Validation of ACQUITY UPLC™ Methods
Tanya JenkinsAndrew AubinDr. Michael Swartz
Waters Corporation34 Maple StreetMilford, MA, 01757,USA
©2006 Waters Corporation
Agenda
• Test Method Validation
• Why ACQUITY UPLC™?
• Redevelop or Convert your current methods?
• Validation of a Method on ACQUITY UPLC
• Method Validation Software
©2006 Waters Corporation
Why Validate?
• Method validation is completed to insure that an analytical methodology is accurate, reproducible and robust over the specific range that an analyte will be analyzed.
• Method validation provides assurance of reliability.
• FDA Compliance
"The process of providing documented evidence demonstrating that something (the method or procedure) does what it is intended to do; is
suitable for it’s intended purpose."
©2006 Waters Corporation
The Process of Validation
Methods Method
System System
Suitability
Validation
Software
Hardware
©2006 Waters Corporation
USP Analytical Performance Characteristics
Precision
Accuracy
Limit of Detection
Limit of Quantitation
Specificity
Linearity
Robustness
Range
MethodValidation
©2006 Waters Corporation
Validation Characteristics Vs.Type of Analytical Method
* May be required, depending on the nature of the specific test.
Analytical Performance Parameter Quant.
Category 2: Impurities Category 3:Specific Tests
Accuracy Yes Yes * NoPrecision Yes Yes Yes NoSpecificity Yes Yes * YesLOD No No * NoLOQ No Yes * NoLinearity Yes Yes * No
Robustness Yes Yes Yes NoRange Yes Yes * No
*NoYesYesNo
No
NoNo
Category 1:Assays Limit Tests
Category 4:I.D.
©2006 Waters Corporation
When Do I Revalidate?
• Formulation Changes
• Manufacturing Batch Changes
• Changes in Incoming Raw Material
• Changes in Method
• To Take Advantage of New Technology– Cost/Benefit Economic Exercise– Columns, Instrumentation, Methods
"Validation is a constant, evolving process and should be considered during method development!"
©2006 Waters Corporation
Why UPLC™?
• Speed – batch release depends on the time it takes to complete chromatographic analysis
• Sensitivity – need to ensure that impurities can be reproducibly quantified
• Resolution – required for reproducible quantitation and for ensuring that new impurities are detected
©2006 Waters Corporation
Smaller ParticlesThe Enabler of Productivity
Optimal velocity range
©2006 Waters Corporation
Same Resolution and Selectivity withIncreased Speed - Constant L / dp
2.5 µm – 75 mmF = 500 µL/min
Injection = 2.5 µLRs (2,3) = 2.34
5 µm – 150 mmF = 200 µL/min
Injection = 5.0 µLRs (2,3) = 2.28
3.5 µm – 100 mmF = 300 µL/min
Injection = 3.3 µLRs (2,3) = 2.32
1.7 µm – 50 mmF = 600 µL/min
Injection = 1.7 µLRs (2,3) = 2.29
AU
0.00
0.10
0.20
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
AU
0.00
0.10
0.20
Minutes0.00 2.00 4.00 6.00 8.00 10.00
AU
0.00
0.10
0.20
Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
AU
0.00
0.10
0.20
Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.10
ImprovementResolution – SameSpeed – 3XPressure – 3X
ImprovementResolution – SameSpeed – 9XPressure – 9X
©2006 Waters Corporation
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
HPLC vs. UPLCSpeed, Sensitivity and Resolution
2.1 x 150 mm, 5 µmRs (2,3) = 4.29
12 3
HPLC
20.00
0.26
Abs
orba
nce
at 2
70 n
m
0.00
Minutes0.40 0.80 1.20 1.60 2.00 2.50
2.1 x 50 mm, 1.7 µmRs (2,3) = 4.281
23
8X Speed3.4X SensitivitySame Resolution
0.26
Abs
orba
nce
at 2
70 n
m
0.00
UPLC
Faster, More Sensitive Methods
Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
3
2.1 x 100 mm, 1.7 µmRs (2,3) = 6.38
1
2
4.5X Speed2X Sensitivity1.5X Resolution
4.50
0.26
Abs
orba
nce
at 2
70 n
m
0.00
UPLC
Faster, More Sensitive, Higher Resolution Methods
Transferring from HPLC to UPLC:
Redevelop or Convert Existing Methods
©2006 Waters Corporation
Redevelop or Convert Existing Methods?
