NOVEL SIMULTANEOUS SEPARATION AND QUANTITATIVE DETERMINATION OF FOUR SARTANS IN PRESENCE OF...

NOVEL SIMULTANEOUS SEPARATION AND QUANTITATIVE DETERMINATION OF FOUR SARTANS

IN PRESENCE OF HYDROCHLOROTHIAZIDE BY ISOCRATIC RP-HPLC

Under the guidance of:Dr. Panchumarthy Ravisankar M. Pharm., Ph.D.

Professor & HOD Dept. of Pharmaceutical Analysis and Quality AssuranceVignan Pharmacy CollegeVadlamudi. Presentation by:

K. Manjusha (12AB1R0043)V. Laya Sri (12AB1R0044)B. Vijaya Kumar (12AB1R0009)K. Rajyalakshmi (12AB1R0037)K. Avinash kumar (12AB1R0039)

2

CONTENTS: AIM AND OBJECTIVE

INTRODUCTION DRUGS PROFILE LITERATURE SURVEY PLAN OF WORK MATERIALS AND METHODS RESULTS CONCLUSION REFERENCES

Vignan Pharmacy College,Vadlamudi.

3

AIM:

The core AIM of the present study is to develop a novel, rapid,

precise and accurate RP-HPLC method for simultaneous

separation and quantification of Hydrochlorothiazide along with

four sartans Telmisartan(TELM), Losartan(LOSA),

Olmesartan(OLME) and Valsartan(VALS).

To develop Novel methods for separation and quantification of

all the above said drugs on single chromatographic system

without any minor changes in detection wavelength and mobile

phase composition.


4

OBJECTIVE:


To develop rapid, sensitive and economical analytical methods

based on spectrophotometric & RP-HPLC techniques for estimation

and separtion of Hydrochlorothiazide with Telmisartan, Losartan,

Olmesartan and Valsartan in pharmaceutical dosage forms.

To develop method with shorter run time and better sensitivity.

Reducing the solvent consumption to make it more eco-friendly.

To validate the method for different parameters like Accuracy,

Precision, Linearity, specificity, Robustness according to

International Conference on Harmonization ICH Q2(R1)

guidelines[5] .

To apply the developed RP-HPLC method in the analysis of

pharmaceutical formulations.

5

INTRODUCTION


HPLC is a versatile tool for the qualitative and quantitative analysis

of drugs and pharmaceuticals, chemical and biological materials has

become indispensable in pharmacokinetics studies.

Of the many instrumental methods available for quantification RP -

HPLC is selected as a tool for developing novel analytical procedure.

Method development is done for the products as follows:

- New products

- Existing products

Alternate methods for existing products are developed for better

precision and ruggedness.

Method development:

Vignan Pharmacy College,Vadlamudi. 6

DRUG PROFILE

7

Drug Name : HYDROCHLOROTHIAZIDE

Structure :

IUPAC : 6-chloro-1,1-dioxo-3,4-dihydro-2H-1,2,4-

benzothiadiazine-7-sulfonamide

Category : Anti hypertensive

Molecular Formula : C7H8ClN3O4S2

Molecular Weight : 297.74 g/mol

Brand names : Apo-Hydro, Aquazide H, Hydrodiuril,

HydroSaluric, Hydrochlorot, Microzide,

Esidrex, and Oretic


8

Drug name : VALSARTAN

Structure :

IUPAC Name :(S)-3-methyl-2-(N-{[2'-(2H-1,2,3,4-tetrazol-5- yl)biphenyl-4-yl]methyl}pentanamido)butanoic acid

Molecular weight :435.519 g/mol

Molecular formula : C24H29N5O3

Brand names : Diovan, Valent -H


9

Drug name : LOSARTAN

Structure :

IUPAC Name :(2-butyl-4-chloro-1-{[2'-(1H-tetrazol-5-yl)biphenyl-

4-yl]methyl}-1H-imidazol-5-yl)methanol

Molecular weight :422.91 g/mol

Molecular formula :C22H23ClN6O

Brand names : Cozaar, Losagard, Zargo-H


10

Drug name : OLMESARTAN

Structure :

