precision) was not required as this time. However, in the future

should the need arise to transfer this method to an outside laboratory, this aspect of method

validation will need to be addressed at that time as part of the transfer process. All precision

studies were executed using actual expired finished product for each formulation. The “worst

case” formulation for each formulation was shown to be that of the lowest strength for the ACE

inhibitor as each product tested was shown to be dosed similar across each strength for the

respective product. All testing was also performed using expired samples so that no product

currently available on the market would be affected by the results of any validation experiment.

Therefore, some of the assay values obtained for each study were shown to be outside of their

usual specification values due to the products being well past their date of expiration. The

repeatability was assessed by six preparations of the finished product. The acceptance criteria

was a°0RSD of less than or equal to 2.0° o. The intermediate precision was assessed by the

analysis of another six sample preparations of the same product by a different chemist, on a

different day, using different chromatographic systems, solutions, mobile phase, standards, and

columns. The acceptance criteria for intermediate precision are the same as repeatability. The

41

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Table 51: Assay Precision Results for Benazepril HCI/HCTZ 5 mg/6.25 mg Tablets on UPLCRepeatability: JSM Intermediate Precision: JU

UPLC System # 03 Asset # 16874 UPLC System # 04 Asset # 16875Sample # Column Serial # 1233031215536 Column Serial # 1233031215541Benazepril HCTZ Benazepril HCTZ

1 99.189 95.474 97.704 95.4352 99.369 95.780 97.73 1 95.4063 99.251 95.495 97.760 95.3314 98.516 95.788 98.405 94.2005 98.903 95.498 97.801 95.0826 99.206 96.218 97.786 95.384

Mean 99.1 95.7 97.9 95.1%RSD 0.3 0.3 0.3 0.5

%Difference (Benazepril) 1.2%Difference (HCTZ) 0.6

Table 52: Assay Precision Results for Benazepril HC1/HCTZ 5 mg/6.25 mg Tablets on HPLCRepeatability (JSM) Intermediate Precision (JU)

HPLC System # 01 Asset # 16930 HPLC System #41 Asset # 16938Sample # Column Serial # 12030309136 Column Serial # 117303014072Benazepril HCTZ Benazepril HCTZ

1 99.660 96.121 97.953 95.7502 99.869 96.333 98.020 95.7413 99.745 96.007 98.080 95.6994 99.361 96.583 98.506 95.6165 99.601 96.244 98.070 95.6496 99.915 96.954 98.130 95.930

Mean 99.7 96.4 98.1 95.7%RSD 0.2 0.4 0.2 0.1

%Difference (Benazepril) 1.6%Difference (HCTZ) 0.7

The test method for the assay method uses a weighed portion from a homogenous sample

grind. However, the content uniformity method uses individual whole intact tablets. Because

there is a difference between the two extraction procedures, one being from a sample grind and

the other being an entire tablet, the precision of the content uniformity preparation was evaluated

as part of the method validation activities. During routine testing, the content uniformity of the

finished product is the assay of 10 individual units (i.e. tablets). To be in accordance with the

analytical method, the validation studies for content uniformity were executed using 10

44

individual tablets for each finished product. The ruggedness studies (precision) were executed

by two different chemists on two different days, using different equipment, columns, solutions,

standards, and mobile phase preparation. The acceptance criteria for the day 1 ruggedness was

to obtain an acceptance value (AV) of not more than 15.0. The acceptance criteria for the day 2

ruggedness was the same as for day 1, but the difference in mean between day 1 and day 2 was

not more than 2° o. All results met the pre-set acceptance criteria for precision of content

uniformity. The content uniformity sample preparation was executed at the same time as the

precision samples for assay. Therefore, the aforementioned analyst, system and column number

are applicable to this analysis as well.

Table 53: Content Uniformity Results for {JPLCDay 1 AV Day 2 AV %Difference BetweenSample

(JSM) (JU) MeansFosinopril Sodium 10mg Tablets 11.1 11.8 0.0

Fosinopril SodiumJHCTZ 10 mg/12.5 mg6.8 7.0 0.8Tablets_(Fosinopril)

Fosinopril SodiumJHCTZ 10 mgll2.5 mg2.5 2.7 0.2Tablets_(HCTZ)

Benazepril HC1 5 mg Tablets 5.8 5.2 0.0Benazepril HC1/HCTZ 5 mg/6.25 mg

3.0 2.7 0.9Tablets_(Benazepril)Benazepril HC1IHCTZ 5 mg/6.25 mg

5.2 5.1 0.3Tablets_(HCTZ)

Table 54: Content Uniformity Results for HPLCDay 1 AV Day 2 AV %Difference BetweenSample

(JSM) (JU) MeansFosinopril Sodium 10 mg Tablets 11.3 12.4 0.1

Fosinopril Sodiumi’HCTZ 10 mg/12.5 mg7.1 7.4 0.8Tablets_(Fosinopril)

Fosinopril SodiumIHCTZ 10 mg/12.5 mg2.5 2.7 0.7Tablets_(HCTZ)

Benazepril HC1 5 mg Tablets 5.7 6.3 1.4Benazepril HC1/HCTZ 5 mgl6.25 mg

2.9 2.6 0.7Tablets_(Benazepril)Benazepril HC1/HCTZ 5 mg/6.25 mg

4.6 0.8Tablets_(HCTZ)

45

The specificity of the analytical method was evaluated by preparations of the placebo and

for each finished product. The chromatograms of the placebo and diluent injections were then

analyzed for possible interferences, which could cause bias on any analytical data. The

acceptance criterion for the assay method was no placebo peak occurring at the same time of the

corresponding active peak above 100 of the target sample concentration.

Figure 20 Chromatogram of the Blank (Diluent) on UPLC0.05

0.0C,,

• C

0.02D

0.01

0.00

-0.01

-0.020.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Mnutes

Figure 21 Chromatogram of the Fosinopril Sodium Tablets Placebo for Assay (UPLC)0.05

C’,

0.0

0.03

0.02D

0.01

0.00

-0.01

-0.020.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Mnutes

46

Figure 22 Chromatogram of the Fosinopril SodiumIHCTZ Tablets Placebo for Assay (UPLC)0.05

tf)

0.04

C)

0.03-

CCu

0.02D

0.01

0.00

-0.01

-0.020.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

vinutes

Figure 23 Chromatogram of the Benazepril HC1 Tablets Placebo for Assay (UPLC)0.05

U,U,

0.0

0.03

u-S0.02 :

0.01 —

0.00

-0.01

-0.020.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Mnutes

Figure 24 Chromatogram of the Benazepril HC1/HCTZ Tablets Placebo for Assay (UPLC)0.05

0.04

0.03 Atypical baseline disturbance. This is not aresult of the specificity solution.

c—I0.02 U,D

0.01

0.00

-0.01

-0.020.00 2.00 4.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Mnutes

47

Figure 25 Chromatogram of the Blank (Diluent) on HPLC0.05’

D

D

0.04

0.03

0.02

0.01

0.00

-0.01

-0.020.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 3400 36.00 38.00 40.00

Mnutes

Figure 27 Chromatogram of the Fosinopril Sodium/HCTZ Tablets Placebo for Assay (HPLC)0.05’

D

-0.02’ -

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00Mnutes

Figure 26 Chromatogram of the Fosinopril Sodium Tablets Placebo for Assay (HPLC)0.05

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Mnutes

48

Figure 28 Chromatogram of the Benazepril HC1 Tablets Placebo for Assay (HPLC)

Mnutes

Figure 29 Chromatogram of the Benazepril HC1/HCTZ Tablets Placebo for Assay (HPLC)0.05

0.04

0.03

0.02

RedDye0.01

0.00

-0.01

-0.020.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Mnutes

The specificity of the assay method was further characterized through forced degradation

studies. During these studies samples were exposed various extreme conditions to demonstrate

that degradation of the samples could be observed by the analytical method. All samples were

injected using a photo-diode array (PDA) detector and analyzed for peak purity. The purity

analysis using PDA detection allows for the analyst to observe the angle of inflection of the peak

in comparison to the underlying baseline. Therefore, if an unknown or related compound peak

were to co-elute with the active peak of interest, the purity angle would be greater than the

threshold angle. The acceptance criterion for all forced degradation samples was that the purity

angle for the main component peak must be less than the threshold angle for the method to be

49

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

considered stability indicating. The target degradation for each condition is not more than 15%;

however, a higher percentage of degradation was considered acceptable on a case-by-case basis.

However, if no degradation was observed for the “worse-case” conditions outlined in the table

below then the condition was considered not to be a degradation pathway of the product. Each

condition was tested using a sample of the raw materials alone, the finished product blend, and

the placebo and compared against control samples.

Table 55: Forced Degradation Conditions for Fosinopril Sodium Tablets and FosinoprilSodium/HCTZ TabletsCondition Time

5 days under the short and long wavelengths(Fosinopril)Ultraviolet Degradation of Solutions3 hours under the short and long wavelengths(HCTZ)

Ultraviolet Degradation of Dry Powders 5 days under the short and long wavelengthsThermal Degradation of Dry Powders 5 days at 105°CHumidity Exposure of Dry Powders 5 days at Room TemperatureDry Powders Under Moisture Saturated

3 hours at 90°CHeadspace (MSH)Acidic Hydrolysis O.1N HC1 in water bath at 60°C for 10 minutes

0.0 iN NaOH at room temperature for 10Alkaline Hydrolysisminutes

Oxidation 10% H202in water bath at 60°C for 5 minutesOxidation (less harsh) 3% H202 in water bath at 60°C for 5 minutes

Table 56: Forced Degradation Conditions for Benazepril HC1 Tablets and BenazeprilHC1 HCTZ TabletsCondition Time

5 days under the short and long wavelengths(Benazepril)Ultraviolet Degradation of Solutions3 hours under the short and long wavelengths(HCTZ)

Ultraviolet Degradation of Dry Powders 5 days under the short and long wavelengthsThermal Degradation of Dry Powders 2 days at 105°C

Humidity Exposure of Dry Powders 5 days at Room TemperatureDry Powders Under Moisture Saturated

3 hours at 90°CHeadspaceAcidic Hydrolysis iN HC1 in water bath at 60°C for 2 HoursAlkaline Hydrolysis 0. iN NaOH at room temperature for 5 minutesOxidation 10% H202 in water bath at 60°C for 1 Hour

50

As shown by the forced degradation experiments below both the UPLC and the HPLC

methods were demonstrated as being stability indicating for the assay methods. In all conditions

the purity angle was shown to be less than the purity threshold at the baseline, indicating there

were no underlying degradations peaks under the active peak. Both Fosinopril and Benazepril

were shown to be the most sensitive to thermal degradation when in the finished product dosage

form. However, this degree of degradation was not observed by the raw materials alone thus

indicating that the placebo actually catalyzes this heat degradation. Both the Fosinopril and

Benazepril finished product blends degraded in the moisture saturated headspace, which is

another form of heated condition when under humidity. Both the Fosinopril and Benazepril in

their respective formulations degraded into their primary related compounds of Fosinoprilat and

Benazeprilat. Due to this heat sensitivity of the finished product, the sample should always be

stored under ambient conditions without excessive heat. Both the Fosinopril and Benazepril

(raw materials and finished products)- were shown to be sensitive to hydrolysis in both acidic

and basic conditions. This was to be expected as the primary related compounds (Fosinoprilat

and Benazeprilat) are the result of hydrolysis reactions. The HCTZ was shown to be the most

sensitive to UV degradation when in the sample solution. However, some degradation can be

observed in the chromatograms of the dry powders over 5 days, though all peaks were under the

limit of quantitation (LOQ). The HCTZ raw material and finished product was shown to degrade

into the 4-amino impurity primarily through acidic hydrolysis. This would probably be observed

during basic hydrolysis should a more harsh condition have been explored. HCTZ was shown to

be stable against thermal degradation unlike the aforementioned ACE inhibitors. Further

discussion regarding the degradation of each product will be provided in the related compounds

section of this report.

Table 57: Forced Degradation Results for Fosinovril Sodium Tablets on UPLCRaw Material I Blend

Condition Time Temp % Purity Threshold % Purity ThresholdDegradation Angle Angle Degradation Angle Angle

Control NA NA NA 0.112 1.028 NA 0.138 1.031UvSolutions 3Hrs RT 1 0.122 1.028 1 0.117 1.028UV Solutions 5 Days RT 0 0.777 1.028 4 0.575 1.027

UV (Dry) 5 Days RT 0 0.767 1.028 3 0.595 1.03 1Thermal (Dry) 5 Days 105°C 1 0.609 1.027 21 0.504 1.053Humidity(Dry) 5 Days RT 1 0.536 1.028 2 0.550 1.026

MSH 3 Hrs 90°C 2 0.337 1.028 7 0.309 1.03 1Acidic

10Mm 60°C 33 0.124 1.041 10 0.115 1.032(0.INHC1)Alkaline

10Mm RT 12 0.114 1.033 2 0.103 1.029(0.0 iN NaOH)Oxidation

5Mm 60°C 30 0.126 1.041 33 0.129 1.039(10% H202)Oxidation

5Mm 60°C 13 0.311 1.028 23 0.442 1.036(3%H2O2) I I

Analysts: JSM and JU

Note: All %degradation is based on the assay of the control sample.

Table 58: Forced Degradation Results for Fosinopril SodiumIHCTZ Tablets (Fosinopril) onUPLC

Raw Material BlendCondition Time Temp % Purity Threshold % Purity Threshold

Degradation Angle Angle Degradation Angle AngleControl NA NA NA 0.112 1.028 NA 0.115 1.029

UV Solutions 3 Hrs RT 1 0.122 1.028 0 0.100 1.028UV Solutions 5 Days RT 0 0.777 1.028 11 0.517 1.05 1

UV (Dry) 5 Days RT 0 0.767 1.028 1 0.668 1.035Thermal (Dry) 5 Days 105°C 1 0.609 1.027 25 0.485 1.157Humidity (Dry) 5 Days RT 1 0.536 1.028 0 0.437 1.027

MSH 3 Hrs 90°C 2 0.337 1.028 4 0.264 1.027Acidic

10Mm 60°C 33 0.124 1.041 5 0.137 1.030(0.IN HC1)Alkaline

10Mm RT 12 0.114 1.033 0 0.104 1.028(0.0 iN NaOH)Oxidation

5Mm 60°C 30 0.126 1.041 29 0.117 1.043(10% H2O2)Oxidation

5Mm 60°C 13 0.311 1.028 7 0.309 1.032(3%H202)I


52

Table 61: Forced Deadation Results for Benazepril HC1/HCTZ Tablets (Benazepril) on UPLCRaw Material Blend


Control NA NA NA 0.102 1.006 NA 0.140 1.006UV Solutions 3 Hrs RT 1 0.129 1.005 0 0.264 1.051UV Solutions 5 Days RT 0 0.113 1.006 3 0.193 1.022

UV(Dry) 5Days RT 0 0.112 1.005 1 0.151 1.012Thermal (Dry) 5 Days 105°C 0 0.143 1.007 25 0.136 1.0 18Humidity(Dry) 5 Days RT 1 0.118 1.006 0 0.127 1.007

MSH 48Hrs 90°C 37 0.102 1.013 58 0.227 1.165MSH 3 Hrs 90°C 0 0.122 1.005 0 0.149 1.006

1)2Hrs 60°C 4 0.110 1.005 13 0.098 1.005

(0.1NNaOI-I)5Mm RT 8 0.110 1.005 1 0.136 1.004

Oxidation 1 Hr 60°C 0 0.139 1.004 11 0.114 1.005(10/o H202)Analysts: JSM and JU

Table 62: Forced Degradation Results for Benazepril HCI/HCTZ Tablets (HCTZ) on UPLCRaw Material Blend


Control NA NA NA 0.230 1.008 NA 0.282 1.011UV Solutions 3 Hrs RT 3 0.314 1.216 0 0.286 1.080UV Solutions 5 Days RT 77 0.43 1 1.045 57 0.372 1.046

UV(Dry) 5 Days RT 0 0.537 1.006 1 0.171 1.010Thermal (Dry) 5 Days 105°C 0 0.286 1.011 0 0.184 1.016Humidity (Dry) 5 Days RT 1 0.224 1.008 1 0.273 1.0 12

MSH 48Hrs 90°C 0 0.297 1.011 8 0.236 1.070MSH 3 Hrs 90°C 1 0.06 1 1.006 0 0.059 1.008

2 Hrs 60°C 4 0.062 1.005 5 0.067 1.006

(0.1NNaOH)5Mm RT 0 0.320 1.006 0 0.113 1.007

Oxidation1 Hr 60°C 1 0.107 1.005 0 0.076 1.006(10 /o H202)


54

Table 63: Forced Degradation Results for Fosinopril Sodium Tablets (HPLC)Raw Material Blend

0/Condition Time Temp % Purity Threshold Purity ThresholdDegradation Angle Angle Degradation Angle Angle

Control NA NA NA 0.190 1.148 NA 0.284 1.236UVSolutions 3Hrs RT 0 0.217 1.156 0 0.157 1.132UV Solutions 5 Days RT 0 0.353 1.412 4 0.403 1.438

UV (Dry) 5 Days RT 0 0.332 1.352 1 0.347 1.375Thermal (Dry) 5 Days 105°C 0 0.3 12 1.329 21 0.371 1.428Humidity (Dry) 5 Days RT 0 0.326 1.334 1 0.340 1.387

MSH 3Hrs 90°C 2 0.659 1.119 7 0.748 1.150Acidic

10 Mm 60°C 33 0.250 1.206 9 0.208 1.202(0.1N HC1)Alkaline 10Mm RT 12 0.204 1.175 1 0.174 0.148(0.0 iN NaOH)

Oxidation5 Mm 60°C 31 0.217 1.204 33 0.248 1.214(10% H202)

Oxidation 5Mm 60°C 13 0.787 1.117 22 0.847 1.138(3% H202) IAnalysts: JSM and JU

Table 64: Forced Degradation Results for Fosinopril SodiumlHCTZ Tablets (Fosinopril) onHPLC

r Raw Material BlendCondition Time Temp % Purity Threshold % Purity Threshold

Degradation Angle Degradation Angle AngleControl NA NA NA 0.190 1.148 NA 0.309 1.276

UVSolutions 3Hrs RT 0 0.217 1.156 1 0.168 1.173UV Solutions 5 Days RT 0 0.353 1.412 11 0.369 1.400

UV (Dry) 5 Days RT 0 0.332 1.352 1 0.352 1.393Thermal (Dry) 5 Days 105°C 0 0.3 12 1.329 26 0.498 1.552Humidity (Dry) 5 Days RT 0 0.326 1.334 0 0.338 1.350

MSH 3Hrs 90°C 2 0.659 1.119 4 0.524 1.167Acidic 10 M 60°C 33 0.250 1.206 5 0.185 1.174(0.1N HC1)

Alkaline 10Mm RT 12 0.204 1.175 0 0.200 1.165(0.0 IN NaOH)Oxidation 5Mm 60°C 31 0.217 1.204 29 0.250 1.211(10% H202)Oxidation 5Mm 60°C 13 0.787 1.117 7 0.561 1.170(3%H202)


55

Table 67: Forced Degradation Results for Benazepril HC1/HCTZ Tablets (Benazepril) on HPLCRaw Material Blend


Control NA NA NA 0.186 1.029 NA 0.192 1.028UV Solutions 3 Hrs RT 0 0.208 1.043 0 0.208 1.035UV Solutions 5 Days RT 0 0.191 1.026 0 0.186 1.025

UV(Dry) 5 Days RT 0 0.188 1.027 2 0.194 1.026Thermal (Dry) 5 Days 105°C 0 0.189 1.025 24 0.177 1.03 1Humidity (Dry) 5 Days RT 0 0.197 1.033 0 0.193 1.03 1

MSH 48 Hrs 90°C 63 0.174 1.062 57 0.169 1.050MSH 3 Hrs 90°C 0 0.164 1.038 0 0.164 1.035

1H2Hrs 60°C 3 0.107 1.016 12 0.110 1.025

(0.1NNaOH)5Mm RT 8 0.103 1.019 0 0.110 1.019

lHr 60°C 0 0.114 1.016 10 0.106 1.018


Table 68: Forced Degradation Results for Benazepril HC1/HCTZ Tablets (HCTZ) on HPLCRaw Material Blend


Control NA NA NA 0.095 1.015 NA 0.082 1.017UV Solutions 3 Hrs RT 3 0.088 1.016 0 0.082 1.020UV Solutions 5 Days RT 78 0 113 1.059 56 0.627 1.057

UV(Dry) 5 Days RT 1 0.099 1.014 0 0.094 1.017Thermal (Dry) 5 Days 105°C 1 0.100 1.014 1 0.092 1.017Humidity (Dry) 5 Days RT 1 0.092 1.017 0 0.079 1.019

MSH 48 Hrs 90°C 1 0.094 1.014 8 0.098 1.018MSH 3 Hrs 90°C 1 0.068 1.016 0 0.065 1.018

1)2Hrs 60°C 4 0.105 1.013 4 0.090 1.027

(0.1NNaOH)5Mm RT 1 0.113 1.011 0 0.093 1.017

Oxidation1 Hr 60°C 0 0.103 1.012 0 0.115 1.011(10/o_H202)


The robustness of the filtration technique used in the analytical method was evaluated as

part of the validation process. The filter study was conducted using O.45-.tm Millex-HV

polyvinylidenedifluoride (PVDF) filter, a 0.45-.tm Whatman glass microfiber filter (GMF) and

centrifugation. The minimum discard volume for each filter was determined as well. The

acceptance criteria for the filter study was a percent different of not more than 2.00o between the

final method specified discard volume and the subsequent filter portions. The filter study was

57

executed using both the external standard and the finished product. Though the external standard

is not filtered during routine testing, it is filtered during the filter validation to determine if the

filter binds the main component as a result of a placebo interaction. The finish product sample

was centrifuged in glass and plastic centrifuge tubes for comparison purposes. As the UPLC and

the HPLC methods were shown to be equivalent, the filter validation was analyzed using only

the HPLC method. As shown in the following tables each of the filters may be considered

equivalent after a discard volume of not less than the first 5 mL of filtrate. The centrifugation

was shown to be equivalent to filtration whether using glass or plastic centrifuge tubes and stored

for an extended period of time in the tubes before being tested.