• New methods are developed from the beginning
• Existing HPLC methods can be redeveloped or converted to UPLC
• Redevelopment of a method starts at the beginning of the method development process
• Conversion uses the current HPLC method as a starting point
• Redeveloping or converting the method will require validation
• Tools exist to help convert the current method
• This is the time to improve the method!
©2006 Waters Corporation
Converting MethodsGeometrically Scaling from HPLC to UPLC
d2Target
d2 Original
Target Flow Rate = Original Flow Rate x
Target injection volume =
Original injection volume X Target Column VolumeOriginal Column Volume
Gradient Volume = Flow Rate x Time
Column Volume = π x r2 x L
Gradient Duration (cv) = Gradient VolumeColumn Volume
©2006 Waters Corporation
Converting MethodsACQUITY UPLC Calculator
©2006 Waters Corporation
Converting MethodsChoosing an ACQUITY UPLC Column
ACQUITY UPLC BEH C18
ACQUITY UPLC BEH C8
ACQUITY UPLC BEH Shield RP18
ACQUITY UPLC BEH Phenyl
USP L11
USP L1
USP L7
USP L1
Revisions are currently under way to formally include 1.7μm particles in the USP listings
©2006 Waters Corporation
Converting MethodsReversed-Phase Column Selectivity Chart
Retentivity(ln [k] acenaphthene)
SunFire™ C18
YMC-Pack™ PolymerC18™
Hypersil® CPS Cyano
YMC-Pack™ CN
Waters Spherisorb® S5 P
Hypersil® BDS PhenylNova-Pak® Phenyl
YMC-Pack™Phenyl
Hypersil® PhenylInertsil® Ph-3
YMC-Pack™ Pro C4™
YMCbasic™
Symmetry® C8YMC-Pack™ Pro C8™
Nova-Pak®C8
XTerra® MS C18 Symmetry® C18
YMC-Pack™Pro C18™
Inertsil® ODS-3
YMC-Pack™ ODS-A™
Nova-Pak®C18
YMC J'sphere™ODS–L80 Nucleosil® C18
Waters Spherisorb® ODS2
Waters Spherisorb® ODS1Resolve® C18
µBondapak® C18
YMC-Pack™ ODS–AQ™
YMC J'sphere™ ODS–H80YMC J'sphere™ ODS–M80
Inertsil® CN-3
Waters Spherisorb® S5CN
Nova-Pak® CN HP
SymmetryShield™ RP8
SymmetryShield™ RP18
XTerra® RP8
XTerra® RP18
-0.6
-0.3
0
0.3
0.6
0.9
1.2
1.5
1.8
2.1
2.4
2.7
3
3.3
3.6
-1.5 -0.5 0.5 1.5 2.5 3.5
Sele
ctiv
ity(ln
[a] a
mitr
ipty
line/
acen
apht
hene
)
XTerra® MS C8
Luna ®C18 (2)
ACQUITY UPLC BEH C18
XTerra ®Phenyl Luna ™
Phenyl Hexyl
ChromolithTM
RP-18
Atlantis® dC18
Zorbax® XDB C18ACT Ace® C18
Zorbax® SB C18
SunFire ™ C8
Luna®C8 (2)
ACQUITY UPLC Shield RP18
ACQUITY UPLC BEH C8
ACQUITY UPLC BEH Phenyl
©2006 Waters Corporation
Converting MethodsReversed-Phase Column Selectivity Chart
©2006 Waters Corporation
Why Redevelop?
• If there are tools available to aid in the method conversion, why redevelop?
• Selectivity differences between column chemistries can make the conversion process difficult.