IUPAC Name : 2,3-dihydroxy-2,2-butenyl-4(1-hydroxy-1- methylethyl)-2-propyl-1-[p-(o-1H-tetrazol-5- ylphenyl)benzyl]imidazole5-carboxylate2,2-carbonate

Molecular weight : 558.59


Brand names : Olmicep-H, Olmax


N

NN

HN

Olmesartan

N

N OH

H3C

O

11

Drug name : TELMISARTAN

Structure :

IUPAC Name : 2-(4-{[4-Methyl-6-(1-methyl-1H-1,3-benzodiazol- 2-yl)-2-propyl-1H-1,3-benzodiazol-1-yl]methyl} phenyl)benzoic acid

Molecular weight : 514.617 g/mol


Brand names : Telmisat-H, Cresar, MicardisVignan Pharmacy College,Vadlamudi.


LITERATURE SURVEY

13

S.NO AUTHORS TITLE STUDY JOURNAL

NAME

YEAR,VOLUME,ISSUE & PG.NO

1

Reem Youssef*, AdnanHbash, Ahmad Hassan

Development and Validation of RP-HPLC Method for the Estimation and Separation ofValsartan, Losartan and Irbesartan in Bulk and Pharmaceutical Formulation.

Separation of 3 sartans using c18 column and its validation according to ICH guidelines.

International Journal of Pharmaceutial Sciences Review and Research.(IJPSRR)

Jan – Feb 2014, 24(2), 311-314.

2

Ganipisetty Lakshmi Aswini*, D.Dachinamoorthy,J.V.L.N.Seshagiri Rao

A Sensitive RP-HPLC Method Development and Validation for the SimultaneousEstimation of Losartan Potassium and Hydrochlorothiazide.

Validation and estimation using mobile phase of acetonitrile and phosphate buffer.

International Journal for PharmaceuticalResearch Scholars. (IJPRS)

2014, V-3, I-2, 408-416.


Vignan Pharmacy College,Vadlamudi.14

S. NO AUTHORS TITLE STUDY JOURNAL

NAME

YEAR,VOLUME,

ISSUE & PG.NO

3

T. M. Kalyankar*, S. J. Wadher, S. S. Pekamwar N.G. Doiphode

Development and Validation of RP- HPLC method forEstimation of Hydrochlorothiazide and Irbesartan inPharmaceutical Preparation.

Analysis using C18 column using methanol as solvent.

InternationalJournal of PharmTech ResearchCODEN (USA): IJPRIF

Jan-March 2014,Vol.6, Issue No.1, 330-336.

4

T. Gopala Swamy,K. Nagaraju,A. Lakshmana Rao

RP-HPLC Method for the Simultaneous Estimation of Telmisartanand Hydrochlorothiazide in Pharmaceutical Dosage Form.

RP-HPLC method was found to be rapid,precise,accurate,Selective.

International Journal of Drug Development & Research.(IJDDR)

October-December 2011,Vol. 3 ,Issue 4,362-368.

S.NO AUTHORS TITLE STUDY JOURNAL NAME

YEAR,VOLUME,

ISSUE & PG.NO

5

Lakshmanarao. A,Bhaskhara Raju .V Simultaneous

Estimation Of

Valsartan And

Hydrochlorothiazide

In Tablets By RP-

HPLC Method.

Column having isocratic mode with mobile phase of acetonitrile and phosphate buffer.

IJPIR Jul-sep 2011,Volume 01, Issue-03,170-174.

6 Hany Mohammed Hafez*, Abdullah Ahmed Elshanawane

Quantitative Determination of three Angiotensin-II-receptor Antagonists in Presence of Hydrochlorothiazide by RP-HPLC in their Tablet Preparations.

C18 column , phosphate buffer is used.

Iranian Journal of Pharmaceutical Research.

2013, 12 (4), 635-643.


16

PLAN OF WORK:


Plan of RP-HPLC technique: Literature collection regarding various aspects like

physicochemical properties of the drugs

Procurement of drug reference standards, reagents, solvents,

other chemicals and formulations.

Establishment of initial chromatographic conditions.

Optimization of the chromatographic conditions to achieve the

system suitability goals and validating the method according to

ICH guidelines.