Table 69: Assay Filter Validation Using the External StandardSample %Fosiuopril %Benazepril %HCTZ

Millex 3 mL 99.2 98.8 100.7Millex5mL 100.3 100.9 101.5Millex7mL 100.5 100.8 101.4MillexlOmL 100.1 101.0 101.3

GMF 3 mL 97.6 87.5 100.3GMF 5 mL 100.3 100.4 101.3GMF7mL 100.5 100.9 101.1

GMF1OmL 100.4 100.4 100.8Analyst: LMG

Table 70: Assay Filter Validation for Fosinopril Sodium 10 mg TabletsSample %Fosinopril

Millex 3 mL 87.0Millex 5 mL 87.9Millex 7 mL 87.6

Millex 10 mL 87.8GMF3mL 82.8GMF5mL 87.8GMF7mL 87.8

GMF1OmL 87.8Centrifuge in glass 88.1

Centrifuge in plastic 87.7Centrifuge in plastic (stored for 4 hrs) 87.6

Analyst: LMG

58

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The robustness of the extraction procedure was evaluated during the validation of the

analytical method. Currently, the extraction technique uses 15 minutes of sonication followed by

15 minutes of shaking on a mechanical shaker. Each portion of the extraction procedure was

evaluated at 10 minutes, 15 minutes, and 30 minutes for the sonication and shaking to

demonstrate that slight variations in the extraction time would not cause any bias on the

analytical results. The acceptance criteria for the extraction study was a difference of not more

than 2.0° o from the method specified extraction time for each time point evaluated.

Table 74: Assay Extraction Validation for Fosinopril Sodium 10 mg TabletsExtraction Time Point (mm) %Fosinopril

10 87.7Sonication 15 87.7

30 87.710 88.1

Shaking 15 87.730 87.7

Analyst: JSM

Table 75: Assay Extraction Validation for Fosinopril SodiumJHCTZ 10 mg/12.5 mg Tablets. Time PointExtraction . %Fosinopril %IICTZ(mm)

10 94.8 96.1Sonication 15 94.8 96.4

30 94.7 96.510 94.9 97.0

Shaking 15 94.9 97.130 94.9 97.0

Analyst: JSM

Table 76: Assay Extraction Validation for Benazepril HC1 5 mg TabletsExtraction Time Point (miii) %Benazepril

10 97.2Sonication 15 97.4

30 97.210 97.4

Shaking 15 97.430 97.5

Analyst: JSM

60

Table 77: Assay Extraction Validation for Benazepril HC1 HCTZ 5 mg/6.25 mg TabletsTime PointExtraction %Benazepril %HCTZ(mm)

10 98.8 96.2Sonication 15 98.7 96.1

30 98.5 95.810 99.1 96.4

Shaking 15 99.0 96.230 98.7 95.9

Analyst: JSM

The robustness of the chromatographic parameters was evaluated for both the UPLC and

HPLC methods. During the robustness studies, small deliberate changes were made to the

method specified chromatographic parameters. The robustness samples prepared by the analysis

of an assay sample spiked with each of the formulation specified related compounds. The

samples were then analyzed using a PDA (photo-diode array detector) to determine the peak

purity and all system suitability criteria were evaluated. For acceptance of the condition, the

purity angle was shown to be less than the purity threshold for the main component peak. The

system suitability criteria for each system suitability specification were also met, which are

outlined in Table 78.

Table 78: System Suitability Criteria

The following changes were made to the chromatographic parameters for the UPLC and the

HPLC during the robustness study outlined in Table 79 and Table 87.

The %RSD of the five replicate injections of the standard preparation must be not more than2.0% for each main component peak of interest.The tailing of the main component peak in the standard preparation must not be more than 2.0for the main component peak of interest.The plate count for the main component peak must be:

• HCTZ NLT 2,000• Fosinopril NLT 10,000• Benazepril NLT 10,000

-

61

Table 79: Robustness Parameters for UPLCParameter VariationFlow Rate ±0.1 mL/min

%ACN in Mobile Phase A ±3%Mobile Phase A pH ± 0.2

Temperature ± 5°CWavelength ±4 mn

Note: There is no column equivalent to the Waters Acquity BEH Phenyl Column used for UPLC at this time.

Table 80: Robustness Results for Fosinopril Sodium Tablets on UPLCPurity ThresholdParameterAngle Angle

Initial 0.236 1.040Flow Rate (0.4 mL/min) 0.670 1.028Flow Rate (0.6 mL/min) 0.667 1.050

Column Temperature 35°C 0.475 1.055Column Temperature 45°C 0.177 1.035

Wavelength 202 nm 0.245 1.041Wavelength 210 nm 0.238 1.040

Mobile Phase A pH 1.9 0.760 1.073Mobile Phase A pH 2.3 0.786 1.050

+3% ACN in Mobile Phase A 0.123 1.020-3%ACNinMobilePhaseA 0.147 1.042

Analyst: JSM, JU, and LMG

Table 81: Robustness Results for Fosinopril SodiumIHCTZ Tablets on UPLCFosinopril HCTZ

Parameter Purity Threshold Purity ThresholdAngle Angle Angle Angle

Initial 0.145 1.057 0.032 1.022FlowRate(0.4mL/min) 0.670 1.031 0.330 1.008Flow Rate (0.6 mL/min) 0.800 1.110 0.027 1.0 14

ColumnTemperature35°C 0.406 1.055 0.334 1.016Column Temperature 45°C 0.109 1.049 0.033 1.016

Wavelength 202 nm 0.133 1.057 0.035 1.023Wavelength2lonm 0.137 1.057 0.032 1.022

Mobile Phase A pH 1.9 0.859 1.073 0.432 1.02 1Mobile Phase A pH 2.3 0.841 1.054 0.3 18 1.022

+3% ACN in Mobile Phase A 0.107 1.021 0.092 1.002-3%ACNinMobilePhaseA 0.285 1.121 0.114 1.015


62

Table 82: Robustness Results for Benazepril HC1 Tablets on UPLCPurity ThresholdParameterAngle Angle

Initial 0.310 1.007Flow Rate (0.4 mL/min) 0.097 1.005Flow Rate (0.6 mE/mm) 0.075 1.008


Wavelength 202 nm 0.317 1.007Wavelength 210 nm 0.293 1.007

Mobile PhaseApH 1.9 0.134 1.011MobilePhaseApH2.3 0.178 1.016

+3% ACN in Mobile Phase A 0.259 1.004-3% ACN in Mobile Phase A 0.235 1.007


Table 83: Robustness Results for Benazepril HC1/HCTZ Tablets on UPLCBenazepril HCTZ


Initial 0.119 1.016 0.030 1.030Flow Rate (0.4 mE/mm) 0.078 1.005 0.473 1.012Flow Rate (0.6 mL/min) 0.085 1.016 0.026 1.019

Column Temperature 35°C 0.167 1.022 0.435 1.040Column Temperature 45°C 0.075 1.006 0.045 1.015

Wavelength 202 nm 0.119 1.016 0.033 1.034Wavelength2lonm 0.119 1.016 0.030 1.030

Mobile PhaseApH 1.9 0.129 1.016 0.712 1.047MobilePhaseApH2.3 0.192 1.015 0.095 1.050

+3% ACN in Mobile Phase A 0.126 1.004 0.070 1.003-3% ACN in Mobile Phase A 0.129 1.016 0.115 1.023

%RSD of Std StdParameter5 Standard Tailing Plate Count

Initial 0.0 1.3 144793Flow Rate (0.4 mL/min) 0.1 1.3 144816Flow Rate (0.6 mL/min) 0.0 1.3 144855

Column Temperature 35°C 0.0 1.3 136477ColumnTemperature45°C 0.1 1.3 119913

Wavelength 202 nm 0.1 1.5 120494Wavelength 210 nm 0.1 1.2 168253

Mobile Phase ApH 1.9 0.1 1.4 201839Mobile Phase A pH 2.3 0.2 1.3 99616

+3% ACN in Mobile Phase A 0.1 1.3 151318-3% ACN in Mobile Phase A 0.1 1.4 152814

Analyst: JSM, JU, anci LMG

Table 84: Robustness Results for System Suitability Criterion for Fosinopril


63

Table 85: Robustness Results for System Suitability Criterion for Benazepril%RSDof Std StdParameter

5 Standard Taffing Plate CountInitial 0.0 1.4 195950

Flow Rate (0.4 mL/min) 0.1 1.4 195958Flow Rate (0.6 mL/min) 0.0 1.4 195928

Column Temperature 35°C 0.3 1.4 145798Column Temperature 45°C 0.1 1.3 266310


Mobile PhaseApH 1.9 0.1 1.6 299316MobilePhaseApH2.3 0.2 1.4 139639

+3%ACNinMobilePhaseA 0.0 1.4 288836-3% ACN in Mobile Phase A 0.1 1.6 281712

Table 86: Robustness Results for System Suitability Criterion for HCTZ%RSD of Std StdParameter

5 Standard Tathng Plate CountInitial 0.0 0.9 5264




Mobile PhaseApH 1.9 0.1 1.1 80445Mobile Phase A pH 2.3 0.1 1.2 4327

+3% ACN in Mobile Phase A 0.1 0.9 5324-3%ACNinMobilePhaseA 0.0 1.0 5139


Table 87: Robustness Parameters for HPLCParameter Variation

Column Equivalency Waters Atlantis dCl8 4.6mm x 150mm 5j.tm columnFlow Rate ± 0.3 mL/min


Temperature ± 5°CWavelength ±4 nm

64

Table 88: Robustness Results for Fosinopril Sodium Tablets on HPLC

Purity ThresholdParameterAngle Angle



Wavelength 202 nm 0.412 1.182Wavelength 2lOnm 0.404 1.183

Mobile PhaseApH 1.9 0.470 1.146Mobile Phase A pH 2.3 0.295 1.278


Column Equivalency 0.137 1.095Analyst: JSM, JU, and LMG

Sodium/H CTZ Tablets on HPLCTable 89: Robustness Results for FosinoprilFosmopril HCTZ


Initial 0.404 1.166 0.111 1.015Flow Rate (0.9 mL/min) 0.3 19 1.092 0.115 1.012FlowRate(1.5mL/min) 0.385 1.466 0.101 1.025


Wavelength202nm 0.368 1.163 0.111 1.015Wavelength2lonm 0.371 1.164 0.111 1.015

Mobile Phase ApH 1.9 0.430 1.115 0.080 1.014Mobile Phase A pH 2.3 0.256 1.266 0.097 1.020

+3% ACN in Mobile Phase A 0.609 1.123 0.079 1.019-3% ACN in Mobile Phase A 0.160 1.122 0.111 1.014

ColumnEquivalency 0.152 1.106 0.160 1.011Analyst: JSM, JU, ann LMG

65

Table 90: Robustness Results for Benazepril HC1 Tablets on HPLCPurity ThresholdParameterAngle Angle



Wavelength 202 nm 0.168 1.033Wavelength2lonm 0.168 1.034

Mobile PhaseApH 1.9 0.143 1.022MobilePhaseApH2.3 0.184 1.045


Column Equivalency 0.146 1.021Analyst: JSM, JU, and LMG

Table 91: Robustness Results for Benazepril HCI/HCTZ Tablets on HPLCBenazepril HCTZ


Initial 0.169 1.031 0.106 1.019FlowRate(0.9mL/min) 0.162 1.013 0.112 1.012Flow Rate (1.5 mL/min) 0.177 1.063 0.093 1.026

ColumnTemperature35°C 0.183 1.051 0.101 1.026Column Temperature 45°C 0.193 1.048 0.109 1.024

Wavelength202nm 0.169 1.032 0.105 1.018Wavelength2l0nm 0.168 1.031 0.105 1.019

Mobile PhaseApH 1.9 0.142 1.022 0.073 1.018Mobile Phase A pH 2.3 0.180 1.04 0.102 1.023

+3% ACN in Mobile Phase A 0.13 1 1.024 0.075 1.022-3% ACN in Mobile Phase A 0.125 1.024 0.102 1.016

Column Equivalency 0.145 1.020 0.157 1.012Analyst: JSM, JU, and LMG

66

Table 92: Robustness Results for System Suitability Criterion for Fosinopril%RSD of Std Std

Parameter5 Standard Tathng Plate Count


ColumnTemperature35°C 0.1 1.1 363075Column Temperature 45°C 0.2 1.1 323859


Mobile PhaseApH 1.9 0.1 1.1 309618Mobile Phase A pH 2.3 0.1 1.1 307949


Column Equivalency 0.1 1.0 258827Analyst: JSM, JU, and LMG

Table 93: Robustness Results for System Suitalj ility CriterI

%RSD of Std StdParameter

5 Standard Taffing Plate Count



Wavelength 202 nm 0.1 1.2 230478Wavelength2lOnm 0.1 1.2 228399

Mobile Phase A pH 1.9 0.1 1.5 206676Mobile Phase A pH 2.3 0.1 1.5 212853


Column Equivalency 0.1 1.0 190542

on for Benazepril


67

Table 94: Robustness Results for System Suitability Criterion for HCTZ%RSDof Std StdParameter

5 Standard Tailing Plate CountInitial 0.1 1.0 5373


Column Temperature 35°C 0.2 1.0 5414ColumnTemperature45°C 0.1 1.0 5619

Wavelength2o2nm 0.1 1.1 5868Wavelength 210 nm 0.1 1.1 4527

Mobile Phase A pH 1.9 0.2 0.9 2292MobilePhaseApH2.3 0.1 1.1 5288


Column Equivalency 0.0 1.1 3438Analyst: JSM, JU, and LMG

The stability of the sample solutions was evaluated as part of the validation experiments

for the analytical method. By defining the period in which the solutions may be considered

acceptable for use, laboratory reference standard may be conserved, as well as time being saved

for the analysts. However, as this analytical method may be used during the testing of four

different products, the solution stability study was executed using every possible standard

preparation that could be used with this method. These standards include preparations that

contain the Fosinopril alone, the Benazepril alone, the Fosinopril and HCTZ, the Benazepril and

HCTZ, and a standard that may contain all three components (Fosinopril, Benazepril, and

HCTZ). The standards and samples were stored under ambient laboratory conditions and under

refrigeration in both clear and amber flasks. The acceptance criteria for the solution stability

study was that the ° o assay for both the standard and the assay sample solution should be not

more than 2.0° o different when compared to the initial sample. As shown in the following tables

the working standard and assay solutions were shown to be stable for up to 7 days following the

day of preparation.

68

Table 95: Solution Stabilit of the Fosino iril Alone Working Standard

Time PointRoom Temperature 1 Refrigeration

Day 0Clear Amber Clear Amber

Day 1Day3Day 7

Table 96: Solution Stabilit of the Fosino .ril HCTZ Working Standard (Fosinoiril)Room Temperature I RefrigerationTime Point

Clear Amber Clear AmberDayODay 1Day

3

_______________

Day_7

_______________

100.1

Room Temperature I RefrigerationTime PointClear Amber Clear Amber

DayO 100.3Day 1 I 99.8 I 99.7 99.9 100.2Day3 I 100.0 100.2 100.2 100.1Day7 100.8 I 100.8 100.7 100.7

Analyst: LMG

Table 98: Solution Stabilit of the Benaze.ril (Alone Workin: StandardRoom Temperature RefrigerationTime Point

Clear I Amber Clear I AmberDayO 100.0Dayl 100.2 100.3 100.1 100.3Day3 99.2 99.1 99.2 99.3Day7 100.7 100.6 100.7 100.7

Analyst: LMG

Table 99: Solution Stabilit of the Benazearil HCTZ Standard BenazeerilRoom Temperature RefrigerationTime Point

Clear I Amber Clear I AmberDayO 100.0Dayl 100.1 100.1 100.0 100.1Day3 99.1 99.0 99.1 99.1Day7 100.6 100.7 100.8 100.8

Analyst: LMG

Analyst: LMG

Table 97: Solution Stabilit of the Fosino .ril/HCTZ Workin: Standard HCTZ)

69

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Table 105: Solution Stability of the Fosinopril SodiumIHCTZ lOmg/12.5mg Tablets(Fosino .ril)

Time PointRoom Temperature I Refrigeration

Day 0Clear Amber Clear I Amber

Day 1Day 3Day 7

Table 106: Solution Stability of the Fosino.ril SodiumJHCTZ lOm: 12.5m: Tablets (HCTZRoom Temperature I RefrigerationTime Point

Clear Amber Clear AmberDayODay 1Day 3Day7

Table 107: Solution Stabilit of the Benaze.ril HC1 5 m TabletsRoom Temperature I RefrigerationTime Point

Clear Amber Clear AmberDay 0Day 1

Day 7

Room Temperature I RefrigerationTime PointClear Amber Clear Amber

DayO

_________________________

Day_1

_____________________________

Day3Day7 100.1 I 100.1 100.1 100.2

Table 109: Solution Stabilit of the Benazeeril HC1 HCTZ Sm: 6.25 m: (HCTZRoom Temperature I RefrigerationTime Point

Clear Amber Clear AmberDayODay 1Day 3Day 7

Analyst: LMG

Table 108: Solution Stabilit of the Benaze.ril HCI/HCTZ Sm: 6.25 m: (Benazeeril)

Analyst: LMG

100.1 I 100.0

Analyst: LMG

Method equivalency studies were executed for each product to assess that the results

generated by the new analytical methods are comparable to those already in place at Sandoz, Inc

in Wilson, NC. For the method equivalency study six samples were prepared for assay using

both the new method and the existing method. For equivalency the percent difference in mean

between the two methods must be not more than 2.000 (absolute). The new assay method on

UPLC and HPLC were shown to be equivalent to the existing methods.

Table 110: Assay Equivalency Results for Fosinopril Sodium 10 mg Tablets on UPLCSample # - New UPLC Method - Sandoz Monograph FOO7QC

1 88.443 88.2382 88.496 88.1883 88.250 87.9834 88.640 88.1435 88.700 87.9806 88.320 88.103

Mean 88.5 88.1%RSD 0.2 0.1

%Difference 0.4

Sample # - New HPLC Method Sandoz Monograph FOO7QC1 88.167 88.2382 88.172 88.1883 88.174 87.9834 88.361 88.1435 88.588 87.9806 88.214 88.103

Mean 88.3 88.1%RSD 0.2 0.1

%Difference 0.2

Table 111: Assay Equivah :ncy Results for Fosinopril Sodium 10 mg Tablets on HPLC

72

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Results of Method Validation Experiments for Related Compounds

The diluent, extraction procedure, and chromatographic parameters for the related

compound procedure are the same as the previously described assay procedure. Therefore,

during the validation activities the related compounds method was validated at the same time.

During the validation for Benazepril HC1 HCTZ Tablets, the HCTZ portion of the combination

product failed to meet acceptance criteria for the related compound 4-Amino. Though the

protocol specifically states that a wider range of acceptance criteria may be used on a case-by-

case basis for the accuracy study, the 4-Amino was spiked into the solution at the 1.000. Because

the 4-Amino is the main degradation component and this level was already considered high, the

justification of a wider acceptance criteria could not be scientifically justified. Since this

recovery problem was not observed in the linearity solutions there appeared to be an extraction

problem from the placebo matrix at such a high concentration. The Benazepril HC1 HCTZ

Tablets contained the highest amount of placebo to active ratio of any formulation tested during

these validation activities. During future developmental work the extraction volume will need to

be increased to optimize the recovery for the 4-Amino related compound. As an increase in the

extraction volume will subsequently lower the sample concentration the injection volume of the

chromatographic methods will need to be increased to compensate for this loss. This will be

pursued at a later date. The actual °orecovery for the 4-Amino related compound in Benazepril

HC1 HCTZ Tablets has been included in this report for information only.

The linearity for related compounds was assessed across the range of the analytical

method for both the assay and the related compounds for main components. The results of these

linearity studies for the active pharmaceutical ingredient (API) was discussed previously and

outlined in Tables 31 and 32. The linearity for each related compound was executed across the

75

range of the method from the limit of quantitation (0.05° o) up to 12000 of the specification value.

The acceptance criteria for the linearity is not less than 0.99 for the correlation coefficient and

the °oy-intercept must be less than 25°c of the specification area for related compounds. As

shown in the Table 118 and 119, all results met the preset acceptance criteria.