• Advances in column technologies may allow for dramatic improvements in retention and peak shape
©2006 Waters Corporation
Reversed-Phase Retention MapNote: Column Particle,Temperature and % Organic Held Constant
pH
0
5
10
15
20
25
30
35
40
0 2 4 6 8 10 12
Ret
enti
on F
acto
r (k
) Acid
Base
Neutral
Note: Retention of neutral analytes not affected by pH
Increased, robust base retention
Increased acid retention
Silica pH Range
Hybrid Particle pH Range
Redeveloping MethodsTaking Advantage of New Column Chemistries
New Hybrid Technology allows for high pH retention of bases
Validation of an ACQUITY UPLC Method
©2006 Waters Corporation
Validation Example of a Redeveloped Method
USP Method for a Topical Anesthetic
©2006 Waters Corporation
HPLC Method Conditions
ConditionsSystem: Alliance® XC System
2487 UV/Vis DetectorEmpower™ CDS
Column: μBondapak C18, 3.9 x 300 mm, 10 μm Sample: Topical AnestheticMobile Phase: 500:500:20 Water:Methanol:0.25M 1-heptanesulfonateFlow Rate: 2.0 mL/minInjection Volume: 10 µLNeedlewash: 5:1:1 Acetonitrile:Water:IsopropanolTemperature: 25°CDetection: 313 nmData Rate: 1 HzFilter Constant: 1 sec
©2006 Waters Corporation
Redevelop or Convert?
• Things to Improve:– Poor retention of Benzocaine– Poor peak shape for Tetracaine– Mobile phase uses ion pair reagent
©2006 Waters Corporation
Redeveloped UPLC Method
Ben
zoca
ine
- 0.2
93
But
ambe
n - 0
.654
Tetra
cain
e - 1
.045
AU
0.00
0.25
0.50
0.75
1.00
Minutes0.15 0.30 0.45 0.60 0.75 0.90 1.05 1.20 1.35 1.50
40 minutes by HPLC
1.5 minutes by UPLC
~ 27X Faster
©2006 Waters Corporation
UPLC Method Conditions
ConditionsSystem: ACQUITY UPLC System
Tunable UV Detector (TUV)Empower CDS
Column: ACQUITY UPLC BEH C18, 2.1 x 50 mm, 1.7 μmSample: 0.20 mg/mL Benzocaine
0.030 mg/mL Butamben and Tetracaine HClMobile Phase: 60/40 10 mM Ammonium Bicarbonate pH 10 / AcetonitrileFlow Rate: 1.0 mL/minInjection Volume: 1 µL PLUNO with 5 μL LoopWeak Wash: 60/40 Water/Acetonitrile 1200 μLStrong Wash: 10/90 Water/Acetonitrile 400 μLTemperature: 40 °CDetection: 0.0 – 0.5 min 220 nm
0.5 – 0.9 min 290 nm0.9 – 1.5 min 307 nm
Data Rate: 20 HzFilter Constant: 0.1 sec
©2006 Waters Corporation
Validation of the ACQUITY UPLC Method
Precision
Accuracy
Limit of Detection
Limit of Quantitation
Specificity
Linearity
Robustness
Range
MethodValidation
©2006 Waters Corporation
Accuracy: Definition
• The closeness of test results obtained by the method to the true value.– Established across the range
©2006 Waters Corporation
Accuracy: Determination
• Drug Substance– Analysis of reference material
• Drug Product– Analysis of synthetic mixtures spiked with known quantities
of components
• Impurities (Quantitation)– Analysis of samples (Drug substances/Drug product) spiked
with known amounts of impurities– If impurities are not available, see specificity
• Additional Option: Drug Product/Drug Substance– Compare results to a second, well-characterized method – Determined concurrently with precision, linearity and
specificity
©2006 Waters Corporation
Accuracy: Determination (Cont.)