Applying the optimized method for estimation of selected drug

candidates in tablets and pharmaceutical dosage forms.

17

MATERIALS AND METHODS


18

Table 1. Commercial brand names of TELMI, LOS , VAL, IRBE and HYDRO combinations used for the present study.

Brand name Formulation Labeled amount (mg) Manufacturer

Telmisat-H (40 mg) Tablets

Telmisartan- 40 mg

Hydrochlorothiazide- 12.5 mgBiocon laboratories

Zargo-H(50 mg) Tablets

Losartan- 50 mg

Hydrochlorothiazide- 12.5 mg

Biocon laboratories

Olmicep-H(40mg)

TabletsOlmesartan –40 mg

Hydrochlorothiazide – 12.5 mg

Cipla Ltd., Mumbai, India.

Valent-H(80 mg)

TabletsValsartan- 80 mg

Hydrochlorothiazide- 12.5 mgLupin Laboratories, Hyderabad.


Table 2. Materials used in the present study

S.No. Materials Procured from1. Telmisartan Aristo Pharmaceuticals Pvt.Ltd., Bombay.

2. Losartan Hetero Labs Ltd., Hyderabad

3. Olmesartan Hetero Labs Ltd., Hyderabad

4. Valsartan Anant Pharmaceuticals, Kamal, Haryana.

5. Acetonitrile HPLC grade Thermo Fisher Scientific India Pvt. Ltd., Mumbai.

6. Water HPLC grade Merck Specialties Pvt. Ltd., Mumbai.

7. Methanol HPLC grade Merck Specialties Pvt. Ltd., Mumbai.

8. Dipotassium hydrogen phosphate Thermo Fisher Scientific India Pvt. Ltd., Mumbai.

9. Potassium dihydrogen phosphate Glaxo Smith Kline Pharmaceuticals Ltd., Mumbai.


20

Table 3. Instruments used in the present study

S.No. Instrument Name of the company and model

1. HPLC

Shimadzu LC-20AT Prominence Liquid Chromatograph with Shimadzu SPD-20A Prominence UV-Vis detector,Welchrom C18

Column (4.6 X 250 mm, 5μm), with Rheodyne manual loop injector (20 μL) and Spinchrom data acquisition software.

2. UV-Vis spectrophotometer

UV-Visible Spectrophotometer ELICO – SL - 210 with Spectra treats(TM) software.

3. Weighing balance Essae vibra AJ (0.001g), Essae-Teraoka Ltd.

4. pH meter Elico LI120 pH meter, Elico India Ltd.

5. Ultrasonicator Ultrasonic bath sonicator, PCI ltd., Mumbai.

6. Vacuum pump Single Stage Vacuum Pump.


21

Preparation of reagents and standards: Preparation of phosphate buffer pH 3.3

Phosphate buffer with 10 mM was prepared by dissolving 6.056 g KH2PO4 in

445 mL of HPLC grade water. To this said solution 55 mL of 0.1M H3PO4 was added to

adjust the pH 3.3.

Preparation of mobile phase:

The above stated prepared phosphate buffer (pH 3.3) acetonitrile were mixed

completely in the proportion of 50: 50 v/v and was filtered through 0.45 µm nylon

membrane filter and degassed by sonication.

Preparation of standard stock solution:

Stock standard solutions containing (1.25, 0.4, 0.5, 0.4, 0.8 mg/mL) of

Hydrochlorothiazide, Telmisartan, Losartan, Olmesartan and Valsartan respectively

were prepared by dissolving (12.5, 40, 50, 40, 80 mg) of each in methanol in 100 mL

volumetric flask respectively. It was then sonicated for 15 minutes and the final volume

of solutions was made up to 100 mL with methanol to get stock standard solutions.Vignan Pharmacy College,Vadlamudi.

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Preparation of sample solution: A composite of ten Telmisat-H, Zargo-H, Olmecip-H and Valent-H

tablets are prepared by grinding them to a fine, uniform size powder,

triturated using mortar and pestle. After calculating the average tablet

weight, amounts of powder equivalent to HCTZ, TELM, LOSA, OLME

and VALS respectively are prepared by dissolving (12.5, 40, 50, 40, 80

mg) respectively of each type of tablets were accurately weighed and

transferred separately to 100 mL volumetric flasks respectively.