Table 118: Linearity Results for UPLCCorrelationAPI Related Compound

Coefficient (R)%Y-Intercept

4-Amino 0.999971833 0 (based on 0.48%)HCTZ CTZ 0.999975923 1 (based on 0.08%)

Dimer 0.999982374 3 (based on 0.16%)S-Amine 0.999965373 0 (based on 0.50%)

Benazepril Benazeprilat 0.999979882 0 (based on 0.50%)R,S-Isomer 0.999976074 0 (based on 0.50%)

RC-A 0.999978 150 0 (based on 1.25%). . RC-B 0.999980969 0 (based on 0.15%)Fosinopril

RC-C 0.999949937 1 (based on 0.15%)RC-D 0.9993 80857 21 (based on 0.15%)

Analyst: JSM

Table 119: Linearity Results for HPLCCorrelationAPI Related Compound

Coefficient (R)%Y-Intercept

4-Amino 0.999983306 2 (based on 0.48%)HCTZ CTZ 0.99995 1391 0 (based on 0.08%)

Dimer 0.999979737 5 (based on 0.16%)S-Amine 0.999969095 0 (based on 0.50%)

Benazepril Benazeprilat 0.9999434 16 0 (based on 0.50%)R,S-Isomer 0.999975574 0 (based on 0.50%)

RC-A 0.999968490 0 (based on 1.25%). . RC-B 0.999950872 0 (based on 0.15%)Fosinopril

RC-C 0.999954204 0 (based on 0.15%)RC-D 0.999402974 2 (based on 0.15%)

Analyst: JSM

The limit of quantitation was assessed for each main component as well as each

respective related compound. The limit of quantitation (LOQ) study was executed by the

preparation of samples in placebo matrix spiked at the respective LOQ for the component of

interest. The sample was injected into each chromatographic system six times and evaluated for

76

repeatability (°0RSD) and mean signal-to-noise. The acceptance criteria for the signal-to-noise

level was to obtain a°0RSD of the six replicate injection of less than lO0o and a mean signal-to-

noise ratio of not less than 10.

Table 120: Limit of luantitation for Fosino .ril Sodium Tablets

Table 121: Limit of luantitation for Fosino eril SodiumJHCTZ Tablets

% LevelComponent

Fosinopril

UPLC HPLCComponent

Level Mean Signal to%RSD

Mean Signal to%RSD

____________________

Noise NoiseFosinopril

UPLC I HPLCMean Signal to Mean Signal to

%RSDNoise Noise

HCTZ 370.05 (625 ,tWmL)

0.03 (1250 g/mL)

I 0.05 (625 p.g/mL)4-Amino

0.03 (1250 ig/mL)

0.05 (625 .tgImL)

__________

0.03 (1250 ig/mL)

0.05 (625 i.iWmL)Dimer0.03 (1250 pg/mL)

CTZ

12

21

40

Analyst: JSM and LMG

Table 122: Limit of uantitation for Benazeeril HC1 Tablets

UPLC I HPLCComponent % Level Mean Signal to I

%RSDMean Signal to

%RSD

__________________________

I Noise I NoiseBenazeprilS-Amine

Benazeprilat

R,S-Isomer

Table 123: Limit of luantitation for Benaze.ril HC1/HCTZ TabletsUPLC I HPLC

ComponentLevel I Mean Signal to

%RSDMean Signal to

%RSDNoise NoiseBenazeprilS-Amine

BenazeprilatR,S-Isomer

Analyst: JSM

The accuracy for related compounds was assessed across the range of the limit of

quantitation (LOQ) up to at least 12000 of specification value of the related compound. The

study was executed by the preparation of three representative samples at three concentrations.

Each sample was spiked with known amounts of raw material, placebo matrix, and each

respective related compound and prepared according to the analytical method for related

compounds. The mean recovery of each level should be within the range outlined in Table 124.

However, due to the low levels of some of the impurities a wider range was considered

acceptable on a case-by-case basis.

Table 124: Acce tance Criteria for Related Compound Accuracy% Level Range<0.5% 80—120%

0.5%-5% 90—110%

Table 125: Accuracy Results for Related Compound A in Fosinopril Sodium TabletsMean Level Result

- Mean Level Result%Level(UPLC) (HPLC)

0.05% 102% 111%2.0% 109% 105%4.0% 108% 105%

Analyst: JSM

Table 126: Accuracy Results for Related Compound B in Fosinopril Sodium TabletsMean Level Result Mean Level Result%Level

(UPLC) (HPLC)0.05% 105% 97%0.3% 101% 98%0.6% 101% 98%

Analyst: JSM

78

Table 127: Accuracy Results for Related Compound C in Fosinopril Sodium TabletsMean Level Result Mean Level Result%Level

(UPLC) (HPLC)0.05% 101% 104%0.3% 101% 100%0.6% 102% 100%

Analyst: JSM

Table 128: Accuracy Results for Related Compound D in Fosinopril Sodium TabletsMean Level Result Mean Level Result%Level

(UPLC) (HPLC)0.1% 86% 79%’0.3% 82% 80%0.4% 80% 78%’

Analyst: JSMNote’: A wider range for the accuracy study was considered acceptable for the RC-D due to the low levels whichwere being recovered from each sample preparation. The RC-D is also a process-related impurity and was neverpresent in the expired samples, or in any forced degradation studies. This compound is further controlled throughthe release of the raw material. As the monitoring of process-related impurities is not a requirement of finishedproduct test methods this related compound will eventually be removed from this method in the future.

Table 129: Accuracy Results for Related Compound A in Fosinopril SodiumfHCTZ TabletsMean Level Result - Mean Level Result%Level

(UPLC) (HPLC)0.05% 104% 100%2.0% 103% 101%4.0% 103% 101%

Analyst: JSM

Table 130: Accuracy Results for Related Compound B in Fosinopril Sodium.IHCTZ TabletsMean Level Result Mean Level Result%Level

(UPLC) (HPLC)0.05% 108% 98%0.3% 102% 101%0.6% 99% 98%

Analyst: JSM

Table 131: Accuracy Results for Related Compound C in Fosinopril SodiumJHCTZ TabletsMean Level Result Mean Level Result%Level

(UPLC) (HPLC)0.05% 99% 96%0.3% 96% 100%0.6% 97% 99%

Analyst: JSM

79

Table 132: Accuracy Results for Related Compound D in Fosinopril SodiumJHCTZ TabletsMean Level Result Mean Level Result%Level

(UPLC) (HPLC)0.1% 85% 91%0.3% 82% 88%0.4% 83% 95%

Analyst: JSM

Table 133: Accuracy Results for 4-Amino in Fosinopnl SodiumJHCTZ TabletsMean Level Result Mean Level Result%Level

(UPLC) (HPLC)0.05% 89% 108%0.24% 84% 89%0.48% 84% 87%

Analyst: JSM

Table 134: Accuracy Results for CTZ in Fosinopril SodiumIHCTZ TabletsMean Level Result Mean Level Result%Level

(UPLC) (HPLC)0.05% 105% 111%0.24% 91% 92%0.48% 90% 91%

Analyst: JSM

Table 135: Accuracy Results for Dimer in Fosinopril SodiumJHCTZ TabletsMean Level Mean LevelMean Level Mean Level%Level

Result (UPLC)Result with

Result (HPLC)Result with

Factor Factor0.05% 44% 131% 38% 115%0.2% 31% 92% 27% 81%0.3% 34% 100% 28% 85%

Analyst: LMG

The accuracy for the HCTZ Dimer impurity failed to meet the acceptance criteria for

accuracy when in the presence of sample matrix. Currently the Dimer is not assessed by the

finished product monographs at Sandoz, Inc in Wilson, NC. The Dimer was added to this

validation based on a statement in the Cambrex Technical Package (the manufacturer of HCTZ)

that though Dimer is a process-related impurity, it could be a possible degradation impurity as

well. After an assessment of all forced degradation and the expired lots tested for Fosinopril

SodiumJHCTZ lOmg/12.5mg Tablets the amount of Dimer appears to be consistent and without

growth. The forced degradation studies demonstrated no increase in the Dimer and it can still be

80

accurately quantitated with a signal-to-noise greater than 10. The Dimer appears to be a process-

related impurity as it was never observed to increase during any forced degradation study.

Injections of the raw material in placebo matrix and raw material alone were analyzed. It was

demonstrated that the low accuracy was a result of the placebo interference in solution as the

Dimer appeared to be fully extracted when in the raw material solutions without placebo. The

raw material in placebo matrix would yield a °oDimer of 0.02%, while the °oDimer in the raw

material alone would yield about 0.06%. Because the placebo used during method validation is

prepared at the highest possible variance, the high amount of placebo used during validation

would never be present in the actual finished product. The Dimer impurity is controlled through

the release of the HCTZ raw material alone, which uses the USP compendial monograph.

Process-related impurities are not required to be monitored in finished product testing

monographs. Therefore, the validation of the Dimer impurity for this finished product method

would not be required.

Table 136: Accuracy Results for S-Amine in Benazepril HC1 TabletsMean Level Result Mean Level Result%Level

(UPLC) (HPLC)0.05% 116% 115%0.5% 114% 113%0.6% 114% 113%

Analyst: JSM

Table 137: Accuracy Results for Benazeprilat in Benazepril HC1 TabletsMean Level Result Mean Level Result%Level

(UPLC) (HPLC)0.05% 93% 103%0.5% 96% 99%0.6% 95% 98%

Analyst: JSM

Table 138: Accuracy Results for R,S-Isomer in Benazepril HC1 TabletsMean Level Result Mean Level Result/oLevel

(UPLC) (HPLC)0.05% 114% 103%0.5% 103% 103%0.6% 109% 103%

Analyst: JSM

81

Table 139: Accuracy Results for S-Amine in Benazepril HC1/HCTZ TabletsMean Level Result Mean Level Result%Level

(UPLC) (HPLC)0.05% 110% 113%0.5% 108% 109%0.6% 108% 109%

Analyst: JSM

Table 140: Accuracy Results for Bena.zeprilat in Benazepril HC1IHCTZ TabletsMean Level Result Mean Level Result%Level

(UPLC) (HPLC)0.05% 101% 106%0.5% 102% 104%0.6% 101% 104%

Analyst: JSM

Table 141: Accuracy Results for R,S-Isomer in Benazepril HC1/HCTZ TabletsMean Level Result Mean Level Result/oLevel

(UPLC) (HPLC)0.05% 81% 103%0.5% 98% 104%0.6% 95% 104%

Analyst: JSM

Table 142: Accuracy Results for 4-Amino in Benazepril HC1/HCTZ Tablets (For Information Only)Mean Level Result Mean Level Result%Level

(UPLC) (HPLC)0.05% 77% 82%1.0% 70% 73%1.2% 72% 74%

Analyst: JSM

Table 143: Accuracy Results for CTZ in Benazepril HC1/HCTZ Tablets (For Information Only)

Mean Level Result Mean Level Result%LevelPLC) (HPLC)

0.05% 99% 111%0.5% 87% 88%0.6% 87% 89%

Analyst: JSM

The precision studies for the related compound method were executed for both

repeatability and intermediate precision. All precision studies were executed using

representative sample preparations consisting of the “worst case” placebo formulation, raw

82

material, and spiked with the respective related compounds for each pharmaceutical formulation.

The repeatability was assessed by six preparations of the spiked representative sample

preparations. The acceptance criteria were as follows in Table 144 for the six sample

preparations.

Table 144: Acceptance Criteria for RC Repeatability% Level %RSD I<0.2% RSD20%

0.2% to < 0.5% RSD 10%0.5%-5% RSD5%

The intermediate precision was assessed by the analysis of another six sample

preparations of the same spiked representative sample preparations by a different chemist, on a

different day, using different chromatographic systems, solutions, mobile phase, standards, and

columns. The acceptance criteria for intermediate precision are the same as repeatability. The

percent difference in mean between the repeatability study and the intermediate precision must

be not more than outlined in Table 145.

Table 145: Acceptance Criteria for RC Intermediate Precision% Level % Difference (Absolute)

NMTO.l% A0.05%0.2—0.3%0.4—0.5% A0.15%0.6—0.8% 0.20%0.9—3.0%

Limit> 3.0% 10% relative

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Table 179: RC Precision Results for R,S-Isomer in Benazepril HC1IHCTZ Tablets on HPLCRepeatability: JSM - Intermediate Precision: LMG

Sample # HPLC System 01 Asset # 16930 IIPLC System 02 Asset # 16926Column Serial # 012030309136 Column Serial # 01203030913633

1 0.543 0.5602 0.544 0.5383 0.543 0.5564 0.545 0.5445 0.549 0.5616 0.547 0.558

Mean 0.545 0.553%RSD 1 2

%Difference 0.01

The specificity of the analytical method was evaluated by preparations of the placebo for

each pharmaceutical formulation. The sample diluent was also analyzed for specificity. The

chromatograms of the placebo and diluent injections were then analyzed for possible

interferences, which could cause bias on any analytical data. The acceptance criterion for the

related compound method was no placebo peak occurring at the same time of a corresponding

related compound peak above the respective limit of quantitation. The specificity of the related

compound method was further characterized through forced degradation studies. During these

studies, samples were exposed to various extreme conditions to demonstrate that degradation of

the samples could be observed by the analytical method. The forced degradation for the related

compound procedure was executed with that of the assay sample (the assay being an

appropriately diluted related compound sample). One could determine which peaks in the

chromatography are the result of active component degradation versus placebo peaks. The

percentage of total related compounds was then calculated for each condition and compared to

that of the assay value to determine the degree of mass balance between the two analytical

methods. The target degradation for each condition is not more than 150 & however, a higher

percentage of degradation was considered acceptable on a case-by-case basis. In some cases,

95

when the percentage of degradation observed was over the 15% target for the raw material, but

within acceptable levels for the finished product formulation, then the degradation pathway was

considered acceptable. This is due to the finished product blend being what is currently available

to the market and is the most indicative of the degradation pathways the true product would

experience under extreme conditions. The results of the forced degradation studies for related

compounds are outlined in Tables 180 - 188 with discussion to follow.

Table 180: Forced De adation Results for Fosino .ril Sodium Raw Material on UPLCII

I-Condition Time

ControlUV (Solutions) 3 HoursUV (Solutions) 5 Days

UV (Dry Powders) 5 DaysThermal (Dry Powders) 5 Days 105°CHumidity (Dry Powders) 5 Days

MSH (Dry Powders) 3 Hours 90°CAcidic (0.1N HC1) 10 Mm 60°C

Alkaline (0.0 iN NaOH) 10 MmOxidation (10%H202) 5 Mm 60°COxidation (3%H202) 5 Mm 60°C

Analyst: JSM and JUOKRC — Max Other Known Related CompoundUKRC — Max Unknown Related CompoundTRC — Total Related CompoundsMSH — Moisture Saturated HeadspaceRT — Room Temperature

Table 181: Forced Degradation Results for Benazepril HC1 Raw Material on UPLCCondition Time Temp %Benazeprilat %OKRC %UKRC %TRC

Control NA NA 0.0 ND ND 0.0UV (Solutions) 3 Hours RT 0.1 ND 0.0 0.1UV (Solutions) 5 Days RT 0.1 ND 0.2 0.5

UV (Dry Powders) 5 Days RT 0.1 ND ND 0.1Thermal (Dry Powders) 2 Days 105°C 0.1 ND ND 0.1Humidity (Dry Powders) 5 Days RT 0.1 ND 0.0 0.1

MSH (Dry Powders) 48 Hours 90°C 20.2 ND 0.7 21.3MSH (Dry Powders) 3 Hours 90°C 0.3 ND 0.0 0.4

Acidic (iN HCI) 2 Hours 60°C 2.8 ND 0.0 2.8Alkaline (0.1N NaOH) 5 Mm RT 6.6 ND 0.0 6.7Oxidation (lO%H202) 5 Mm 60°C 0.1 0.1 0.1 0.4

Analyst: JSM and JU

%RC-A I %OKRC %UKRC ( %TRC

96

Table 182: Forced Degradation Results for HCTZ Raw Material on UPLCCondition Time



MSH (Dry Powders) 3 Hours 90°CAcidic (0.1N HC1) 10 Mm 60°CAcidic (iN HC1) 2 Hours 60°C

Alkaline (0.0 iN NaOH) 10 MmAlkaline (0. iN NaOH) 5 MmOxidation (10%H202) 1 Hour 60°COxidation (10%H2O2) 5 Mm 60°COxidation (3%H2O2) 5 Mm 60°C

Condition TimeControl

UV (Solutions) 3 HoursUV (Solutions) 5 Days


MSH (Dry Powders) 3 Hours 90°C’Acidic (0.1N HC1) 10 Mm 60°C

Alkaline (0.0 iN NaOH) 10 MmOxidation (1 0%H2O2) 5 Mm 60°COxidation (3%H2O2) 5 Mm 60°C





Alkaline (0.O1N NaOH) 10 MmOxidation (1 0%H2O2) 5 Mm 60°COxidation (3%H2O2) 5 Mm 60°C

%4-Amino %OKRC %UKRC I %TRC

Analyst: JSM and JU

Table 183: Forced De adation Results for Fosino .ril Sodium Tablets on UPLC%RC-A I %OKRC %UKRC I %TRC

Analyst: JSM and JU

Table 184: Forced Degradation Results for Fosinopril SodiumJHCTZ Tablets (Fosinopril) onUPLC

%RC-A %OKRC %UKRC %TRC

Analyst: JSM and JU

97

Table 185: Forced Dei adation Results for Fosino .ril SodiumJHCTZ Tablets HCTZ on UPLCCondition Time


UV (Dry Powders) 5 DaysThermal (Dry Powders) 5 DaysHumidity (Dry Powders) 5 Days

MSH (Dry Powders) 3 HoursAcidic (0.1N HC1) 10 Mm

Alkaline (0.OIN NaOH) 10 MmOxidation (1 0%H2O2) 5 MmOxidation (3%H2O2) 5 Mm

Analyst: JSM and JU

Table 186: Forced De adation Results for Benaze.ril HCI Tablets on UPLCCondition Time



48 Hours3 Hours

Acidic (iN HC1) 2 HoursAlkaline (0.1N NaOH) 5 MmOxidation (10%H202) 1 Hour

Table 187: Forced Degradation Results for Benazepril HC1 HCTZ Tablets (Benazepril) onUPLC




48 Hours3 Hours

Acidic (1N HC1) 2 HoursAlkaline (0. iN NaOH) 5 MmOxidation (10%H202) 1 Hour

%4-Amino I %OKRC %UKRC %TRC

%Benazeprilat %OKRC %UKRC %TRC

Analyst: JSM and JU

%Benazeprilat %OKRC %UKRC

Analyst: JSM and JU

98

Table 188: Forced Degradation Results for Benazepril HC1/HCTZ Tablets (HCTZ) on UPLC(For Information Only)

Condition Time Temp %4-Amino %OKRC %UKRC %TRCControl NA NA 0.0 ND ND 0.0

UV (Solutions) 3 Hours RT 0.1 0.1 0.2 0.4UV (Solutions) 5 Days RT 9.9 ND 43.3 63.3

UV (Dry Powders) 5 Days RT ND ND ND NDThermal (Dry Powders) 2 Days 105°C 0.0 ND 0.0 0.1Humidity (Dry Powders) 5 Days RT 0.0 ND ND 0.0

MSH 48 Hours 90°C 1.9 0.1 0.2 2.3MSH 3 Hours 90°C 0.1 ND 0.0 0.2

Acidic (iN HC1) 2 Hours 60°C 2.9 ND 0.1 3.0Alkaline (0. iN NaOH) 5 Mm RT 0.0 ND ND 0.0Oxidation (l0%H202) 1 Hour 60°C 0.2 0.2 0.0 0.5

Analyst: JSM and JU

To begin the assessment of specificity using the related compound method on the

chromatographic system, forced degradation studies were executed using samples for each

placebo, raw material and finished product blend. These degraded samples were then compared

to non-degraded control samples to determine the different degradation pathways of the

respective product. The chromatograms of the control samples, injected on the UPLC, are

illustrated in Figures 30 - 41 below. Chromatograms are also provided for each of the

degradation conditions.

Figure 30: Chromatogram of the Blank (Diluent) - UPLC0.2

0.1;

0.16

0.14

0.1

o.1

0.08

0.06

0.04

0.0

0.0’

-0.00.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Mnutes

99

Figure 31: Chromatogram of the Fosinopril Sodium Raw Material (Control) - UPLC0.05

0.04

oLf)Q

0.03 ó 1

‘-I- A.

0.02 C dD

gAAO)

0.01CO1

AAjA

C

c0.00

0.90-0.01 0U-

-0.020.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

IVnutes

Figure 32: Chromatogram of the Benazepril HC1 Raw Material (Control) - UPLC0.05

0.0

0.03

0.02 I.D

0.01 A

A m0.00 ‘i:;;.

a)N

-0.01 C

-0.020.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

vnutes

Figure 33: Chromatogram of the HCTZ Raw Material (Control) - UPLC0.05

0

0.04 r— c’i

F— Lt 0

0.03 ...

a) I 0E j C PA

0.02 C) 0

Ix Ca 0D —

N AA

0.01Ca)

0.00

-0.01

-0.020.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

vinutes

100

Figure 34: Chromatogram of the Fosinopril Sodium Tablets Placebo (Control) - UPLC0.050

0.040

0.030

0.020

0.010

0.000

-0.0 10

-0.0200.00

c’l

PA

AAcD

x

12.