Solutions were sonicated for 15 min and the solutions were then filtered

through 0.45 µm nylon membrane filters. Aliquots of appropriate volume

(10 mL) were transferred to 100 mL calibrated flasks and diluted to

volume with mobile phase to furnish the mentioned concentration above.


23

RESULTS:

Method Development:

Selection of a common detection wavelength:

The ultra violet spectrum of four Sartans and hydrochlorothiazide were

scanned individually in the region between 200 - 400 nm. The UV

overlain spectra of these five drugs showed that they absorbed at 238

nm. So this wavelength was selected for the determination of sartans.

Figure 1 shows UV overlain spectra of four sartans with HCTZ.


24Vignan Pharmacy College,Vadlamudi.

Figure 1. UV overlain spectra of four sartans and HCTZ.


Method optimization: Numerous trials were performed to obtain the better separations. From all trials, eventually better reproducibility of the results and good

resolution, good peak shape, short runtime, minimal peak tailing were well identified when mobile phase consisting of phosphate buffer mixture properly adjusted to pH 3.3, acetonitrile in the proportion of 50:50 v/v with satisfactory results.

Table 4. Optimized chromatographic conditions

Parameter Chromatographic conditions for 4 sartans & HydrochlorothiazideInstrument Shimadzu LC-20AT Prominence liquid chromatographColumn Welchrom C18 column (4.6 X 250 mm, 5 µm)

Detector Shimadzu SPD-20A prominence UV-VIS detector

Mobile phase 10 mM phosphate buffer (pH 3.3) : Acetonitrile 50:50, v/vFlow rate 1mL/minwave length UV at 238 nmRun time 8 minutesTemperature Ambient temperature (25 0C)Injection volume 20 µL

Name of the drugs HCTZ TELM LOSA OLME VALSRetention time (minutes) 3.300 3.780 4.467 5.247 5.570

Th.Pl (Efficiency) 12,312 12,468 14,715 15,250 17,188 Theoretical plates per meter 246247 247368 294308 305005 343756Resolution - 3.776 4.862 4.931 2.008Tailing factor 1.194 1.116 1.182 1.160 1.154

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Figure 2. Typical chromatogram showing the HCTZ, TELM, LOSA, OLME AND VALS in a synthetic mixture.


27

Method validation: The developed analytical method was validated in pursuance of the guide

lines of ICH. SPECIFICITY: The purpose of this study is to determine the effect of excipients and other

additives that are usually present in the formulations. The test results obtained were compared with the results of standard drug.


Table 5. Results of specificity

Result: The present study shows that the ingredients are not interfering with the developed method.

Name HCTZ TELM HCTZ LOSA HCTZ OLME HCTZ VALS

Mobile

phase

No peaks No

peaks

No peaks No peaks No peaks No

peaks

No peaks No peaks

Placebo No peaks No

peaks

No peaks No peaks No peaks No

peaks

No peaks No peaks

Individual

Separate

standard

solutions.

Peak for HCTZ and

TELM at 3.300

min. and 3.780 min.

respectively.

Peak for HCTZ and

LOSA at 3.300 min.

and 4.467 min.

respectively.

Peak for HCTZ and

OLME at 3.300

min. and 5.247 min.

respectively.

Peak for HCTZ and

VALS at 3.300 min.

and 5.570 min.

respectively.

28

The linearity of the method was studied by analyzing different

concentration of the drugs. According to ICH recommendations at

least five concentrations are to be used. In this study five

Concentrations were chosen, in the ranges of 2.5-12.5, 8-40, 10-50,

8-40, 16-80 µg/mL. Calibration curve is constructed by plotting

concentration on X-axis and the peak areas on Y-axis.

The least square analysis method was adopted for achieving slope,

intercept and correlation coefficient, regression data values.


LINEARITY:


Linearity method

HCTZ LOSA TELM OLME VALS

Conc.

μg/mL

Peak area,

mV.s.

Conc.

μg/mL

Peak area,

mV.s.

Conc.

μg/mL

Peak area,

mV.s.

Conc.

μg/mL

Peak area,

mV.s.

Conc.

μg/mL

Peak area,

mV.s.

0 0 0 0 0 0 0 0 0 0

2.5 217.93 10 209.972 8 205.794 8 175.56 16 234.722

5 435.79 20 416.85 16 410.63 16 382.42 32 466.872

7.5 669.786 30 642.32 24 628.47 24 605.81 48 706.7

10 889.52 40 862.97 32 853.23 32 839.64 64 928.68

12.5 1092.803 50 1080.64 40 1068.11 40 1053.92 80 1168.004

Table 6. Results relating to Linearity data

30

Table 7. Regression analysis data relating to HCTZ, TELM, LOSA, OLME and VALS


Parameter HCTZ LOSA TELM OLME VALS

Detection wavelength(λmax) UV at 238 nm UV at 238 nm UV at 238 nm UV at 238 nmUV at 238 nm

Linearity range (µg/mL) 2.5-12.5 µg/mL 8-40 µg/mL 10-50 µg/mL 8-40 µg/mL 16-80 µg/mL

Regression equation (Y =aX + b) Y = 88.146 X + 0.0586

Y = 27.099 X - 6.5173

Y = 21.431 X - 8.5685

Y = 13.36 X - 25.10

Y = 14.576 X + 1.0686

Slope(a) 88.146 27.099 20.968 13.366 14.576

Intercept(b) 0.0586 6.5173 8.5685 25.101 1.0686

Standard error of slope (Sa) 0.7592 0.1537 0.24642 0.2900 0.06053

Standard error of intercept (Sb) 1.9488 0.9310 1.49213 1.4277 2.9327

Regression coefficient (R2) 0.9997 0.9998 0.9997 0.9981 0.9999

% Relative standard deviation* 1.1875 2.4993 0.5239 0.104 1.050

Percentage range of errors

0.05 significance level

0.01 significance level

1.02904

1.6138

1.15695

1.8144

0.54992

0.86242

0.12232

0.19183

0.36373

0.57042

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Figure 3. Standard chromatogram relating to HCTZ

Figure 4. Standard chromatogram relating to TELM

Figure 5. Standard chromatogram relating to LOSAVignan Pharmacy College,Vadlamudi.

CHROMATOGRAMS

32

Figure 6. Standard chromatogram relating to OLME

Figure 7. Standard chromatogram relating to VALS


33

Figure 8: Standard chromatogram of HCTZ (2.5 μg/ml) and TELM (8 μg/ml)

Figure 9. Standard chromatogram of HCTZ (2.5 μg/ml) and LOSA (5 μg/ml).

Figure 10. Standard chromatogram of HCTZ (2.5 μg/ml) and OLME (8 μg/ml).Vignan Pharmacy College,Vadlamudi.


Fig. 11. Standard chromatogram of HCTZ (2.5 µg/ml) and VALS (16 µg/ml)


Fig. 12. Sample chromatogram of HCTZ (2.5 µg/mL) and TELM (8 µg/mL)

Fig. 13. Sample chromatogram of HCTZ (2.5 µg/mL) and LOSA (5 µg/mL)


Fig. 14. Sample chromatogram of HCTZ (2.5 µg/mL) and OLME (8 µg/mL)

Fig. 15. Sample chromatogram of HCTZ (2.5 µg/mL) and VAL (16µg/mL)

37

Figure 16. Linearity plot pertaining to HCTZ

Figure 17. Linearity plot pertaining to LOSA

Figure 18. Linearity plot pertaining to TELM


0 2 4 6 8 10 12 140

200

400

600

800

1000

1200

f(x) = 88.1460685714286 x + 0.0585714285709855R² = 0.999703310805355

Concentration, µg/mL.

peak

are

a, m

V.s

0 5 10 15 20 25 30 35 40 450

200

400

600

800

1000

1200

f(x) = 27.0988 x − 6.51733333333323R² = 0.999799524859121


peak

are

a, m

V.s

0 10 20 30 40 50 600

200400600800

10001200

f(x) = 21.4305657142857 x − 8.0584761904762R² = 0.999698391175146


peak

are

a, m

V.s

38

Figure 20. Linearity plot pertaining to VALS


Figure 19. Linearity plot pertaining to OLME

0 10 20 30 40 50 60 70 80 900

200

400

600

800

1000

1200

f(x) = 13.3664821428571 x − 25.1009523809527R² = 0.998120619345239

Concentration µg/mL.