Figure 35: Chromatogram of the Fosinopril Sodium Tablets Blend (Control) - UPLC0.05’

D

vnutes

Figure 36: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Placebo (Control) - UPLC0.05

-0.020.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

vInutes

0)

Placebo Peak

4.0 6.0Minutes

8.0

-0.02’0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

D

0.0

0.03

0.02

0.01

0.00

-0.01

F—

U)

N

CU)CD

•1

1AVA”

NPlacebo Peak

- 0

— CDo -

.0

0—

0

101

Figure 37: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Blend (Control) - UPLC0.05’

D

D

D

Figure 38: Chromatogram of the Benazepril HC1 Tablets Placebo (Control) - UPLC0.05’

Figure 39: Chromatogram of the Benazepril HC1 Tablets Blend (Control) - UPLC0.05’

-0.02’0.00 1.00 2.00 3.00 4.00 5.00 6 I’ 7.00 8.00 9.00 10.00 11.00 12.00 13.00

nutes

102

Figure 40: Chromatogram of the Benazepril HC1/HCTZ Tablets Placebo (Control) - UPLC0.05’

Mnutes

Figure 41: Chromatogram of the Benazepril HCI/HCTZ Tablets Blend (Control) - UPLC

D

Sample solutions were prepared as per the proposed method for related compounds using

the raw material, the placebo, and the finished product blend for each product. These solutions

were then placed under UV light at both the short (254 nm) and long wavelengths (313 nm) for 3

hours and 5 days. Because HCTZ is known to degrade under UV conditions, the samples were

analyzed after 3 hours for the HCTZ containing components. All sample solutions were then

analyzed after 5 days for the ACE inhibitor components. As expected the HCTZ has degraded

over 5000 after the 5 days with some degradation observed after only three hours. Further

discussion regarding this condition may be expressed under their respective chromatograms for

clarity.

-0.02.0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Mnutes

103

Figure 42: Chromatogram of the Fosinopril Sodium Raw Material Sample Solution After UVExposure for 5 Days - UPLC

0.04

0fX

oc2D

u01c

oixx

-0.01c

Figure 43: Chromatogram of the Benazepril HC1 Raw Material Sample Solution After UVExposure for 5 Days - UPLC

Figure 44: Chromatogram of the HCTZ Raw Material Sample Solution After UV Exposure for5 Days - UPLC

I EN’ I-I— C)o r

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00Mnutes

1.W 2W 3W 4W aco SW 7W SW aW 1W 11W 12 ia

D

0.040

0.030

0.020D

0.010

13.00

-0.010

0.00

Condition too harsh for HCTZ.HCTZ is very sensitive to UVdegradation

104

Figure 45: Chromatogram of the Fosinopril Sodium Tablets Placebo Sample Solution After UVExposure for 5 Days - UPLC

0.05C

0.04(

-0.01(

-0.02G0.00 1.00 4. 6.00 7 12.00

Figure 46: Chromatogram of the Fosinopril Sodium Tablets Blend Sample Solution After UVExposure for 5 Days - UPLC

D

QOl’

-QOl’

QQJ

Figure 47: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Placebo Sample SolutionAfter UV Exposure for 5 Days - UPLC

cdX5oi

Placebo Peak

OLD aw 4W SW 6.W 7.W OLD aw 1GW 11.W 12W iax

0.;I

QI’,

a,”

0II

105

Figure 48: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Blend Sample SolutionAfter UV Exposure for 5 Days - UPLC

Figure 48: Chromatogram of the Benazepril HC1 Tablets Placebo Sample Solution After UVExposure for 5 Days — UPLC

Figure 49: Chromatogram of the Benazepril HC1 Tablets Blend Sample Solution After UVExposure for 5 Days - UPLC

D

Note: Due to the large amount of placebo degradation observed in this condition separation ofthe placebo peaks from the related compound peaks became difficult using the shorter UPLC

Mrtt

Minutes

106

gradient method. Therefore, a recommendation is made to run all stability samples forBenazepril HC1 Tablets using the more separated HPLC related compound method.

Figure 50: Chromatogram of the Benazepril HC1 HCTZ Tablets Placebo Sample Solution AfterUV Exposure for 5 Days - UPLC

0.05’

D

Note: There is usually a large single placebo peak in the Benazepril/HCTZ Tablet placebo.However, after UV degradation of the sample solution for 5 days this peak was shown to degradeinto many other large placebo peaks. This should be noted when performing investigations onexpired sample solutions stored on the bench top in clear glassware as this degradation mightoccur and bias results.

10.00 11.00 12.00 13.00

Figure 51: Chromatogram of the Benazepril HCI/HCTZ Tablets Blend Sample Solution AfterUV Exposure for 5 Days - UPLC

D

0.000 o . N Condition too harsh for HCTZt

-0.010a)

..009fl

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00Mnutes

107

Figure 52: Chromatogram of the Fosinopril Sodium Raw Material Sample Solution After UVExposure for 3 Hours - UPLC

a)N(aCa,

Minutes

Figure 53: Chromatogram of the BenazeprilExposure for 3 Hours - UPLC

0.05

0.0

0.03

0.02D

0.01

0.00

-0.01

-0.020.00 1.00 2.00 3.00 4.00 5.00

HC1 Raw Material Sample Solution After UV

0

A.1A

A

6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00IVnutes

Figure 54: Chromatogram of the HCTZ Raw Material Sample Solution After UV Exposure for3 Hours - UPLC

0.050 --

D

108

Note: HCTZ degradation in sample solution could be observed after only 3 hours under UVlight. This is the only condition in which the CTZ related compound was shown to increasedramatically along with other unknown HCTZ related compounds that are not observed in anyother degradation condition. The 4-Amino was not observed in this condition as this relatedcompound appears to be a hydrolysis related compound. The Sandoz monograph FOO9QC forFosinopril SodiumIHCTZ Tablets specifically states that CTZ is a process-related impurity andshould NOT be quantitated for related compounds. However, as shown by this study the CTZ isclearly a degradant as well. This monograph should be updated to include this impurity in thecalculations for total related compounds.

Figure 55: Chromatogram of the Fosinopril Sodium Tablets Placebo Sample Solution After UVExposure for 3 Hours - UPLC

0.050

0.040

O

-o.oic P1acebo Peak

-0.0200.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Mnutes

Figure 56: Chromatogram of the Fosinopril Sodium Tablets Blend Sample Solution After UVExposure for 3 Hours - UPLC

0 050

D

109

Figure 57: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Placebo Sample SolutionAfter UV Exposure for 3 Hours - UPLC

0.050

0.040

0.030 cftu!,

CDCSC .

0.020 uD

N )AVAtA0.010 — CD

a a0.000

-CD

a-

-0.010 Placebo Peak

-0.0200.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

Minutes

Figure 58: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Blend Sample SolutionAfter UV Exposure for 3 Hours - UPLC

0.050

D

Note: The CTZ does not appear as it did in the raw material sample solution alone. Thisindicates that the presence of the placebo matrix may help to shield the HCTZ from UVdegradation when in the sample solution.

I.

110

-0.020.00 1.00 2.00 3.00

iil

4.00 5.00 6.00Mnutes

CD

cc

7.00 8.00 9.00 10.00 11.00 12.00 13.00

Figure 60: Chromatogram of the Benazepril HC1 Tablets Blend Sample Solution After UVExposure for 3 Hours - UPLC

D

Mnutes

Figure 59: Chromatogram of the Benazepril HC1 Tablets Placebo Sample Solution After UVExposure for 3 Hours - UPLC

0.050

0.040

0.030

0.020

0.01

0.000

-0.01

-0.02’0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Figure 61: Chromatogram of the Benazepril HC1 HCTZ Tablets Placebo Sample Solution AfterUV Exposure for 3 Hours - UPLC

0.05’

5.00 6.00 7.00Mnutes

10.00 11.00 12.00 13.00

111

Figure 62: Chromatogram of the Benazepril HC1/HCTZ Tablets Blend Sample Solution AfterUV Exposure for 3 Hours - UPLC

0.05’

- A

A -

A A -

N

a)

-0.02.0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Mnutes

Figure 63: Chromatogram of the Fosinopril Sodium Raw Material Dry Powders After UVExposure for 5 Days — UPLC

ü0E

JiD

101(

-G01(

OXD 1.W 2(0 3OJ

Figure 64: Chromatogram of the Benazepril HC1 Raw Material Dry Powders After UVExposure for 5 Days UPLC

0.050)CD

7(0 aX) 10(0

D

0.04

0.03

0.02

0.01

0.00

-0.01

-0.020.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Fvnutes

112

Figure 65: Chromatogram of the HCTZ Raw Material Dry Powders After UV Exposure for 5Days—UPLC

0.05

0.04‘4,

0)0.03

E0.02 N

DC)

0.01A

0.00 jA U)

-0.01

-0.020.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Mnutes

Figure 66: Chromatogram of the Fosinopril Sodium Tablets Placebo Dry Powders After UVExposure for 5 Days UPLC

0.05

0.00)

0.03

0.02 I:D I

.1 .o AAA0.01 II

U) 4A

0.00 AAA A U)

-0.01

-0.020.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Fvnutes

Figure 67: Chromatogram of the Fosinopril Sodium Tablets Blend Dry Powders After UVExposure for 5 Days UPLC

0.05’

D

Mnutes13.00

113

Figure 68: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Placebo Dry Powders AfterUV Exposure for 5 Days UPLC

D

Figure 69: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Blend Dry Powders AfterUV Exposure for 5 Days — UPLC

D

D

9.00 10.00 11.00 12.00 13.00

-0.02’0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Finutes

Figure 70: Chromatogram of the Benazepril HC1 Tablets Placebo Dry Powders After UVExposure for 5 Days — UPLC

0.05.

Mnutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

114

Figure 71: Chromatogram of the Benazepril HC1 Tablets Blend Dry Powders After UVExposure for 5 Days — UPLC

Figure 72: Chromatogram of the Benazepril HC1/HCTZ Tablets Placebo Dry Powders AfterUV Exposure for 5 Days UPLC

0.05’

D

Figure 73: Chromatogram of the Benazepril HC1/HCTZ Tablets Blend Dry Powders After UVExposure for 5 Days — UPLC

D

Note: Recommdation is to use the HPLC related compound method when running analysisusing Benazepril HC1 Tablets or Benazepril HC1IHCTZ Tablets for better separation.

13.00

Minutes

Mnutes

115

Figure 75: Chromatogram of the Benazepril HC1 Raw Material Powder Exposed to Humidityfor 5 Days at Room Temperature - UPLC

0.05

CD

Figure 74: Chromatogram of the Fosinopril Sodium Raw Material Powder Exposed toHumidity for 5 Days at Room Temperature - UPLC

0.,;,

iax

0.04

0.03

0.02 •1.D

0.01 A

A0.00

0

N-0.01

-0.020.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Mnutes

Figure 76: Chromatogram of the HCTZ Raw Material Sample Powder Exposed to Humidity for5 Days at Room Temperature - UPLC

0’.’

0’’

0.”’

0.’ I

0.01’

0.iii

-0.01’

-a.’’1 2w aoo 4.cO 5:w 6.w 7.w &w 9.00 10.00 11.00 1200

116

Figure 77: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder Exposed toHumidity for 5 Days at Room Temperature - UPLC

0..;’

D

-(loll

Figure 78: Chromatogram of the Fosinopril Sodium Tablets Blend Powder Exposed toHumidity for 5 Days at Room Temperature — UPLC

I

_

Placebo Peak

1.3 2 3.W 4. 5. OW 7.W 8W 9.W 1O.W 1l.W laW

Figure 79: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Placebo Powder Exposed toHumidity for 5 Days at Room Temperature - UPLC

0.

D

(101’

-001’

a’,’

0.01’

0.i-i

O.i’’

0.11

-0. i’lOW

117

Figure 80: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Blend Powder Exposed toHumidity for 5 Days at Room Temperature - UPLC

O.i;i

D

001’

0.11 I

-001’

- ,Ø,

oa

Figure 81: Chromatogram of the Benazepril HC1 Tablets Placebo Powder Exposed to Humidityfor 5 Days at Room Temperature - UPLC

D

Figure 82: Chromatogram of the Benazepril HC1 Tablets Blend Powder Exposed to Humidityfor 5 Days at Room Temperature - UPLC

D

0..c.

O.i’i

tw zo ao 4W SW 7W &W aW flW 11W 12W iac

-0.02’0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

vnutes

118

0.0a,C,(U0

Figure 85: Chromatogram of the Fosinopril Sodium Raw Material Powder After ThermalDegradation at 105°C for 5 Days- UPLC

0c6

cmi

k03

ox

G011

-0O1(

lW 2W 3W 4W SW 6.cO 7W &W co 1W 11W 12W 13

Figure 83: Chromatogram of the Benazepril HCI/HCTZ Tablets Placebo Powder Exposed toHumidity for 5 Days at Room Temperature - UPLC

0.05

0.04

0.03

0.02

0.01iA(U

0.00 cr

-0.01

-0.020.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Fvnutes

Figure 84: Chromatogram of the Benazepril HC1/HCTZ Tablets Blend Powder Exposed toHumidity for 5 Days at Room Temperature - UPLC

0.051

0.04’

0.03’

0.02’D

0.01’

0.00i

-0.01’

-0.02’0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

vnutes

D

119

Figure 86: Chromatogram of the Benazepril HCI Raw Material Powder After ThermalDegradation at 105°C for 2 Days- UPLC

0.OOC

-0.01c

-0.02C

-

5.00 6.00 7.00

Figure 87: Chromatogram of the HCTZ Raw Material Sample Powder After ThermalDegradation at 105°C for 5 Days- UPLC

0.’

0.’ I

0. II

0.0I

0.01’

0.11,

-0.01,

-0.1.1

OGD

Figure 88: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder After ThermalDegradation at 105°C for 5 Days- UPLC

0....

0.00 1.00 2.00 3.00

Mnutes8.00 9.00 10.00 11.00 12.00 13.00

120

Figure 89: Chromatogram of the Fosinopril Sodium Tablets Blend Powder After ThermalDegradation at 105°C for 5 Days- UPLC

D

Note: A large unknown Fosinopril related compound peak was observed at 5.3 minutes, elutingbeside the large baseline disturbance caused by the placebo degradation under heat. This relatedcompound has been observed in the expired samples used during method validation as well.This related compound appears to be secondary degradation of the RC-A peak and would not becaptured by the existing Sandoz monographs.

Figure 90: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Placebo Powder AfterThermal Degradation at 105°C for 5 Days- UPLC

D

MnA

Ues

121

Figure 91: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Blend Powder AfterThermal Degradation at 105°C for 5 Days- UPLC

O.i;i

D

0.’ I

0. l.

o.l,’

Coil

CIII

-0.01’

-0OW

Note: The large RC peak at 5.3 minutes observed in the combination formulation as well.Figure 92: Chromatogram of the Benazepril HC1 Tablets Placebo Powder After ThermalDegradation at 105°C for 5 Days- UPLC

‘-LI-nj

QQI

cicD

oDic

ac

.uO1(

010 1.W 2W

IJ.

Figure 93: Chromatogram of the Benazepril HC1 Tablets Blend Powder After ThermalDegradation at 105°C for 2 Days- UPLC

3W 4W SW 6W 7W SW 0 lOW 11.W 12W 13(X

122

-0.020.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

rvnutes

Figure 94: Chromatogram of the Benazepril HC1/HCTZ Tablets Placebo Powder After ThermalDegradation at 105°C for 2 Days- UPLC

Figure 95: Chromatogram of the Benazepril HC1/HCTZ Tablets Blend Powder After ThermalDegradation at 105°C for 2 Days- UPLC

After MoistureFigure 96: Chromatogram of the Fosinopril Sodium Raw Material PowderSaturated Headspace at 90°C for 30 minutes - UPLC

0.05

D

0.0

II0.03 d

0.02

‘ L A

A

‘A’L CD0.01

A

CC

0.00

-0.01

123

CD CDCD0) CDCD C

U CD U r... •)lCD C

(50)C— • Eo C,’C,’

O N AC

C-)AA

-0.02000 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 1000 1100 12.00 13.00

Minutes

C,’CD CD0) CDCD CD

0)

CDis I C

— CD iso CD

A-U)

AA 0C.,

N AYACD

0 -z CisCD

-0.0200.00 1.00 200 3.00 4.00 5.00 6.00 700 8.00 9.00 10.00 11.00 12.00 1300

Minutes

Figure 97: Chromatogram of the Benazepril HC1 Raw Material Powder After MoistureSaturated Headspace at 90°C for 30 minutes - UPLC

0.050

D

Figure 98: Chromatogram of the HCTZ Raw Material Sample Powder After Moisture SaturatedHeadspace at 90°C for 30 minutes - UPLC

0.05

D

0.04

0.03

0.02

0.01

0.00

-0.01

a

A

a

CD

CCs

CD

Figure 99: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder After MoistureSaturated Headspace at 90°C for 30 minutes - UPLC

0.050

0.04

0.030

0.02

0.010

0.000

-0.0 10

IAYA A A

Placebo Peak

124

. II

Placebo Peak

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10:00 11:00 12.00Minutes

13.00

Figure 102: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Blend Powder AfterMoisture Saturated Headspace at 90°C for 30 minutes - UPLC

0.0 10

0.000

-0.0 10

Figure 100: Chromatogram of the Fosinopril Sodium Tablets Blend Powder After MoistureSaturated Headspace at 90°C for 30 minutes - UPLC

0.050

0.040

0.030

0.020

0.0 10

0.000

-0.0 10

-0.0200.00

Figure 101: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Placebo Powder AfterMoisture Saturated Headspace at 90°C for 30 minutes - UPLC

0.040

0.030

0.020

0.010

0.000

-0.0 10

0.050

0.040

0.030

0.020

-0.0200.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Minutes

125

0.040

0.030

0.020

0.010

0.000

-0.010

-0.0200.00

Figure 104: Chromatogram of the Benazepril HC1 Tablets Blend Powder After MoistureSaturated Headspace at 90°C for 30 minutes - UPLC

Figure 103: Chromatogram of the Benazepril HCI Tablets Placebo Powder After MoistureSaturated Headspace at 90°C for 30 minutes - UPLC

0.050

D

Minutes

D

Figure 105: Chromatogram of the Benazepril HC1/HCTZ Tablets Placebo Powder AfterMoisture Saturated Headspace at 90°C for 30 minutes - UPLC

0.050

0.040

0.030

0.020D

0.0 10

0.000

-0.0 10

-0.0200.00 1.00 2.00 3.00 4 it 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

Minutesii

126

Figure 106: Chromatogram of the Benazepril HC1/HCTZ Tablets Blend Powder After MoistureSaturated Headspace at 90°C for 30 minutes - UPLC

0.16

0.14

0.12

D0.10

0.08

0.06

0.04

0.02

9.00 10.00 11.00 I’ 13.00

D

Figure 107: Chromatogram of the Fosinopril Sodium Raw Material Powder After AcidHydrolysis - UPLC

0.050

D

Figure 108:- UPLC

0.20

0.18

Chromatogram of the Benazepril HC1 Raw Material Powder After Acid Hydrolysis

•1

I:)

A A /.,

2.00 3.00 4.00 5.00

0.0

-0.020.00 1.00

It)

C

C(V

900 10.00 11.00 1200 13.006.00 7.00 8 00Minutes

127

Figure 109: Chromatogram of the HCTZ Raw Material Sample Powder After Acid Hydrolysis -

UPLC0.20

0.18

0.16

0.14

0.12

0.10D

0.08•

0.06

0.04

0.02

0 .0

-0,020.00 1 ‘0 2.00 3.00 4 ii

Figure 110: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder After AcidHydrolysis - UPLC

0.050

CD

Placebo Peak

-0.0200.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Minutes

Figure 111: Chromatogram of the Fosinopril Sodium Tablets Blend After Acid Hydrolysis -

UPLC

4-Amino observed after hydrolysis.

c) 0

U,C.) U)

C

N

5.00 6’’ 7.00 :1. 9.00 10.00 ‘ 11.00 12’’M flutes

DO

0.040

O.03C

0.020

0.0 10

0.000

-0.0 10

D

3.00 4.00 5.00 6.00 700 8.00 9.00 10.00 11.00 12.00 13.00Minutes

128

0.010

0.0 00

-0.0 10

-0.0200.00

Figure 114: Chromatogram of the Benazepril HC1 Tablets Placebo Powder After AcidHydrolysis — UPLC

0.20

-0.00.00 1.00

Note: As observed with the theimal degradation the different ink peaks in the Benazepril HC1placebo are shown to degrade after hydrolysis reactions in acid. This should be noted as

Figure 112: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Placebo Powder After AcidHydrolysis - UPLC

0.050

0.040o

CD

0.030 l C

-I •

0.020D ——

0.010 CD

0.000

-0.0 10Placebo Peak

-0.02 00.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12

Minutes

Figure 113: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Blend Powder After AcidHydrolysis - UPLC

0.050

0.040

0.030

0.02 0

1.00 2.00 3.00 4.00 5.00 6.00 7,’ II

Minutes

0.18

0.16

0.14

0.12

D0.10

0.08

0.06

0.04

0.02

0.00

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00Minutes

13.00

129

-0.02000 6.00 7.00 8.00 9.00 10.00 11.00 ‘I 13.00

Minutes

Figure 116: Chromatogram of the Benazepril HC1/HCTZ Tablets Placebo Powder After AcidHydrolysis - UPLC

0.20

separation of these placebo peaks from related compound peaks is difficult and the HPLCmethod should be employed for this product.