Peak

are

a m

V.S

0 10 20 30 40 50 60 70 80 900

200400

600800

1000

12001400

f(x) = 14.5761107142857 x + 1.06857142857177R² = 0.999931002891458

concentration, µg/mL.

peak

are

a, m

V.s

39

Precision: The repeatability and intermediate precision experiments were conducted

by determining the intra - day and inter - day precision of the method for

HCTZ with TELM, HCTZ with LOSA, HCTZ with OLME and HCTZ with

VALS.

The intraday precision was done by repeating the assay thrice for the 3

levels in the same day, under the same experimental conditions

Inter - day precision was done by taking over the assay on 3 different days,

three times on the each day for the 3 concentration levels respectively.

The results of precision study were stated in terms of % RSD which was

calculated and results were within the acceptable criteria of NMT 2.0. The

results of precision study are presented in table 8.Vignan Pharmacy College,Vadlamudi.


Accuracy/Recovery studies: The accuracy of the method was assessed by standard addition method.

Recovery tests were carried out by analyzing mixtures of HCTZ

with TELM, HCTZ with LOSA, HCTZ with OLME and HCTZ with

VALS with different compositions.

Known amounts of standards drugs were added to a pre-analyzed sample

at 3 different levels 80 %, 100 % and 120 % and the mixed standard

solutions were analyzed in triplicate at every level as per suggested

method.

The percent of individual combination of mixtures recovery and % RSD

results of accuracy data is shown in table 8.

41

Robustness: The robustness of developed analytical method was proven by the analysis of HCTZ

with TELM, HCTZ with LOSA, HCTZ with OLME and HCTZ with VALS under

different experimental conditions such as making deliberate changes in

chromatographic conditions like flow rate (± 0.2 ml/min), detection wavelength (±5

nm) and mobile phase composition (±5 %). All results were well within acceptable

limits.

LOD and LOQ:

Limit of Detection is the lowest concentration in a sample that can be detected, but

not necessarily quantified under the stated experimental conditions.

Limit of quantification is the lowest concentration of analyte in a sample that can be

determined with acceptable precision and accuracy.

LOD and LOQ were calculated using following formula LOD = 3.3σ/S and

LOQ = 10σ/S, where SD = standard deviation of response (peak area) and S = slope of

the calibration curve. The LOD and LOQ results are shown in the following table.Vignan Pharmacy

College,Vadlamudi.

Formulation Telmisat – H(40mg)

Zargo – H(50mg)

Olmecip(40mg)

Valent - H

Composition TELM HCTZ LOSA HCTZ OLME HCTZ VALS HCTZ

Linearity (μg/mL)

8 - 40 2.5 – 12.5

5 - 50 2.5 – 12.5

8 – 40 2.5 – 12.5

16 - 80 2.5 – 12.5

LOD & LOQ(μg/mL)

0.570 & 1.889

0.295 & 0.982

0.736 & 2.43

0.299 & 0.986

1.195 & 3.958

0.292 & 0.983

0.116 & 0.352

0.296 & 0.980

Assay ± SD (n = 6)

99.95 ± 1.13

98.16 ± 0.38

99.85 ± 1.16

98.21 ± 0.45

99.90 ± 1.10

99.19 ± 0.16

100.12 ± 0.15

99.59 ±0.12

Mean % recovery 100.02± 0.68

100.01 ± 0.15

100.06 ± 0.20

99.95 ± 0.09

100.41 ± 0.35

99.93 ± 0.11

100.05± 0.14

99.96 ± 0.15

Precision Intra day

(n=6)

0.125 0.131 0.346 0.129 0.435 0.171 0.312 0.213

Interday

(n=3)

0.165 0.321 0.326 0.434 0.657 0.322 0.319 0.435

Table 8. Summary of various validation parameters

Formulation TELMISAT – H(40mg)