Figure 115: Chromatogram of the Benazepril HC1 Tablets Blend Powder After Acid Hydrolysis-UPLC

0.20

0.18

0.16

0.14

0.12

0.10D

0.08

0.06

0.04

0.02

0.00

S-Amine is listed as a process-related impurity

1.00 2.00 3.00 4.00 5.00

D

11.00 12.00 13.00

130

Figure 119: Chromatogram of the Benazepril HC1 Raw Material Powder After BasicHydrolysis - UPLC

:

0.00 0 60 700

M in isle n

Note: The Benazeprilat was shown to increase as a result of basic hydrolysis with a secondarydegradation into the related compound peak at 3.6 minutes. This degradation is similar to the

131

Figure 117: Chromatogram of the Benazepril HC1/HCTZ Tablets Blend Powder After AcidHydrolysis — UPLC

0.2.

2.00 3.00 4 00Mnutes

7.00 8.00 9.00 10.00 11.00 12.00 13.00

Figure 118: Chromatogram of the Fosinopril Sodium Raw Material Powder After BasicHydrolysis - UPLC

0.050

C01

0.040 Ci •

0.030 I, •

0.020 .ILI_ LL AVAA 0) CD

0.010 AAAAA c

0.0000.0C

-0.010Ii-

-0.0200.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

Minutes

.00 0.00 1000 1100 1200 1300

secondary degradation observed of the RC-A in the related compound at 5.3 minutes in theFosinopril containing samples.

-0.020.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 1100 12.00 13.00

.inutes

Figure 121: Chromatogram of the FosinoprilHydrolysis - UPLC

0.050

Sodium Tablets Placebo Powder After Basic

It

Figure 120:- UPLC

0.05

0.04

0.03

0.02

0.01

0.00

-0.01

Chromatogram of the HCTZ Raw Material Sample Powder After Basic Hydrolysis

F—0Lr,

F.coa,

aSIt) U)

U)PAa)a)

o E —

A.0 0

aS- —o N AA

EC

132

Figure 122: Chromatogram of the Fosinopril Sodium Tablets Blend Powder After BasicHydrolysis - UPLC

0.050 —

Figure 123: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Placebo Powder AfterBasic Hydrolysis - UPLC

0.050

-0.0200.00

CC.) a:, ‘—•

c-’l

.•

A

LAVA

lCDop

1.00 2.00 3.00 4.00 5.00 600 700 8.00 9.00 10.00 11.00 1200 13.00Minutes

I• II

D

0.040

0.030

0.020

0.010

0.000

-0.0 10

c’JU)

C.,

N

I

NPlacebo Peak

Figure 124: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Blend Powder After BasicHydrolysis - UPLC

0.050

-0.0200.00 i ii ii . ii Ii 6.00 7.00 8.00 9.00 10.00 11.00

Minutes

133

Figure 125: Chromatogram of the Benazepril HC1 Tablets Placebo Powder After BasicHydrolysis - UPLC

0.20

Figure 126: Chromatogram of the Benazepril HC1 Tablets Blend Powder After BasicHydrolysis - UPLC

0.2’

D

0.18

0.16

0.14

0.1

0.11

0.0;

0.0.

0.04

0.0

0.0.

-0.0

‘I

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00Mnutes

134

0)C..

.2C.)

0

0)

U,

CCs

C .in

CCU

AA

/PA

A..n

CDAAA

j

-0.0200.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

135

Minutes

Figure 127: Chromatogram of the Benazepril HCI HCTZ Tablets Placebo Powder After BasicHydrolysis - UPLC

0 20

0.18

0.16

0.14 C_.11 0)

CD0.12 i 0)

c’J0.10 I c.J -

I l0.08

Cs0.06 f i004 c

0.02

0.00 A

-0.020.00 1.00 2.00 3.00 4.00 5.00 600 700 8.00 9.00 10.00 11.00 12.00 1300

Minutes

Figure 128: Chromatogram of the Benazepril HC1 HCTZ Tablets Blend Powder After BasicHydrolysis - UPLC

0.20

0.18

0.16

0.14

0.12

0.10D

0.08

0.06

0.04

0.02

0.00

-0.020.00 1.00

Minutes

Figure 129: Chromatogram of the Fosinopril Sodium Raw Material Powder After Oxidation -

UPLC0.050

2.Ot 3.00 4.00 5.00 6.00 7 II 8.00 9.00 10.00 11.00 12.00 13.00

0.040

0.030

0.020

0.0 10

0.000

-0.0 10

c.’JU,

ci2:

C

C

Figure 130: Chromatogram of the Benazepril HCI Raw Material Powder After Oxidation -

UPLC

Figure 131: Chromatogram of the HCTZ Raw Material Sample Powder After Oxidation -

UPLC

13.00

Chromatogram of the Fosinopril Sodium Tablets Placebo Powder After Oxidation -Figure 132:UPLC

0.050

0.040

0.030

0.020D

0.0 10

0.000

-0.0 10

-0.0200.0

cD

‘A

A.

aU,

Placebo Peak

4. 6.00 7.00 8Minutes

11.00

136

0C’,0

•0• a5

Figure 135: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Blend Powder AfterOxidation - UPLC

0.050

0.040

0.030

0.020D

0.0 10

0.000

-0.0 10

-0.0200.0’

137

Figure 133: Chromatogram of the Fosinopril Sodium Tablets Blend Powder After Oxidation -

UPLC0.05’

0.04’

0.03’

0.02’

0.01’

0.00’

-0.01’

-0.02’0.00 1.00 2.00 3.00 4.00 500 6.00 700 8.00 900 1000 11 00 1200 13.00

Minutes

Figure 134: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Placebo Powder AfterOxidation - UPLC

0.050

D‘C

0.040

0.030

0.020

0.0 10

0.000

-0.0 10

U,

C’,

C)

“A

.- 0

‘P1acebo Peak

-0.02 00.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

Minutes13.00

ii 2.’e ,, 4,, I, .00 7.00 8.00 9.00 , ii ii 1 I’

Minutes

Figure 136: Chromatogram of the Benazepril HC1 Tablets Placebo Powder After Oxidation -

UPLC0.20

0.18

0.16

0.14

0.12

0.10

0.08

0.06

0.04

0.02

0.00

-0.020.00

Minutes

Figure 137: Chromatogram of the Benazepril HC1 Tablets Blend Powder After Oxidation -

UPLC0.20

0.18

0.16

0.14

0.12

0.10

0.08

0.06

0.04

0.02

0.00

-0.020.00

Figure 138: Chromatogram of the Benazepril HCI/HCTZ Tablets Placebo Powder AfterOxidation - UPLC

D

Mnutes

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 1300

-0.00.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

138

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MSH (Dry Powders) 48 HoursAcidic (iN HC1) 2 Hours

Alkaline (0. iN NaOH) 5 MmOxidation (10% H202) 1 Hour

Condition

_____________________________________




Alkaline (0.O1N NaOH) 10 MmOxidation (1 0%H202) 5 Mm 60°COxidation (3%H202) 5 Mm 60°C

Condition Time Temp %RC-A %OKRC %UKRC %TRCControl




Alkaline (0.0 iN NaOH) 10 MmOxidation (10%H202) 5 Mm 60°COxidation (3%H202) 5 Mm 60°C

Table 191: Forced De adation Results for HCTZ Raw Material on HPLC

I I________%4-Amino %OKRC) %UKRC

Analyst: JSM and JU

Table 192: Forced De adation Results for Fosino .ril Sodium Tablets on HPLC%RC-A %OKRC %UKRC %TRC

Analyst: JSM and JU

Table 193: Forced Degradation Results for Fosinopril SodiumIHCTZ Tablets (Fosinopril) onHPLC

Analyst: JSM and JU

140

Table 194: Forced De adation Results for Fosino.ril SodiumJHCTZ Tablets HCTZ on HPLCCondition Time




Alkaline (0.0 iN NaOH) 10 MmOxidation (10%H2O2) 5 Mm 60°COxidation (3%H202) 5 Mm 60°C





Alkaline (0.1N NaOH) 5 MmOxidation (10% H2O2) 1 Hour

Analyst: JSM and JU





Alkaline (0.1 N NaOH) 5 MmOxidation (10% H2O2) 1 Hour

%4-Amino %OKRC %UKRC %TRC

Analyst: JSM and JU

Table 195: Forced Dei adation Results for Benaze.nl HC1 Tablets on HPLC

____I


Table 196: Forced Degradation Results for Benazepril HC1/HCTZ Tablets (Benazepril) onHPLC


Analyst: JSM and JU

141

Table 197: Forced Degradation Results for Benazepril HCI/HCTZ Tablets (HCTZ) on HPLC(For Information Oni





Alkaline (0. iN NaOH) 5 MmOxidation (10% H202) 1 Hour

To begin the assessment of specificity on the HPLC forced degradation control samples

for each placebo, raw material and finished product blend were prepared for comparison

purposes. These chromatograms are illustrated in Figures 140 244 below. The comments

included in the UPLC section regarding the degradation pathways are applicable to the HPLC

chromatograms as well and will therefore not be repeated. Due the longer gradient of the related

compounds analysis, the placebo peaks and related compound peaks observed in the Benazepril

HC1 containing products are much more well separated. A recommendation is made to utilize

the HPLC related compound method when running related compound analysis, for Benazepril

HC1 products. This separation is not an issue during assay analysis as the actual Benazepril peak

is not impacted at the assay concentration as shown by the passing purity during the forced

degradation studies for assay. The UPLC or HPLC method are both considered to be acceptable

for analysis of Fosinopril containing products.

%4-Amino I %OKRC I %UKRC

Analyst: JSM and JU

142

Figure 140: Chromatogram of the Blank (Diluent) — HPLC0.050

-0.010

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00Mnutes

Figure 141: Chromatogram of the Fosinopril Sodium Raw Material (Control) - HPLC0.05

0.04

0U,C)

0.03C’1

C)0.02 a

0.01A

0.00

-0.010(0

-0.020.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 3400 36.00 38.00 40.00

Mnutes

Figure 142: Chromatogram of the Benazepril HC1 Raw Material (Control) - HPLC0.05

0.0

0.03

0.02D

0.01

0.00 —

a)-0.01

a)-0.02

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00Finutes

143

Figure 143: Chromatogram of the HCTZ Raw Material (Control) - HPLC0.05

-0.01

-0.020.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 3400 36.00 38.00 40.00

Anutes

Figure 144: Chromatogram of the Fosinopril Sodium Tablets Placebo Control - HPLC0.050

-0.010

—0.020,0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Mnutes

Figure 145: Chromatogram of the Fosinopril Sodium Tablets Blend Control - HPLC0.05 —

0.0F.—

-

0.03

0)

0.02

A

-0.01

-0.020.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

flutes

144

0)

0F—0)F—

c-I0I

C.)

Figure 146: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Placebo Control - HPLC0.05

0.0

0.03

0.02D

0.01

0.00

Placebo Peak

-0.020.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 3400 36.00 38.00 40.00

vInutes

Figure 147: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Blend Control - HPLC0.05’

-0.02’ —

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00Mnutes

Figure 148: Chromatogram of the Benazepril HC1 Tablets Placebo Control - HPLC0.05’

D

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Mnutes

145

Figure 149: Chromatogram of the Benazepril HC1 Tablets Blend Control - HPLC0.05’

0.04’

0.03’

0.02’D

0.01’

0.00’

-0.01’

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Mnutes

Figure 150: Chromatogram of the Benazepril HC1/HCTZ Tablets Placebo Control - HPLC0.05’

0.04’

D

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00nutes

Chromatogram of the Benazepril HCI/HCTZ Tablets Blend Control - HPLC

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.001nutes

The raw material, placebo, and blend samples for each product were prepared as outlined

in the proposed method. The sample solutions were then exposed to ultraviolet (UV) light at

both the short and long wavelengths for 3 hours and 5 days. The HCTZ was shown to be the

146

(0

0)d(‘I

0

0)0CO

-0.02’0.00

Figure 151:

most susceptible component to the UV light degradation when in the sample solution. The

HCTZ degraded 300 after only 3 hours of exposure to the UV light, and over 5000 when exposed

for 5 days. The large degree of degradation in the 5 days samples could possibly contain

secondary degradants and is therefore not a representation of the primary degradation pathways.

The 3 hour time point was utilized for the analysis of the primary degradation pathway of the

HCTZ component only. Both the Fosinopril and Benazepril components were shown to be

stable against UV degradation when in the sample solution.

Figure 152: Chromatogram of Fosinopril Sodium Raw Material Sample Solution After UVLight Exposure for 5 Days - HPLC

aw

D

2W 4W 6W aw iaw 12W 14W 1BW laW 21W 22W 24W 21W W 21W 21W 34(1) W W 42C(

Figure 153: Chromatogram of HCTZ Raw Material Sample Solution After UV Light Exposurefor 5 Days —HPLC

D

Mnutes

147

Figure 154: Chromatogram of HCTZ Raw Material Sample Solution After UV Light for 3Hours - HPLC

0.050

S

,0.040 ;:;

0.030

0020 -

LjJJLL0.010

0.000 ._.__q3

-0.010

0.00 2.00 4.00 6.00 8,00 0.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00Minutes

Figure 155: Chromatogram of Benazepril HC1 Raw Material Sample Solution After UV Lightfor 5 Days - HPLC

D

0.040

0.030

0.020

0.010

0.000

-0.010

-0.0200.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Mnutes

H

Figure 156: Chromatogram of Fosinopril Sodium Tablets Placebo Sample Solution After UVLight Exposure for 5 Days — HPLC

ac

D

cn,‘r

ElQON

QW

-Q01( ,,.

QD 2D 4W 6W 6W 1QW 2(1) 14W 16W 18(X) aIW W 24W W (D 32W W 34(X) W QJ fl(X

148

Figure 157: Chromatogram of Fosinopril Sodium Tablets Blend Sample Solution After UVLight Exposure for 5 Days - HPLC

0....

0.01’

0.”

-0.01’

Figure 158: Chromatogram of Fosinopril SodiumJHCTZ Tablets Placebo Sample Solution AfterUV Light Exposure for 5 Days - HPLC

D

Qolel

-QOl ‘I

0.ii

_0.l,I • -

OW 2W 4W 6W SW 10C0 12W 14W 16W 18W aLW 22W 24W W W 32W QW 34W 32W 32W 42C(

Q

Qeø I

QlcII

QIlI

-UQW 2W 4W 6W SW 1Q03 1203 14W 1603 1603 2CO 22W 2403 Q) ZW 3303 32W 34CC 32W 3203 42CC

Figure 159: Chromatogram of Fosinopril SodiumJHCTZ Tablets Blend Sample Solution AfterUV Light Exposure for 5 Days - HPLC

D

-0D1-

00

003 20) 4W SCE) 60) lOW 12W 14W 16W 16W 21W 720) 24W W W 32W 03 34W W W 42CC

149

Figure 160: Chromatogram of Fosinopril SodiumJHCTZ Tablets Placebo Sample Solution AfterUV Light Exposure for 3 Hours - HPLC

0.05’

0.0’

0.03’

0.02’

0.01’

0.00’

-0.01’

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

nutes

Figure 161: Chromatogram of Fosinopril Sodium/HCTZ Tablets Blend Sample Solution AfterUV Light Exposure for 3 Hours - HPLC

0.05’

0.0’

0.03’

0.02’D

0.01’

0.00i

-0.01.

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Mnutes

150

Figure 162: Chromatogram of Benazepril HC1 Tablets Placebo Sample Solution After UV LightExposure for 5 Days - HPLC

0.05.

Mnutes

Figure 163: Chromatogram of Benazepril HC1 Tablets Blend Sample Solution After UV LightExposure for 5 Days - HPLC

D

Mnutes

Figure 164: Chromatogram of Benazepril HC1IHCTZ Tablets Placebo Sample Solution AfterUV Light Exposure for 5 Days - HPLC

0.050

0.040

0.030

0.020D

0.010

0.000

-0.010

-0.0200.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Mnutes

-0.02.0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Much better separation in HPLC method withR,S-Isomer being accurately quantitated(though under the LOQ).

I I I I I16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00

IFIIIIIIII1U C’)

FTIl_LI

H

Large amount of placebo degradation.

151

Figure 165: Chromatogram of Benazepril HC1/HCTZ Tablets Blend Sample Solution After UVLight Exposure for 5 Days - HPLC

D

Figure 166: Chromatogram of Benazepril HC1/HCTZ Tablets Placebo Sample Solution AfterUV Light Exposure for 3 Hours - HPLC

0.05’ -__________

D

Figure 167: Chromatogram of Benazepril HC1/HCTZ Tablets Blend Sample Solution After UVLight Exposure for 3 Hours - HPLC

D

Mnutes

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Mnutes

10.00 12.00 14.00 16.00 18.00 20.00 22.00nutes

152

The raw material, placebo, and blend samples dry powders for each product were

weighed into individual 1 OO-mL volumetric flasks. The dry powder samples were then placed in

a UV light chamber at both the short and long wavelengths for 5 days. After the 5 day period all

samples were prepared as outlined in the proposed method and analyzed for related compounds.

All raw material, placebo, and finished product blends were shown to be stable against

degradation due to UV light when in the powder form.

Figure 168: Chromatogram of Fosinopril Sodium Raw Material Dry Powder After StorageUnder UV Light for 5 Days — HPLC

uu1

c.o4

A::

.oo1

nny.

OW 2W 4W W aco flW 12(0 140) 1&W laW aico co 24(0 co iw o 34co o co gao:

Figure 169: Chromatogram of HCTZ Raw Material Dry Powder After Storage Under UV Lightfor 5 Days HPLC

after storage under UV light for 5 days.

0.00 2.00 4.bO 6.00 8.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34:00 36.00 38:00 4.00Mnutes

153

Figure 170: Chromatogram of Benazepril HC1 Raw Material Dry Powder After Storage UnderUV Light for 5 Days — HPLC

U.UU if) CO)- co-

0040 (o1— 0)

0.030

EE—_L1-0.010

a)flil)fl

0.00 2.00 4.00 6.00 8.00 1000 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00vinutes

Figure 171: Chromatogram of Fosinopril Sodium Tablets Placebo Dry Powder After StorageUnder UV Light for 5 Days — HPLC

0.04.

00

0.02.

0.01

0.0.

-0.01.

-0.02.000 2.00 4.00 6.00 8.00 1000 12 00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Mnute

0 02.00. 2.00 4.00 600 800 10.00 12 00 14.00 16.00 1800 20.00 22.00 2400 2600 2800 30.00 32.00 34.00 3600 38.00 4000

Mnues

154

Figure 173: Chromatogram of Fosinopril SodiumIHCTZ Tablets Placebo Dry Powder AfterStorage Under UV Light for 5 Days HPLC

Mnutes

Figure 175: Chromatogram of Benazepril HC1 Tablets Placebo Dry Powder After StorageUnder UV Light for 5 Days - HPLC

D

Figure 174: ChromatogramStorage Under UV Light for

.002000 200 400 6.00 8.00 10.00 1200 14 00 1600 18.00 2000 22 2400 26.00 2800 30.00 32.00 34.00 36.00 38.00 40.00

SodiumJHCTZ Tablets Blend Dry Powder After

Mrn,t.s

Mnutes

155

Figure 176: Chromatogram of Benazepril HC1 Tablets Blend Dry Powder After Storage Under

UV Light for 5 Days - HPLC

LJ’1-0.010

..flfl9l’

2.00 4.00 6.00 8.00 1000 12.00 14.00 16:00 18.00 20:00 22:00 24:00 26.00 28:00 30:00 32:00 34.00 36.00 38.00 40.00Mnutes

Figure 177: Chromatogram of Benazepril HC1/HCTZ Tablets Placebo After Storage Under UV

Light for 5 Days — HPLC

0.050 —

______________________ ____________________________________________________________________________

0.04

0.030 -

0.020

:

-0.0100(U-0.020

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00Mnutes

Figure 178: Chromatogram of Benazepril HC1/HCTZ Tablets Blend Dry Powder After Storage

Under UV Light for 5 Days - HPLC

-0.010U

U-

D

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00Mnutes

156

The raw material, placebo, and blend samples dry powders for each product were

weighed into individual 100-mL volumetric flasks. The sample dry powders were then placed in

a covered desiccator with water in the bottom. All samples were then exposed to the humid

conditions for 5 days under ambient laboratory conditions. After the 5 day period, all samples

were prepared as outlined in the proposed method and analyzed for related compounds. All raw

materials, placebo, and finished product blend powders tested were shown to be stable against

degradation due to humidity at room temperature.

Figure 179: Chromatogram of the Fosinopril Sodium Raw Material Powder Exposed toHumidity for 5 Days at Room Temperature — HPLC

L. U1i\-OD1(

QC’10

QOE —

-üO1(-

cJ’(, ..

1) 2W 4W W aw law 120) 14W 160) 1&W 21W 720) 240) .Q) W iW 0) 340) W W 41(

D

O0) 2(E) 4.0) 6) &03 120) 12(X) 14(E) 160) 160) 31(X) W 24.0) 0) 0) W 0) 340) 310) W (X

Figure 180: Chromatogram of the HCTZ Raw Material Sample Powder Exposed to Humidityfor 5 Days at Room Temperature - HPLC

D

157

Figure 181: Chromatogram of Benazepril HC1 Raw Material After Storage Under Humidity for5 Days-HPLC

0.16

0.14

0.12

0.10

008

006

004

0 02

Zoom Plot (5% of Highest Peak)

0 00

0.00 500 1 15.00 3000 3500 4000

Figure 183: Chromatogram of the Fosinopril Sodium Tablets Blend Powder Exposed toHumidity for 5 Days at Room Temperature - HPLC

2000 2500A n..tes

SampleName Bena RM RT Hurmdity_RC; Vial 46: Injection 1; Result Id 50050; Date Acquired Thursday,February 24. 2011 2:07:28 AM EST

Figure 182: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder Exposed toHumidity for 5 Days at Room Temperature - HPLC

-0.02.0.00 2 . 4 .. •. •. 10.00 12.00 14.00 16.00 18,00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

•bwte4

Mn.8e

158

Figure 184: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Placebo Powder Exposedto Humidity for 5 Days at Room Temperature - HPLC

iLJL-001( “I

C

n tar.10

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 26.00 30.00 32.00 34.00 36.00 3800 40.00Mwte

Figure 186: Chromatogram of Benazepril HC1 Tablets Placebo After Storage Under Humidityfor 5 Days - HPLC

Mnules

Figure 185: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Blend Powder Exposed toHumidity for 5 Days at Room Temperature - HPLC

D

Auto-Scaled Chrom atogram

20 00U iriutes

SarripleName Bena Place RT Humidity RC; Vial 33; h-ij 1; nj Id 26039; Result Id 50038; Date AcquiredWednesday. February 23, 2011 5:16:05 PM EST

159

Figure 187: Chromatogram of Benazepril HC1 Tablets Blend After Storage Under Humidity for5 Days - HPLC


01

o 14

0 12

01

008

00 hA AA AAAA

000 5 1’ .. 1500 2000 25 3000 3500U flutes

SampleName Bena Blend RT Humidity_RC; Vial 39; Injection 1; Result Id 50044; Date AcquiredWednesday. February 23. 2011 9:21:23 PM EST

Figure 188: Chromatogram of Benazepril HC1 HCTZ Tablets Placebo After Storage UnderHumidity for 5 Days - HPLC

Auto-Scaled Chrom atogram

0 06D

0 04

0 00

000 •. . .• .. 2000 , ‘‘ 40.00N flutes

SampleName Bena HCTZ Place RT Humidity RC; Vial 34; lnj 1; lnj Id 26095; Result Id 50039; Date AcquiredWednesday, February 23. 2011 5:56:57 PM EST

Figure 189: Chromatogram of Benazepril HC1/HCTZ Tablets Blend After Storage UnderHumidity for 5 Days - HPLC


018

0 16

0 14

0 12

0 10

006

::: J I0 02

00 A A h

000 500 10.00 15.00 2000 2500 300 35 4000U flutes

SampleName Bena_HCTZ_Blend RT Hurr1dity RC; Vial 40; Injection 1; Result Id 50045; Date AcquiredWednesday. February 23, 2011 10:02:12 PM EST

160

Figure 190: Chromatogram of the Fosinopril Sodium Raw Material Powder After ThermalDegradation at 105°C for 5 Days- HPLC

0.050

0.040

0.030

0.020

0.010

0.000

-0.01c

-0.0200.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00

Mnutes

Figure 191: Chromatogram of the Benazepril HC1 Raw Material Powder After ThermalDegradation at 105°C for 2 Days- HPLC

0.05.

D

Mnutes

38.00 40.00

ucuc

(N

*..JI

Jill- I I P I I I I I I I I I I I I I I I I

Q(D 2W 4W 6W 8W W 2(1) 14W 18W 18W 21(0 W 24W 21(0 21W 1W W 340) 21W 21W 41(1

50

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Figure 192: Chromatogram of the HCTZ Raw Material Sample Powder After ThermalDegradation at 105°C for 5 Days- HPLC

D

161

Figure 193: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder After ThermalDegradation at 105°C for 5 Days- HPLC

uw., —

c)Q01

C)L003

003 -

D

QOl

Q03c’J

-001 - CC (

0(1) 2W 403 6(1) 8W 1QW 12W 1403 1603 18W 22(1) 72(0 2403 W W 32(0 QW 34(1) 32W 3203 41(X

Figure 194: Chromatogram of the Fosinopril Sodium Tablets Blend Powder After ThermalDegradation at 105°C for 5 Days- HPLC

0I;I

Ii’,

UI”

aIID

(101’

Urn

401’

OW 2(0 4(0 6(0 60) 10(1) 120) 14.11) 160) 16(1) 720) 72(1) 24(0 72(0 720) 32(0 (D 34(1) 32W 32W 4)JX

Figure 195: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Placebo Powder AfterThermal Degradation at 105°C for 5 Days- HPLC

Qi;i

(103 203 4(1) 6W 8W 1Q03 120) 14(0 16(0 18W ala) 72(0 24(0 72W 72(1) 32(1) W 34(0 3203 3203 42(1

162

Figure 196: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Blend Powder AfterThermal Degradation at 105°C for 5 Days- HPLC

Figure 197: Chromatogram of the Benazepril HC1 Tablets Placebo Powder After ThermalDegradation at 105°C for 2 Days- HPLC

0.05’

D LLJ:LIj:r7*l-0.02’

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 2000 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00nutes

Figure 198: Chromatogram of the Benazepril HC1 Tablets Blend Powder After ThermalDegradation at 105°C for 2 Days- HPLC

0.051

D

0.04.

0.03’

0.02’

0.01’

0.00’

-0.01’

Note: The Benazepril containing finished product blends are highly susceptible to thermal degradation. However all peaks are much more well separated in the HPLC method than the UPLCmethod.

0.04’

0.03’

0.02’

0.01’

0.00I

-0.01’

Mnutes

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

nutes

163

Figure 199: Chromatogram of the Benazepril HC1 HCTZ Tablets Placebo Powder AfterThermal Degradation at 105°C for 2 Days- HPLC

0.05’

Figure 200: Chromatogram of the Benazepril HC1/HCTZ Tablets Blend Powder After ThermalDegradation at 105°C for 2 Days- HPLC

D

Note: The Benazepril containing finished product blends are highly susceptible to thermal degradation. However all peaks are much more well separated in the HPLC method than the UPLCmethod.

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Mhiutes

nutes

164

Figure 201: Chromatogram of the Fosinopril Sodium Raw Material Powder After MoistureSaturated Headspace at 90°C for 48 Hours - HPLC

0.011

0.00I

-0.01’

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Mnutes

Figure 202: Chromatogram of the Benazepril HC1 Raw Material Powder After MoistureSaturated Headspace at 90°C for 48 Hours - HPLC

0.05’

D

0.0.

0.03.

0.02’

0.01’

0.00’

-0.01’

0.05’

0.04’

0.03.

0.02’

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

nutes

165

Figure 203: Chromatogram of the HCTZ Raw Material Sample Powder After MoistureSaturated Headspace at 90°C for 48 Hours - HPLC

0.05’

D

0.04.

0.03’

0.02’

0.01’

0.00.

-0.01.

Fvnutes

Figure 204: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder After MoistureSaturated Headspace at 90°C for 48 Hours - HPLC

0.01

0.00

-0.01

CD

‘1•

E0

CD CDc,. )c)c, .

-

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

0.05’

0.0

0.03

0.02

-0.020.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Mnutes

166

Figure 205: Chromatogram of the Fosinopril Sodium Tablets Blend Powder After MoistureSaturated Headspace at 90°C for 48 Hours - HPLC

D

0.05’

0.04’

0.03’

0.02’

0.011

0.00’

-0.01’

Figure 206: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Placebo Powder AfterMoisture Saturated Headspace at 90°C for 48 Hours - HPLC

0.05’

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Fvnutes

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Nnutes

167

I •0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Mnutes

Figure 208: Chromatogram of the Benazepril HC1 Tablets Placebo Powder After MoistureSaturated Headspace at 90°C for 48 Hours - HPLC

D

Figure 207: Chromatogram of the Fosinopril Sodium!HCTZ Tablets Blend Powder AfterMoisture Saturated Headspace at 90°C for 48 Hours - HPLC

0.040

0.030

0.020D

0.010

0.000

-0.0 10

Mnutes

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 3400 36.00 38.00 40.00

168

Figure 209: Chromatogram of the Benazepril HC1 Tablets Blend Powder After MoistureSaturated Headspace at 90°C for 48 Hours - HPLC

C,,

-0.02C i i0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Mnutes

Figure 210: Chromatogram of the Benazepril HC1IHCTZ Tablets Placebo Powder AfterMoisture Saturated Headspace at 90°C for 48 Hours - HPLC

D

169

a.’0.056

0.04C

0.03C

0.02C

0.01c

0.OOC

-0.01c

D

0.00)C)

-0.02t0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

vnutes

Figure 211: Chromatogram of the Benazepril HC1/HCTZ Tablets Blend Powder After MoistureSaturated Headspace at 90°C for 48 Hours - HPLC

D

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00vinutes

Figure 212: Chromatogram of the Fosinopril Sodium Raw Material Powder After AcidHydrolysis - HPLC

0.05 —

0.04

cc0.03 (N

cc

0.02D - AA

rj a0.01 I—

(Nci0.00 AVA AA

-o.oi Placebo Peak 0C

0U

-0.020.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

nuteS

170

Figure 213: Chromatogram of the Benazepril HC1 Raw Material Powder After Acid Hydrolysis- HPLC

0.05’

0.0

0.03’

0.02’D

0.01’

0.00I

-0.01’

-0.02’ -- -_______

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00Minutes

Figure 214: Chromatogram of the HCTZ Raw Material Sample Powder After Acid HydrolysisHPLC

0.05’

0.041

0.03’

0.02’

0.01’

0.00i

-0.01

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Minutes

171

Figure 215: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder After AcidHydrolysis - HPLC

0.05

0.04 (C(‘1

003-• C

cc002 -C

cc w10

0.01 cc

t0.00 A AA

-0.01

-0.020.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Mnutes

Figure 216: Chromatogram of the Fosinopril Sodium Tablets Blend After Acid Hydrolysis —

HPLC0.050

CD CD

0.040CD

(N H00.030

0 1. -“fl- - ,! Jjj ..‘0.020

cc c—

—

D -D •

I 0 LL _—LX0.010

0.000

a.0-0.010U)0

IL—0.020

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00Fnutes

172

Figure 217: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Placebo Powder AfterAcid Hydrolysis - HPLC

D

Figure 218: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Blend Powder After AcidHydrolysis - HPLC

D

Inutes

Fvnutes

173

Figure 219: Chromatogram of the Benazepnl HC1 Tablets Placebo Powder After Acid

Hydrolysis - HPLC0.050 —

________________________________ ___________________________________________________________

CDC,

0)

g0.040 co L()• 0)

. c•,0030 o

c;ioI1; I

0.000

0.020C>

Da>

0.010 1•i

-0.010

-0.020. I i’’

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00Mnutes

Figure 220: Chromatogram of the Benazepril HC1 Tablets Blend Powder After Acid Hydrolysis- HPLC

0.050—

III 0 CD

III L 0)0040 III tf)

III C)

III0

c .g.fl

L1LihJj*L

0.030I

D a>

0.010 —

0000

0

-0.0 10

-0.020 •. .

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00Mnutes

174

Figure 221: Chromatogram of the Benazepril HC1/HCTZ Tablets Placebo Powder After AcidHydrolysis — HPLC

0.050

CO Ci C)CO- 0

If)0.04c’JC%J

(-).C._I U)

C’41... —.

_

C0.03

II diF— 0.

— a) C) Ia0.020 U’ 0 C

D U) o0 N .0F— m a) CO —

0.010 C) COC%J0

010.000 .0 diU) Ci

C) diCa

-0.01

-0.0200.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Minutes

Figure 222: Chromatogram of the Benazepril HC1 HCTZ Tablets Blend Powder After AcidHydrolysis - HPLC

0.050

D

Minutes

-0.02’ — -_________

_______ _______

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 2200 2400 2600 2800 30.00 32.00 34.00 36.00 38.00 40.00

Figure 223: Chromatogram of the Fosinopril Sodium Raw Material Powder After BasicHydrolysis - HPLC

0.050

-0.0200.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 1800 20.00 22.00 2400 26.00 28.00 30.00 32.00 34.00 3600 3800 4000

Minutes

175

Figure 224: Chromatogram of the Bena.zepril HCI Raw Material Powder After Basic Hydrolysis-HPLC

0.05 p

0.04’

D

D

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Mhiutes

Figure 225: Chromatogram of the HCTZ Raw Material Sample Powder After Basic HydrolysisHPLC

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

,inutes

176

Figure 226: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder After BasicHydrolysis - HPLC

0.050

0.04

0.030

0.020D

0.010

0.000—I

-0.010 0

0LL-0.020

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00Mnutes

Figure 227: Chromatogram of the Fosinopril Sodium Tablets Blend Powder After BasicHydrolysis - HPLC

0.05’

D

-0.01’

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

nutes

177

0.041)0

0

a,a)

C’4

0.0a)0

a-

I4

tc0ric-iLL jjC4)

cI

CNC-)

0.0C00

LL

Figure 228: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Placebo Powder AfterBasic Hydrolysis HPLC

0.05

0.04

0.03

0.02

0.01

0.00

-0.01

-0.020.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

rvnutes

Figure 229: Chromatogram of the Fosinopril Sodium/HCTZ Tablets Blend Powder After BasicHydrolysis - HPLC

U,

(N 1— 4a a,0

(N

AL

0.04C

0.03C

0.02C

0.01c

0.OOC

-0.01c

CC,,

) (-‘ICDC,,

.0

0.00 2.00 4.00 6.00 8.00 1000 12:00 14.00 16:00 18.00 20:00 22.00 24.00 26:00 28.00 30100 32.00 34.00 36.00 3800Mnutes

178

Figure 230: Chromatogram of the Benazepril HC1 Tablets Placebo Powder After BasicHydrolysis - HPLC

0.05’

0.0’

0.03’

0.02’D

0.01’

0.00I

-0.01’

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

v1nutes

Figure 231: Chromatogram of the Benazepril HC1 Tablets Blend Powder After BasicHydrolysis - HPLC

0.05’

0.04’

0.03’

0.02’D

0.01’

0.00I

-0.01’

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Ivinutes

179

Figure 232: Chromatogram of the Benazepril HC1/HCTZ Tablets Placebo Powder After BasicHydrolysis - HPLC

0.05’

Fnutes

Figure 233: Chromatogram of the Benazepril HC1/HCTZ Tablets Blend Powder After BasicHydrolysis - HPLC

0.050—

0020 I I0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

nutes

D

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

D

7’

0.04J

0.030

0.020

0.010

0.000

-0.0 10

180

Figure 234: Chromatogram of the Fosinopril Sodium Raw Material Powder After OxidationHPLC

D

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

rvnutes

Figure 235: Chromatogram of the Benazepril HC1 Raw Material Powder After Oxidation —

HPLC

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Fvnutes

181

Figure 236: Chromatogram of the HCTZ Raw Material Sample Powder After OxidationHPLC

0.05

0.04

c)CN

0.03co U)

oU) LL0.02 w

N C0.01

A

0.00 A!áYA AVA

-0.020.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Fvnutes

Figure 237: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder After Oxidation —

HPLC0.05

0.0

0.03

C’4

0.02 0)

0.0

0.01

0.00

-0.01

-0.020.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

nutes

182

Figure 238: Chromatogram of the Fosinopril Sodium Tablets Blend Powder After Oxidation —

HPLC0.05’

0.0’

0.031

0.02’D

0.01’

0.00’

-0.01’

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Fvnutes

Figure 239: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Placebo Powder AfterOxidation - HPLC

0.05

(‘4

0.04

700. (‘4

(‘4 0

a,0) 0 0

0.02O

00.01 -

0

0.00

-0.01

-0.020.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

1vnutes

183

Figure 240: Chromatogram of the Fosinopril SodiumJHCTZ Tablets Blend Powder AfterOxidation - HPLC

D

0.05’

0.04

0.03

0.02

0.01

0.00

-0.01

Mnutes

Figure 241: Chromatogram of the Benazepril HC1 Tablets Placebo Powder After - HPLC0.05*

-

________ _______-

-

________

-

D

0.04*

0.03’

0.02’

0.01*

0.00’

-0.01’

-0.020.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Mnutes

184

Figure 242: Chromatogram of the Benazepril HC1 Tablets Blend Powder After Oxidation —

HPLC

D

Figure 243: Chromatogram of the Benazepril HC1/HCTZ Tablets Placebo Powder AfterOxidation - HPLC

O.O5

vinutes

-0.02’0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

rvnutes

185

Figure 244: Chromatogram of the Benazepril HC1IHCTZ Tablets Blend Powder AfterOxidation - HPLC

D

4LOO

The robustness of the filtration technique used in the analytical method was evaluated as

part of the validation process for the related compounds analytical method. The filter study was

conducted using 0.45-jim Millex-HV PVDF filter, a 0.45-pm Whatman GMF with GMF filter,

and centriftigation. The minimum discard volume for each filter was determined for each filter

as well. The filter is considered free from bias if the absolute difference of individual impurity

between the filtered sample and the centrifuged samples (i.e. unfiltered) is not more than 0.100

for the observed level O.10o to less than 0.5%, not more than 100o (relative) for the observed

level 0.5 to 5.0%, and no additional peak above the reporting level is found. The finish product

sample was also centrifuged in glass and plastic centrifuge tubes as well. The samples in the

plastic centrifuge tubes were placed in glass vials immediately following centrifugation for 4

hours to determine if the plastic would cause a bias on the related compound results due to

extractable/leachables from the plastic. As the UPLC and the HPLC methods were shown to be

equivalent, the filter validation was analyzed using only the HPLC method. The 0.45-im

Millex-HV PVDF filter was shown to be acceptable after all discard volumes. The 0.45-tim

rvlnutes

186

Whatman GMF filter was shown to be acceptable after a minimum discard volume of at least 5

mL of filtrate. All centrifugation techniques were shown to be acceptable as well.

Table 199: Filter Stud Results for Fosino .ril Sodium 10 m: Tablets

Discard Volume %RC-A %OKRC %UKRC %TRC

_________________________

Mifiex Mifiex

lOmLCentrifuged in GlassCentrifuged in Plastic

Centrifuged in Plastic (4 hrs)


Centrifuged in Plastic (4 hrs)

Analyst: LMG

Table 200: Filter Study Results for Fosinopril Sodium/HCTZ 10 mg/12.5 mg TabletsFosino • ru)

Discard Volume %RC-A %OKRC %UKRC %TRC

_________________________

Mifiex Mifiex

Analyst: LMG

Table 201: Filter Study Results for Fosinopril SodiumlHCTZ 10 mg/12.5 mg Tablets(HCTZ

Discard Volume %4-Amino %OKRC %UKRC %TRC

_______________________

Mifiex Millex

lOmLCentrifuged in Glass

• Centrifuged in PlasticCentrifuged in Plastic (4 hrs)

187

Table 202: Filter Stud Results for Benaze.ril HC1 5 m Tablets

Table 204: Filter_Stud Results for Benazeeril HC1 HCTZ 5 m 6.25 m: Tablets HCTZ)

Discard Volume

10 mLCentrifuged in Glass

Centrifuged in PlasticCentrifuged in Plastic (4 hrs)

The robustness of the chromatographic parameters was evaluated for both the UPLC and

HPLC methods. During the robustness studies, small deliberate changes were made to the

method-specified chromatographic parameters. The robustness samples prepared by the analysis

of representative related compound blend samples were spiked with each of the formulation

specified related compounds. The samples were then analyzed using all system suitability

Discard Volume I %Benazeprilat %OKRC %UKRC I %TRC

_________________________

Milex Millex


Centrifuged in Plastic (4 hrs)Analyst: LMG

Table 203: Filter Stud Results for Benazeeril HC1/HCTZ 5 mg/6.25 m: Tablets Benaze.ril

Discard Volume %Benazeprilat %OKRC %UKRC %TRC

__________________________

Mifiex Millex

10 mLCentrifuged in Glass

Centrifuged in PlasticCentrifuged in Plastic (4 hrs)

%TRCGMF

0.20.20.20.2

188

criteria. For acceptance of the condition the results were shown to be within the limits set in

Table 205. The system suitability criteria are outlined in Table 206. Due the method being

sensitive to the amount of acetonitrile in mobile phase, instruction in the method will be provided

stating that the mobile phase must be prepared exactly as written.