ZARGO – H (50mg)

OLMECIP – H (40mg)

VALENT – H (80 mg)

Robustness, % Assay ± % RSD

Flow rate(± 2

mL/min)

0.8 mL/min

99.81 ± 1.05

97.94 ± 0.46

99.98 ± 1.04

98.93 ± 0.49

98.70 ± 1.02

98.96 ± 0.25

99.91 ± 0.29

100.96 ± 0.23

1.2mL/min

100.12± 0.97

99.54 ± 0.23

98.76 ±1.32

98.04 ± 0.36

100.03 ± 1.38

99.97 ± 0.35

100.54 ± 0.77

98.74 ± 0.71

Detection wave

Length(± 5 nm)

233 nm 99.97 ± 1.46

99.43 ± 0.29

98.42±1.46

97.95 ± 0.98

100.21 ± 1.37

99.96 ± 0.84

100.45 ± 0.97

99.98 ± 0.54

243nm 97.98 ±1.09

98.04 ±0.76

100.05 ± 1.03

98.94 ± 1.08

98.79 ± 0.37

98.76 ± 1.09

99.94 ± 1.43

98.47 ± 1.11

Mobile phase

compos -ition (±

5%)

45:55v/v

98.74 ±o.79

99.42 ±0.99

99.93 ± 1.42

97.92 ± 0.68

98.91 ± 0.94

100.07 ± 0.93

99.01 ± 0.61

98.76 ± 0.37

55:45v/v

99.96 ±1.2

97.81 ± 0.27

98.54 ± 0.86

98.86 ± 0.30

98.12 ± 1.57

98.72 ± 1.09

99.49 ± 0.74

100.03 ± 1.18

44

CONCLUSION:• Statistical analysis lucidly proves that it is feasible for analysis of said ARB’s in their

formulations in single run without changing the mobile phase composition and

chromatographic conditions.

• This method was very fast, cost effective, precise, accurate sensitive, highly efficient and

robust than the existing adopted methods. It is noticed that the method is free from

interferences of the excipients and additives utilized in the preparation of above stated ARB’s

after validation.

• This new RP-HPLC method satisfactorily separated all the above said combination drugs

with short retention time (eight minutes and elution window of only two minutes), optimum

peak shape, good separation and best reproducible results. Hence, it is apt to conclude that it

is successfully feasible for the application of routine analysis of said ARB’s agents

individually or binary combinations of HCTZ with TELM, HCTZ with LOSA, HCTZ with

OLME and HCTZ with VALS in quality control laboratories for usual analysis.


45

REFERENCES:

1. Development and Validation of RP-HPLC Method for the Estimation and Separation of

Valsartan, Losartan and Irbesartan in Bulk and Pharmaceutical Formulation,

Youssef ,IJPSRR , Jan – Feb 2014, 24(2), 311-314.

2. Quantitative Determination of three Angiotensin-II-receptor Antagonists in Presence of

Hydrochlorothiazide by RP-HPLC in their Tablet Preparations. Hany Mohammed

Hafez, Abdullah Ahmed, Elshanawane, Lobna Mohammed Abdelaziz, Iranian Journal of

Pharmceutical Research, 2013; 12(4): 635–643.

3. RP-HPLC Method for the Simultaneous Estimation of Telmisartan and

Hydrochlorothiazide in Pharmaceutical Dosage Form, Gopala Swamy, IJDDR,

2011,Vol. 3 ,Issue 4,362-368.

4. RP-HPLC method for simultaneous estimation of Telmisartan and Hydrochlorothiazide

in tablet dosage form. Wankhede SB, Tajne MR, Gupta KR, Wadodkar SG, Indian Journal

of Pharmaceutical Sciences, 2007; 298-300.

5. ICH Q2 (R1): Validation of analytical procedures (definitions and terminology) 2005: 9-

10.Vignan Pharmacy College,Vadlamudi.

We Heart fully thank our guide Dr. P. Ravisankar sir .

I also thank Hetero Labs Limited unit-III, Jeedimetla,

Hyderabad for providing gift sample of pure drugs.

ACKNOWLEDGEMENT


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