Table 205: Acceptance Criteria for RC Robustness% Level % Difference (Absolute)

NMTO.l% ASO.05%0.2—0.3% A0.l0%0.4—0.5% A0.l5%0.6 — 0.8% A 0.20%0.9—3.0%

Limit> 3.0% A 10% relative

The following changes were made to the chromatographic parameters for the HPLC and

the UPLC during the robustness study outlined in the tables below. Because the robustness

studies were executed over one week (7 days), the main degradants of the active ingredients were

shown to have the most variation among the conditions (RC-A for Fosinopril, 4-Amino for

HCTZ, and Benazeprilat for Benazepril). The large percent change observed for these three

related compounds can be attributed to the degradation of the respective API and not due to the

variation of the robustness parameter. The UPLC column being used for the chromatographic

method is a Waters Acquity BEH Phenyl 1 .7-im 2.1 mm x 100 mm column, for which there is

no column equivalent. However, the HPLC utilizes a Waters Atlantis T3 3-i.tm 4.6 mm 150 mm

column which has several options for a column equivalency study. The column that was chosen

189

Table 206: System Suitability CriteriaThe %RSD of the five replicate injections of the standard preparation must be not more than2.0% for each main component peak of interest.The tailing of the main component peak in the standard preparation must not be more than 2.0for the main component peak of interest.The plate count for the main component peak must be greater than:

• HCTZ NLT 2,000• Fosinopril NLT 10,000• Benazepril NLT 10,000

for this study is a chromatographic column that is already common and readily available at the

Sandoz Wilson, NC site. The column used for the HPLC column equivalency study was a

Waters Atlantis dCl8 5-urn 4.6 mm x 150 mm column. As the HCTZ portion for the Benazepril

HC1 HCTZ Tablets was shown not to pass accuracy acceptance criteria for the 4-Amino related

compound, the robustness for the HCTZ related compounds was not assessed during the

robustness studies. Because the assay and related compound robustness was run in the same

sequence, the previously discussed system suitability parameters are applicable to the related

compounds robustness. The resolution between closely eluting related compounds is an

important assessment of system suitability. Therefore, the resolution between the closest

compounds observed in each Resolution Solution is included in this report to determine the

allowable variations within the analytical method.

Table 207: Resolution of Fosinopril Sodium Resolution Solution on UPLCCondition RC-A & RC-B

Initial 3.9Wavelength 202 nm 3.9Wavelength2l0nm 3.9

Slow Flow (0.9 mL/min) 3.2Fast Flow (1.5 mL/min) 3.3

Low Temp (35°C) 3.9High Temp (45°C) 3.6Low ACN (-3%) 3.9High ACN (+3%) 2.9

LowpH(l.9) 3.9HighpH(2.3) 4.1

190

Table 208: Resolution of Fosinopril Sodium Resolution Solution on HPLCCondition RC-A & RC-B

Initial 13.3Wavelength 202 nm 13.2Wavelength 210 nm 13.4


Low Temp (35°C) 10.8High Temp (45°C) 10.3Low ACN (-3%) 10.7High ACN (+3%) 12.6

LowpH(1.9) 12.3HighpH(2.3) 11.0

Column Equivalency 6.3

All conditions are considered to be acceptable for the analysis of Fosinopril Sodium

Tablets on both the UPLC and the HPLC. The recommendation given in the USP chapter titled

“Integration of Chromatograms” states that variations of the mobile phase composition in

gradient elutions are not advised. However, these robustness studies demonstrate that slight

variations in the amount of acetonitrile in mobile phase A will not impact the results of the

related compound analysis.

Table 209: Resolution of Fosinopril SodiumJHCTZ Resolution Solution on UPLCCondition CTZ & HCTZ RC-A & RC-B

Initial 1.4 3.9Wavelength 202 nm 1.4 3.9Wavelength 210 mn 1.4 3.9

Slow Flow (0.9 mL/min) 1.4 3.2Fast Flow (1.5 mL/min) 1.4 3.3

Low Temp (35°C) 1.7 3.9High Temp (45°C) 1.4 3.6Low ACN(-3%) 1.2 3.9HighACN(+3%) 1.5 2.9

LowpH(1.9) 1.4 3.9HighpH(2.3) 1.3 4.1

The 4-Amino and CTZ have greater than 7.0 resolution on the UPLC method.

191

ci,

C CD CD cj C 0 CD CD CD p

C) C 0 Cl)

CD C) 0 Cl)

CD 0 CD C) C) CD CD Cl)

(6)

C Tj

0 Cl)

C

Table 212: Resolution of Benazepril HC1 Resolution Solution on HPLCCondition Benazepril & R,S-Isomer

Initial 2.9Wavelength 202 nm 2.9Wavelength 210 nm 2.9


Low Temp (3 5°C) 2.6High Temp (45°C) 2.8Low ACN (-3%) 2.6High ACN (+3%) 2.9

LowpH(1.9) 2.5HighpH(2.3) 3.1

Column Equivalency 2.5

All conditions are considered to be acceptable for the analysis of Benazepril HC1 Tablets

on both the UPLC and the HPLC. However, based on the forced degradation studies for

specificity the use of the HPLC method for the determination of related compounds in

Benazepril HC1 Tablets is highly recommended.

Table 21_Reso1ution of Benazepril HC1/HCTZ Resolution Solution on UPLCBenazepril & Res. 4-Amino & CTZ &

Condition R,S-Isomer S-Amine HCTZInitial 2.0 1.8 1.4

Wavelength 202 nm 2.1 1.8 1.4 —

Wavelength 210 nm 2.1 1.8 1.5Slow Flow (0.9 mL/min) 2.0 1.6 1.4Fast Flow (1.5 mL/min) 2.3 1.6 1.4

LowTemp(35°C) 2.1 1.6 1.7High Temp (45°C) 2.2 1.7 1.4Low ACN (-3%) 2.1 2.3 1.2High ACN (+3%) 1.6 0.3 1.4

LowpH(l.9) 1.9 3.1 1.4HighpH(2.3) 2.3 3.1 1.3

193

Table 214: Resolution of Benazepril HC1IHCTZ Resolution Solution on HPLCBenazepril & R,S- S-Amine & 4- 4-Amino & CTZ &

Condition Isomer Amino CTZ IICTZInitial 2.9 2.8 2.4 2.9

Wavelength 202 nm 2.9 2.8 2.4 2.9Wavelength2l0nm 2.9 2.8 2.4 2.8

Slow Flow (0.9 mL/min) 2.5 2.6 2.3 2.7FastFlow(1.5mL/min) 3.1 2.7 2.5 3.0

Low Temp (35°C) 2.6 3.3 2.9 3.2High Temp (45°C) 2.7 2.3 2.2 2.8Low ACN (-3%) 2.7 Co-Elution 3.4 3.0High ACN (+3%) 2.9 6.3 1.7 2.7

LowpH(1.9) 2.6 1.7 1.9 2.9HighpH(2.3) 3.1 3.8 2.6 3.0

Colunm Equivalency 2.5 Co-Elution 2.3 1.5

The mobile phase composition must be prepared exactly as specified in the analytical

method for the analysis of Benazepril HC1 HCTZ Tablets on both the HPLC and the UPLC. The

method specified column much also be used for the HPLC analysis of Benazepril HC1/HCTZ

Tablets due to the co-elution of the S-Amine and the 4-Amino related compounds. Even though

the 4-Amino being an HCTZ degradation product is not analyzed by this new method at this

time, the co-elution of the 4-Amino with the S-Amine in the finished product would cause an un

due bias on the result of the S-Amine. Based on the forced degradation studies for specificity,

the use of the HPLC method for the determination of related compounds in Benazepril

HCI HCTZ Tablets is highly recommended.

194

Table 215: Robustness % Change for Fosinopril Sodium Tablets on UPLC

Analyst: JSM, JU and LMG

Table 216: Robustness Results for Fosinopril SodiumIHCTZ Tablets on UPLCParameter 4-Amino CTZ Dimer RC-A RC-B RC-C RC-D

. .. AS A< AS AS AS A<%Difference Based on Initial A S 0.15%0.10% 0.05% 0.3% 0.10% 0.10% 0.10%

Flow Rate (0.47 mL/min) 0.04 0.00 0.01 0.1 0.00 0.03 0.03Flow Rate (0.60 mL/min) 0.08 0.00 0.01 0.2 0.00 0.03 0.01

Column Temperature 35°C 0.03 0.01 0.01 0.0 0.01 0.03 0.04Column Temperature 45°C 0.03 0.00 0.01 0.1 0.00 0.00 0.01

Wavelength 202 nm 0.03 0.10 0.01 0.0 0.01 0.03 0.01Wavelength 210 nm 0.02 0.08 0.01 0.0 0.01 0.02 0.01

Mobile Phase ApH 1.9 0.06 0.01 0.02 0.1 0.00 0.04 0.02Mobile Phase A pH 2.3 0.14 0.02 0.04 0.3 0.00 0.06 0.04

-3% ACN in Mobile Phase A 0.10 0.02 0.03 0.2 0.00 0.06 0.05+3% ACN in Mobile Phase A 0.07 0.01 0.01 0.1 0.00 0.06 0.01


Table 227: Robustness Results for Benazepril HC1 Tablets on UPLC


Parameter %RC-A %RC-B %RC-C %RC-D%Difference Based on Initial A5 0.3% AS 0.10% A 5 0.15% A 5 0.10%

Flow Rate (0.47 mL/min) 0.1 0.01 0.02 0.01Flow Rate (0.60 mL/min) 0.2 0.01 0.04 0.01


Wavelength 202 nm 0.0 0.01 0.03 0.03Wavelength 210 nm 0.0 0.02 0.02 0.01

Mobile Phase A pH 1.9 0.1 0.00 0.05 0.00Mobile Phase A pH 2.3 0.3 0.01 0.06 0.03

-3% ACN in Mobile Phase A 0.2 0.00 0.06 0.03+3% ACN in Mobile Phase A 0.1 0.00 0.06 0.1

Parameter S-Amine Benazeprilat R,S-Isomer%Difference Based on Initial LA 5 0.20% A 5 0.3% AS 0.15%

A flow rate of slower than 0.50 mL/min cannot be used with this product.Flow Rate (0.60 mL/min) 0.01 0.1 0.00

Column Temperature 37°C 0.01 0.1 0.04Column Temperature 45°C 0.00 0.0 0.01

Wavelength 202 nm 0.05 0.0 0.00Wavelength 210 nm 0.07 0.0 0.01

Mobile PhaseApH 1.9 0.00 0.1 0.02Mobile Phase A pH 2.3 0.01 0.1 0.02

-3% ACN in Mobile Phase A 0.00 0.1 0.05+3% ACN in Mobile Phase A 0.01 0.1 0.01

195

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Table 220: Robustness Results for Fosino ri! SodiumJHCTZ Tablets on HPLCParameter 4-Amino CTZ Dimer RC-A RC-B RC-C RC-D

1rntia0.15% 0.10% 0.05% 0.3% 0.10% 0.10% 0.10%

Flow Rate (1.0 mL/min) 0.17 0.01 0.04 0.6 0.08 0.09 0.07Flow Rate (1.5 mL/min) 0.04 0.01 0.02 0.1 0.01 0.00 0.02

Column Temperature 35°C 0.01 0.02 0.02 0.1 0.00 0.00 0.00Column Temperature 45°C 0.01 0.01 0.02 0.0 0.01 0.01 0.02

Wavelength 202 nm 0.01 0.09 0.01 0.0 0.01 0.00 0.03Wavelength 210 nm 0.01 0.07 0.01 0.0 0.01 0.00 0.05

Mobile PhaseApH 1.9 0.15 0.01 0.05 0.3 0.00 0.00 0.00Mobile Phase A pH 2.3 0.10 0.02 0.05 0.2 0.00 0.00 0.03

-3% ACN in Mobile Phase A 0.00 0.01 0.01 0.0 0.00 0.00 0.00+3% ACN in Mobile Phase A 0.13 0.01 0.04 0.2 0.01 0.00 0.02

Column Equivalency 0.15 0.01 0.04 0.2 0.00 0.00 0.05Analyst: JSM, JU and LMG

Note: The slow flow parameter found outside of the acceptance criteria is due to degradation of the Fosinopril andHCTZ in the sample solution and not due to the change in the chromatographic parameter.

Table 221: Robustness Results for Benazepril HC1 Tablets on HPLCParameter S-Amine Benazeprilat R,S-Isomer

Initial A0.20% A0.3%Flow Rate (0.9 mL/min) 0.01 0.0 0.00Flow Rate (1.5 mL/min) 0.00 0.0 0.00

Column Temperature 35°C 0.01 0.0 0.00Column Temperature 45°C 0.01 0.0 0.00

Wavelength 202 nm 0.05 0.0 0.00Wavelength 210 rim 0.07 0.0 0.00

Mobile PhaseApH 1.9 0.01 0.1 0.02MobilePhaseApH2.3 0.01 0.1 0.00

-3% ACN in Mobile Phase A 0.00 0.0 0.02+3% ACN in Mobile Phase A 0.04 0.1 0.01

Column Equivalency 0.00 0.1 0.01Analyst: JSM, JU and LMG

197

Table 222: Robustness Resuits for Benazepril HCI/HCTZ Tablets on HPLCParameter 4-Amino CTZ Dirner S-Amine Benazeprilat R,S-Isomer

Initial NA NA NA A 0.20% A 0.20% A 0.20%Flow Rate (0.9 mL/min) NA NA NA 0.01 0.02 0.00 —

Flow Rate (1.5 mL/min) NA NA NA 0.00 0.03 0.00 —

Column Temperature 35°C NA NA NA 0.00 0.01 0.00Column Temperature 45°C NA NA NA 0.01 0.00 0.00

Wavelength 202 nm NA NA NA 0.04 0.00 0.01Wavelength 210 nm NA NA NA 0.07 0.00 0.00

Mobile PhaseApH 1.9 NA NA NA 0.01 0.09 0.02Mobile Phase A pH 2.3 NA NA NA 0.00 0.06 0.00

-3% ACN in Mobile Phase A NA NA NA 0.02 0.01 0.02+3% ACN in Mobile Phase A NA NA NA 0.03 0.07 0.01

Column Equivalency NA NA NA 0.03 0.08 0.01Analyst: JSM, JU and LMG

The stability of the sample solutions was evaluated as part of the validation experiments

for the analytical method for related compounds. As the standard for the related compound

method is the same one used for the assay method, the solution stability study for the standard

preparation was not repeated. The solution stability for the samples was executed using the

lowest label strength for the each finished product dosage form as this is considered to be “worst

case.” The acceptance criteria out lined in Table 223 are based on each time point when

compared to the initial day of preparation.

Table 223: Solution Stability Acceptance Criteria for Related Compounds Samples%Level - %Difference

Observed Level < 0.5% ± 0.1% absoluteObserved Level 0.5% to < 5.0% 10% relative

Observed Level> 5.0% 5% relativeFor test solution, no additional peak ± 0.1% with respect to initial should be observed

Table 224: Solution Stability of Fosinopril Sodium 10 mg Tablets (Clear Flasks — RoomTemperature)

Time Point %RC-A %UKRC %OKRC %TRCDayO(initial) 7.230 0.195 ND 7.581

Day 1 7.259 0.190 0.011 7.586Day3 7.358 0.191 0.018 7.709Day7 7.487 0.193 0.043 7.818

%Difference (Max) 4 0.0 0.0 3Analyst: LMG

198

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Table 245: Solution Stability of Benazepril HC1/HCTZ 5 mg/6.25 mg Tablets (Clear Flasks —

Refrigeration - HCTZ)Time Point %4-Amino %UKRC %OKRC %TRC

DayO (initial) 0.061 0.026 0.064 0.183Day 1 0.064 0.027 0.064 0.179Day3 0.065 0.032 0.063 0.128Day 7 0.064 0.034 0.064 0.165

%Difference (Max) 0.0 0.0 0.0 0.0Analyst: LMG

Table 246: Solution Stability of Benazepril HC1/HCTZ 5 mg/6.25 mg Tablets (Amber Flasks —

Refrigeration - HCTZ)Time Point %4-Aniino %UKRC %OKRC %TRC

Day 0 (initial) 0.061 0.026 0.064 0.183Day 1 0.061 0.026 0.062 0.168Day3 0.059 0.031 0.059 0.118Day7 0.057 0.034 0.060 0.147

%Difference (Max) 0.0 0.0 0.0 0.0Analyst: LMG

A method equivalency study was executed to determine if the new UPLC/HPLC methods

could elicit equivalent results to the existing analytical methods currently in placebo at Sandoz,

Inc in Wilson, NC. Six related compounds samples were prepared for each product as per the

respective analytical methods. The results were then compared in order to determine the

equivalency. The individual and total related compounds were calculated in all sample

preparations. The number and amount of related compounds were analyzed for comparability

between each of the methods.

Table 247: Method Equivalency for Fosinopril Sodium 10 mg Tablets Related Compounds onUPLC

Method New UPLC Method Sandoz Monograph FOO7QC%RC-A 7.7 7.3

%Max OKRC 0.0 ND%Max UKRC’ 0.2 ND

%TRC 8.2 7.3Note’: The current Sandoz Monograph for Fosinopril Sodium Tablets instructs the user not tointegrate any peak occurring before RC-A in the chromatogram. However, as demonstrated bythis study an unknown Fosinopril peak at about 15.1 minutes on HPLC occurs before RC-A(23.0 minutes on HPLC) in the new methods. Therefore, this peak would be undetected in theexisted monograph at Sandoz. Therefore, the new related compound method has shown to be

203

superior to that already in place in that an additional related compound is now able to beaccurately quantitated and reported.

Table 248: Method Equivalency for Fosinopril Sodium 10 mg Tablets Related Compounds onHPLC

Method New HPLC Method Sandoz Monograph FOO7QC%RC-A 7.5 7.3

%Max OKRC 0.0 ND%Max UKRC 0.2 ND

%TRC 7.8 7.3

Table 249: Method Equivalency for Fosinopril SodiumJHCTZ 10 mg/12.5mg Tablets RelatedCompounds on UPLC (Fosinopril)

Method New UPLC Method Sandoz Monograph FOO9QC%RC-A 4.1 4.0

%Max OKRC 0.0 0.0%Max UKRC 0.2 ND

%TRC 4.5 4.1

Table 250: Method Equivalency for Fosinopril SodiumIHCTZ 10 mg/12.5mg Tablets RelatedCompounds on HPLC (Fosinopril)

Method New HPLC Method Sandoz Monograph FOO9QC%RC-A 4.0 4.0

%Max OKRC 0.0 0.0%Max UKRC 0.1 ND

%TRC 4.4 4.1

Table 251: Method Equivalency for Fosinopril SodiumJHCTZ 10 mg/12.5mg Tablets RelatedCompounds on UPLC (HCTZ)

Method New UPLC Method Sandoz Monograph FOO9QC%4-Amino 0.0 0.0

%Max OKRC 0.1 (Dimer) 0.0%Max UKRC 0.0 ND

%TRC 0.1 0.1

Table 252: Method Equivalency for Fosinopril SodiumJHCTZ 10 mg/12.5mg Tablets RelatedCompounds on HPLC (HCTZ)

Method New HPLC Method Sandoz Monograph FOO9QC%4-Amino 0.1 0.0

%Max OKRC 0.1 (Dimer) 0.0%Max UKRC ND ND

%TRC 0.1 0.1

204

Table 253: Method Equivalency for Benazepril 5 mg Tablets Related Compounds on UPLCMethod New UPLC Method Sandoz Monograph BOO6QC

%Benazeprilat 1.1 1.1%Max OKRC ND ND%Max UKRC ND ND

%TRC 1.1 1.1

Table 254: Method Equivalency for Benazepril 5 mg Tablets Related Compounds on HPLCMethod New HPLC Method Sandoz Monograph BOO6QC

%Benazeprilat 1.1 1.1%Max OKRC 0.0 ND%Max UKRC 0.1 ND

%TRC 1.2 1.1

Table 255: Method Equivalency for Benazepril HC1/HCTZ 5 mg/6.25mg Tablets RelatedCompounds on UPLC (Benazepril)

Method New UPLC Method Sandoz Monograph BOO7QC%Benazeprilat 1.5 1.5%Max OKRC 0.0 ND%Max UKRC ND ND

%TRC 1.5 1.5

Table 256: Method Equivalency for Benazepril HC1/HCTZ 5 mg/6.25mg Tablets RelatedCompounds on HPLC (Benazepril)

Method New IIPLC Method Sandoz Monograph BOO7QC%Benazeprilat 1.5 1.5%Max OKRC 0.0 ND%Max UKRC 0.0 ND

%TRC 1.5 1.5

Results of Method Validation Experiments for Dissolution

The linearity for dissolution was assessed across the range of the analytical method. The

linearity was performed for Fosinopril across the range of 200% of the highest concentration

level (88.8 g/mL) down to 20°o of the lowest concentration level (2.2 jig/mL). The linearity for

HCTZ was performed for HCTZ from 200°c of the target concentration (27.8 j.ig/mL) down to

500 of the target concentration (0.7 ig/mL). The acceptance criteria for the linearity is not less

than 0.99 for the correlation coefficient and the °oy-intercept must be less than 5° o of the target

sample concentration. As shown in the table below, all results met the preset acceptance criteria.

205

Table 257: Dissolution Linearit Results for HPLCComponent Correlation Coefficient (R) %Y-Intercept

5 (based on 10mg)Fosinopril Sodium 0.999975415 2 (based on 20 mg)

1 (based on 40 mg)Hydrochiorothiazide 0.999989297 0 (based on 12.5 mg)

Analyst: LMG

The accuracy for dissolution was assessed across the range of 50° o of the lowest analyte

concentration (5 jig/mL) up to 200°c of the highest analyte concentration (80 ig/mL) for Fosi

nopril. The accuracy study was executed for HCTZ from 500o of the lowest analyte concentra

tion up to 200° o of the highest analyte concentration. The study was executed by the preparation

of three representative samples at the specified concentrations to cover the range for each label

claim. Each sample was spiked with known amounts of raw material in placebo matrix and pre

pared in a matter representative of the dissolution procedure. The acceptance criteria for the dis

solution accuracy study are 9500 - 10500 for each individual preparation and 9700- 103° o for the

mean recovery at each level. As shown in the following tables, all aspects of the accuracy study

met all preset acceptance criteria.

206

Table 258: Dissolution Accuracy Results for Fosinopril Sodium Tablets for HPLC%Level (based on 10 mg) Individual Results Mean Level Result

102%50% 101% 101%

101%

103%100% 104% 103%

103%101%

200% 101% 101%101%100%

400% 102% 101%101%101%

800% 100% 100%100%

Analyst: LMG

Table 259: Dissolution Accuracy Results for Fosinopril SodiumJHCTZ Tablets for HPLC(Fosinopril)

%Level (based on 10 mg) Individual Results Mean Level Result

102%50% 101% 102%

102%101%

100% 101% 101%102%101%

200% 101% 101%102%101%

400% 101% 101%- 101%

Analyst: LMG

207

Table 260: Dissolution Accuracy Results for Fosinopril SodiumJHCTZ Tablets for HPLC(HCTZ)

%Level (based on 10 mg) Individual Results Mean Level Result100%

50% 100% 100%100%99%

100% 99% 100%100%99%

200% 99% 99%99%

Analyst: LMG

The precision studies for the dissolution method were executed for both repeatability and

intermediate precision. All precision studies were executed using actual finished product for

each formulation. The “worst case” formulation for each formulation was shown to be that of

the lowest strength for the Fosinopril Sodium (Fosinopril Sodium 10 mg Tablets and Fosinopril

SodiuniJHCTZ lOmg/12.Smg Tablets). All testing was also performed using expired samples so

that no product currently available on the market would be affected by the results of any

validation experiment. Therefore, some of the dissolution results obtained for some studies were

shown to be outside of their usual specification values due to the products being well past their

date of expiration. The repeatability was assessed by the dissolution of six tablets from each

finished products. The intermediate precision was assessed by the dissolution of another six

tablets of the same product by a different chemist, on a different day, using different

chromatographic systems, solutions, mobile phase, standards, and columns. The acceptance

criteria for intermediate precision was that the percent difference in mean between the

repeatability study and the intermediate precision must be not more than 5.00o for the means.

208

Table 261: Dissolution Precision Results for Fosinopril Sodium 10 mg Tablets on HPLCRepeatability: LMG Intermediate Precision: JSM

Sample # HPLC System Asset # 16930 HPLC System 55 Asset #Column Serial # 01193022914058 Column Serial #01193018814093

1 85.57 84.302 86.54 86.113 86.69 84.634 85.90 84.365 86.80 86.196 84.78 83.95

Mean 86 85%RSD 0.9 1.1

%Difference 1

Table 262: Dissolution Precision Results for Fosinopril SodiumJHCTZ 10 mgl2.5 mg Tabletson HPLC (Fosinopril)

Repeatability: LMG Intermediate Precision: JSMSample # HPLC System Asset # 16930 HPLC System 55 Asset #

Column Serial # 01193022914058 Column Serial #011930188140931 92.45 92.012 91.46 91.053 89.40 89.624 94.00 90.135 91.15 88.816 91.37 89.37

Mean 92 92%RSD 1.7 1.3

%Difference 0

Table 263: Dissolution Precision Results for Fosinopril SodiumIHCTZ 10 mgl2.5 mg Tabletson HPLC (HCTZ)

Repeatability: LMG Intermediate Precision: JSMSample # HPLC System 02 Asset # 16930 HPLC System 55 Asset #

Column Serial # 01193022914058 Column Serial #011930188140931 98.53 99.232 99.37 98.033 99.01 98.004 100.40 97.725 96.71 96.456 100.46 98.88

Mean 99 98%RSD 1.4 1.0

%Difference 1

The specificity of the analytical method was evaluated by preparations of the placebo and

dissolution media with analysis on each chromatographic system. The chromatograms of the

209

placebo and media injections were then analyzed for possible interferences, which could cause

bias on any analytical data. The acceptance criterion for the dissolution method was no placebo

peak occurring at the same time of the corresponding active peak above 100 of the target sample

concentration.

Figure 245:0.050

0.040

0.030

0.020

0.0 10

0.000

-0.010

Chromatogram of the Blank (Dissolution Medium)

-0.02 00.00

Figure 246:o oso

0 040

0 030

0 020D

0010

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18Miii utes

Chromatogram of the Blank (Prepared per Standard preparation with methanol).

2.00 4 00 6 00 6 00 10 00U in uteS

Figure 247: Chromatogram of the Fosinopril Sodium Tablets Placebo Powder.0 050 —

0 040

0 030

0 020

0010

0 000

-0 010

-0 0200 00

0 000

-0 010

-0 0200 00 1200 1400 1600 18

10 00Minutes

210

Figure 248: Chromatogram of the Fosinopril SodiumIHCTZ Tablets Placebo Powder.o 050

0 040

0 030

0 020D

0010

0 000

-0 010

-0 020000 10.00

Minutes

As the previous dissolution method at Sandoz, Inc in Wilson, NC utilizes the same

parameters as this method (with the exception of the chromatographic parameters), the filter

study has been previously performed. The previous validation demonstrated that the Fosinopril

Sodium will bind on the filters, and therefore, the centrifugation of the samples is a must without

filtration. The robustness of the degassing procedure was evaluated as per the various techniques

employed. The dissolution for each product was executed using non-degassed media, dissolution

media that was helium sparged for 15 minutes, and dissolution media that was degassed using

vacuum degassing for 15 minutes.

Table 264: Dissolution Results for the Degassing Procedure for Fosinopril Sodium 10 mgTablets

Sample # Non-Degas Helium Sparge Vacuum Degas72.91 85.56 83.2082.40 86.54 84.0184.90 86.69 85.0985.71 85.90 83.2786.92 86.80 83.8978.52 84.78 82.06

123456

Analyst: LMG

211

Table 265: Dissolution Results for the Degassing Procedure for Fosinopril SodiumJHCTZ 10m: 12.5 mg Tablets Fosino .ril)

_____Sample

# Non-Degas Helium Sparge Vacuum Degas87.84 92.45 73.3989.86 91.46 88.4189.16 89.40 87.7289.56 94.00 90.2591.08 91.15 90.8888.43 91.37 91.30

MeanAnalyst. LMG

1234

116

Table 266: Dissolution Results for the Degassing Procedure for Fosinopril SodiumIHCTZ 10mg /12.5 mg Tablets (HCTZ)

Sample # Non-Degas Helium Sparge Vacuum Degas1 98.54 98.53 100.852 100.97 99.37 97.683 99.65 99.01 96.694 98.37 100.40 98.545 98.81 96.71 101.826 96.87 100.46 99.15

Mean 99 99 99Analyst: LMG

The robustness of the sampling technique was assessed through using a manual sample

collection or an automated sampling apparatus. The results were then compared to determine if

the two techniques may be used inter-changeably.

Table 267: Dissolution Results for the Sampling Procedure for Fosinopril Sodium 10 mgTablets

Sample # Manual Automated1 85.57 85.792 86.54 87.873 86.69 88.754 85.90 86.115 86.80 89.556 84.78 88.09

Mean 86 88%Difference 2

Analyst: LMG

212



Analyst: L 4G

Table 269: Dissolution Results for the Sampling Procedure for Fosinopril SodiumJHCTZ 10 mg/12.5 mg Tablets (HCTZ)



Analyst: LMG

The robustness of the chromatographic parameters was assessed by varying different

parameters of the chromatographic system to determine the effect on the dissolution results.

The various chromatographic parameters are outlined in Table 270. The results are considered

acceptable if the mean result for each condition is within 50 of the initial. All n.ms met the

established system suitability criteria.

Table 270: Dissolution Chromatographic Robustness ParametersParameter VariationFlow Rate ± 0.3 mL/min


Temperature ± 5°CWavelength ±4 nm

Column Equivalency Waters Atlantis dCl8 5itm 4.6mm x 150mm

Table 268: Dissolution Results for the Sampling Procedure for Fosinopril SodiumJHCTZ 10 mg/12.5 mg Tablets (Fosinopril)

213

Table 271: Dissolution Robustness Results for Fosinopril Sodium 10mg Tablets

Analyst: LMG

Table 272: Dissolution Robustness Results for Fosinopril SodiumJHCTZ lOmg/12.5mg TabletsParameter Mean (%) Fosinopril Mean (%) HCTZ

Initial 91 101Flow Rate (0.9 mE/mm) 90 98Flow Rate (1.5 mL/min) 89 98

Column Temperature 35°C 90 98Column Temperature 45°C 90 98

Wavelength2o2nm 91 101Wavelength 210 nm 91 101

Mobile Phase A pH 1.9 89 98Mobile Phase A pH 2.3 91 97

+3% ACN in Mobile Phase A 94 96-3% ACN in Mobile Phase A 92 96

Column Equivalency 89 98Analyst: LMG

The stability of the sample and standard solutions was evaluated as part of the validation

experiments for the analytical method for dissolution. The solution stability for the samples was

executed using the lowest label strength for the each finished product dosage form as this is

considered to be “worst case.” The acceptance criteria for the dissolution solutions is that the

standard and the sample solution must be not more than 2° o different when compared to that of

the initial sample. The solution stability was also executed using a photo-diode array (PDA)

detector and analyzed for peak purity. For the peak to be considered pure, the purity angle must

Parameter Mean (%)Initial 86

Flow Rate (0.9 mL/min) 84Flow Rate (1.5 mL/min) 84

Column Temperature 35°C 85Column Temperature 45°C 85

Wavelength 202 nm 86Wavelength 210 nm 86

Mobile Phase A pH 1.9 84Mobile Phase A pH 2.3 85

+3% ACN in Mobile Phase A 88-3% ACN in Mobile Phase A 86

Column Equivalency 85

214

be less than that of the purity threshold angle. The samples and standards were stored in clear

test tubes and glassware under ambient laboratory conditions. As shown in the following tables,

the standards and samples containing HCTZ can only be used for two days following the day of

preparation. The Fosinopril standard and samples may be considered stable for up to three days

following the day of preparation.

Table 273: Dissolution Solution Stability for the Fosinopril Sodium StandardTime Point - %Fosinopril

Day 0 (initial) 99%Day 1 99%Day3 98%

Analyst: LMG

Table 274: Dissolution Solution Stability for the Fosinopril SodiumJHCTZ Standard

Time Point %Fosinopril %HCTZ

Day 0 (initial) 101% 100%Day 1 99% 99%Day2 101% 98%Day 3 99% 96%

Analyst: LMG

Table 275: Dissolution Solution Stability for Fosinopril Sodium 10 mg TabletsTime Point %Fosinopril

Day 0 (initial) 87%Dayl 85%Day2 86%Day3 85%

Time Point %Fosinopril %HCTZ

Day 0 (initial) 94% 100%Day 1 93% 99%Day2 93% 98%Day3 93% 96%

Analyst: LMG

Table 276: Dissolution Solution Stability for the Fosinopril SodiumlHCTZ lOmg/12.5 mg Tablets

Analyst: LMG

A method comparison study was executed to determine the equivalency between the

current Sandoz, Inc at Wilson, NC method and the new propose method. Six samples of the

215

lowest label claim for each product were tested using the dissolution parameters described by

each method. The acceptance criteria for the method comparison was that a percent difference of

not more than 500 between each of the methods for equivalency to be established. As shown in

the tables below all three methods yield results within the acceptance criteria and may therefore

be used interchangeably.

Table 277: Method E.uivalency for Fosino ml Sodium lOm: Tablets

Sample New Method Sandoz Monograph FOO7QC(%Fosinopril) (%Fosinopril)

85.57 I 87.4386.54 87.2486.69 85.3185.90 85.6186.80 88.5884.78 87.09

New Method I Sandoz Monograph FOO9QCSample #(%Fosinopril) I (%Fosinopril)

92.45 I 93.1191.46 I 92.4189.40 92.0994.00 92.0591.15 I 92.8891.37 92.35

New Method I Sandoz Monograph FOO9QCSample #(%HCTZ) (%HCTZ)

98.53 95.0599.37 I 96.7999.01 96.97100.40 I 94.4196.71 93.86100.46 I 93.93

2

4

23

5

1

3

5

Analyst: LMG

Table 278: Method E.uivalenc for Fosino .ril SodiumJHCTZ lOm: 12.5m: Tablets

Analyst: L.3

Table 279: Method E.uivalenc for Fosino .ril SodiumJHCTZ lOm: 12.5m: Tablets

216

Discussion of the UPLC Dissolution Method and Trouble-Shooting

The method validation for Fosinopril Sodium Tablets and Fosinopril SodiumIHCTZ

Tablets was originally intended for both the HPLC and UPLC chromatographic systems.

However, as the validation for the methods was executed, a multitude of problems were

encountered for the UPLC validation. The first problem encountered was during the use of the

curve feature for the gradient. A convex curve during the gradient analysis for Fosinopril and

HCTZ was used to reduce the run time of both the HPLC and UPLC methods. The HPLC

method was shown to have low°0RSD for both components of interest, but the Fosinopril peak

in the UPLC gradient analysis had very high variability. During replicate injections of the

standard solution, the UPLC method was shown to have as much as 450 o difference between the

highest and lowest standard areas in 10 replicate injections from the same vial. This difference

between replicate standard injections was shown to decrease after changing the sampling rate of

the UPLC from 5 points per second to 20 points per second. However, the °odifference observed

between the highest and lowest standard area 10 replicate injections was still shown to be 2.5° o

for Fosinopril. Meanwhile the HCTZ peak was always shown to be very consistent with°0RSD

of 0.1 regardless of the sampling rate. The HCTZ of the method was shown to elute during the

initial isocratic portion of the gradient, while the Fosinopril eluted on the convex gradient curve.

A review of over-laid chromatograms demonstrated that the absorbance observed on the gradient

curvature varied between injections. This variance was in direct proportion to the area if the

Fosinopril peak. The gradient was changed to that of a linear gradient and the °odifference

observed for the replicate standard injections improved significantly to 0.8° o.

Because the HPLC method also utilized a gradient curve, this issue was never observed

as a problem. The HPLC chromatographic system utilizes a quaternary solvent pump, while the

217

UPLC system uses a binary in-line high pressure mixing solvent pump. Sandoz at Wilson, NC

has recently purchased three new H-Class chromatographic systems. The new H-Class systems

are a hybrid system that use both HPLC and UPLC technology. The H-Class systems also use

the quaternary pumping system, the same as used on the HPLC systems. The same gradient

curve that was being uses on the UPLC system was then programmed into the H-Class system to

determine if the different pumps could possibly be the cause of the high variability. The

°odifference between the highest and lowest standard areas for 10 replicate injections was shown

to be only 0.3%, which was the lowest observed using UPLC. The Waters Corporation was

involved during this investigation and are actively trying to determine in their own laboratories

the cause for the differences observed between the two systems when using a curve during a

gradient elution.

The UPLC method was then changed to a linear gradient, as this gave the most

repeatability between replicate standard injections. However, during the method precision

studies another problem was observed. When the method precision was executed, the same

samples were injected using the new HPLC method, the new UPLC, and the existing Sandoz

Wilson, NC methods. The new HPLC method and the Sandoz methods were shown to give the

same results within acceptance criteria. However, the same samples when injected on the UPLC

system were shown to be 1500 lower for the Fosinopril peak only. The HCTZ peak in the sample

on all three systems gave the same results. A review of the linearity for the UPLC, despite

passing all pre-set acceptance criteria, was in reality shown to be variable at the lowest

concentrations. At times linearity may fail at the higher concentrations due to column over

loading; however, failing at the lower concentration (especially when not even close to a limit of

quantitation) is not common. The results were confirmed on two UPLC systems for authenticity.

218

The same samples were then removed from the UPLC, injected on the HPLC and the results

confirmed the original HPLC data; thus indicating that the abnormal results were directly related

to the UPLC.

The assay CU and related compounds methods were successfully validated using the

UPLC systems, without any incident or problem. Therefore, the issue appeared to be related to

the water dissolution medium. Because the dissolution medium and parameters are pre-set by

the FDA no part of the sample preparation could be altered for a dissolution method. Fosinopril

is known (as previously discussed) to be sparingly soluble in water. The Fosinopril was also

observed during development of the assay related compounds method to form micelles through a

self-association behavior when present in aqueous solutions. Though the concentration of the

Fosinopril in the dissolution samples is much lower than this threshold, one possible explanation

is that the high pressure of the UPLC system could potentially cause this variability of the

Fosinopril in the sample solutions if the Fosinopril were to change as it flowed through the

column. Fosinopril is also a much more hydrophobic compound when compared to that of

HCTZ. Fosinopril is in fact the most lipophilic ACE inhibitor currently on the market. Though

high pressure and temperature would usually increase the solubility of polar compounds the

different nature of this molecule may in fact reduce the solubility. Micelles are also known to

form faster at higher temperatures, which is a known characteristic of Fosinopril and its

lipophilic nature. The assay and related compounds methods uses a diluent that is 50° o by

volume methanol, in which Fosinopril is very soluble. Therefore, this inconsistency between the

HPLC and the UPLC was never an issue.

In the future investigation into the solubility of Fosinopril in water and the effect of

temperature and pressure could be useful. One possible approach is the lowering of the sample

219

compartment temperature to 5°C in order main the solubility of the lipophilic Fosinopril

molecule (remembering that lower temperature may increase hydrophobic molecules rather than

higher temperatures). Another approach would also be to determine if the high pressure mixing

of the binary solvent manager in the UPLC is also causing an effect on the response of the

Fosinopril in the sample matrix. Though this investigation will be resolved to determine the root

cause for this discrepancy between the HPLC and UPLC dissolution methods, a functional

UPLC method in the quality control laboratories may never be a possibility. The prevention of

this occurring again during routine release testing would be very difficult to control. Should this

problem be resolved another occurrence during release testing of marketed products could cause

out of specification (OOS) results, which would cause delays in product release and costs large

sums of money to resolve in the laboratory.

220

CONCLUSION

The analytical methods for the simultaneous analysis of Fosinopril Sodium,

Hydrochlorothiazide, and Benazepril Hydrochloride in their respective finished product dosage

forms has been validated for assay and content uniformity. The same analytical method has been

validated for the analysis of related compound in each of these finished product as well. The

HPLC method is recommended for all products containing Benazepril Hydrochloride for related

compound determination. The UPLC and HPLC methods are both considered acceptable for

related compound analysis of products containing Fosinopril Sodium. The analytical method for

the analysis of all products containing Fosinopril Sodium and Fosinopril Sodium with

Hydrochiorothiazide has been validated. All methods have shown to be linear, accuracy, precise,

specific and robust for their respective products. Assay and related compound methods have

been shown to be stability-indicating and sensitive for the accurate quantitation of the potency

and each known related compound for each active pharmaceutical ingredient of interest.

Future development will be directed toward the completion of a related compound

method for the accurate determination of known related compounds of HCTZ in the Benazepril

HC1/HCTZ Tablets formulation. Investigation is recommended into the cause for failure of the

UPLC method for Fosinopril containing products.

The validation of these analytical methods will allow for a variety of cost savings.

Currently each method is being executed using separate for analytical methods for each finished

product. The products present in combination with HCTZ also use separate testing methods for

each of the main components of the product. The use of this method will allow for the

simultaneous testing of each of the four products validated. For example, the testing of one lot

of Fosinopril SodiumJHCTZ Tablets would need one chemist to analyze the Fosinopril Sodium

221

portion for assay and related compounds, while another chemist would be used for the analysis

of the HCTZ portion. A third chemist would then be utilized for the dissolution testing for the

Fosinopril Sodium and HCTZ. Each chemist would subsequently need their own

chromatographic system (HPLC) and a UV-Vis spectrophotometer for the analysis of the HCTZ

dissolution samples. Now all of these above mentioned systems can be consolidated on only one

HPLC, each using the same column and mobile phase. For added efficiency, the assay and

related compounds sample could utilize the UPLC for faster throughput. The sample diluent was

also changed from a (40:60 v v) 0.2° o phosphoric acid solution and acetonitrile (respectively)

mixture to a (50:50 v v) 0.00 iN hydrochloric acid solution and methanol (respectively) mixture.

Methanol is also known to be a cheaper organic solvent than acetonitrile, which will reduce

solvent costs. By running the assay and related compounds portion of the method on the UPLC

the solvent cost will be further reduced as the flow rate used in the method is only 0.5 mL mm.

The four products validated by this method can use the same standard for any

assay/related compound analysis for Fosinopril Sodium Tablets, Fosinopril SodiumJHCTZ

Tablets, Benazepril HC1 Tablets, and Benazepril HC1IHCTZ Tablets. The standard was shown

to be stable for each component for 7 days following the day of preparation. This will save cost

in the consumption of reference standards used in the laboratory. All four products may also be

tested using the same chromatographic systems, which will consolidate laboratory leaving other

chromatographic systems available for other testing. Because equipment availability can be

limiting factor in the amount of throughput allowed by a laboratory, the consolidation of these

methods will benefit the testing of other products in that equipment availability will now be

increased.

222

The implementation of these new methods for the UPLC and HPLC will allow for cost

saving across the Sandoz laboratory. Through a combination of savings in solvents, reagents,

reference standards, preparation time of the chemists, and equipment availability a higher

throughput of released lots to the commercial market will be observed. The streamlining of these

methods will aid in meeting market demands of these products.

223

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