• Recommended Data– Minimum of 9 determinations over a minimum of 3
concentration levels covering the specified range (e.g. 3 concentrations/3 replicates each)
– Reported as % recovery of known, added amount, or difference between the mean and true value, with confidence intervals
©2006 Waters Corporation
Accuracy/Recovery Results
100.3 ± 1.098.8 ± 0.8101.5 ± 0.9Spiked at 120% of Label
98.8 ± 0.8100.5 ± 0.5100.5 ± 0.6Spiked at 100% of Label
98.8 ± 1.1100.2 ± 1.2100.4 ± 1.2Spiked at 80% of Label
TetracaineButambenBenzocaine
©2006 Waters Corporation
Precision: Definition
• Precision– The measure of the degree of agreement among test results
when the method is applied repeatedly to multiple samplings of ahomogeneous sample
– Expressed as %RSD for a statistically significant number of samples
©2006 Waters Corporation
Precision: Definition/Determination
• Repeatability (Generally the criterion of concern in USP analytical procedures)– Same operating conditions, short time interval– Inter-assay precision
Minimum of 9 determinations covering specified range of procedure (3 levels, 3 reps each), orMinimum of 6 determinations at 100% test conc.
• Intermediate Precision (Experimental design recommended)– Within-lab variations (Random events)– Different days, analysts, equipment
• Reproducibility– Precision between labs– Collaborative studies
©2006 Waters Corporation
Precision - Acceptance Criteria
• Less than 2% relative standard deviation is often recommended.
• Less than 5% RSD can be acceptable for minor components.
• Up to 10% RSD may be acceptable near the limit of quantitation.
©2006 Waters Corporation
Repeatability Results
0.300.280.36%RSD0.00010.00010.0001Std. Dev.0.04520.04570.302Mean
150%
0.620.620.54%RSD0.00020.00020.0001Std. Dev.0.03050.03060.204Mean
100%
0.4850.5610.34%RSD0.00010.00010.0003Std. Dev.0.01500.01520.101Mean
50%
TetracaineButambenBenzocaine
©2006 Waters Corporation
Intermediate Precision Results
0.051.500.70% Diff.
0.220.260.020.360.800.33%RSD
0.0040.0050.0040.0070.030.05Std. Dev.
1.971.961.961.9914.013.9Mean
Analyst 2Analyst 1Analyst 2Analyst 1Analyst 2Analyst 1
Tetracaine %ActiveButamben %ActiveBenzocaine %Active
©2006 Waters Corporation
Reproducibility Results
1.001.511.43% Diff.
1.361.301.080.591.040.51%RSD
0.0270.0260.0210.0120.140.07Std. Dev.
2.002.021.951.9813.814.0Mean
Lab 2Lab 1Lab 2Lab 1Lab 2Lab 1
Tetracaine %ActiveButamben %ActiveBenzocaine %Active
©2006 Waters Corporation
Specificity: Definition
• Specificity (Selectivity)– The ability to measure accurately and specifically the analyte in
the presence of components that may be expected to be present in the matrix
– The degree of interference Active IngredientsExcipientsImpuritiesDegradation ProductsPlacebo Ingredients
©2006 Waters Corporation
Specificity (Selectivity)
• Separation– Resolution
Determination of separation between peaks– Plate Count
Determination of a systems efficiency– Tailing Factor
Calculation referencing peak shape
©2006 Waters Corporation
Specificity: Determination
• Assay– Demonstrate that the results are unaffected by
spiked impurities or excipients (where available)– Compare results to a second well-characterized
procedure– Peak Purity Tests (Diode Array or MS)
©2006 Waters Corporation
Specificity Results
0.603 ± 8.9%0.437 ± 8.9%11.7 ± 0.51.05Tetracaine
0.419 ± 7.7%0.252 ± 9.0%12.9 ± 0.90.66Butamben
2.612 ± 8.4%2.117 ± 15.5%4.5 ± 1.40.37Unknown 2
0.604 ± 2.1%0.225 ± 11.5%11.3 ± 1.80.30Benzocaine
0.498 ± 5.0%3.611 ± 8.9%---0.14Unknown 1
Purity Threshold
Purity AngleResolutionRetention Time (min)
Peak
Results for 6 Replicate Injections
Purity Angle was below the Purity Threshold for all major peaks indicating purity.
Blank injections demonstrated no co-elutions
©2006 Waters Corporation
Specificity Results0.314 min
166.2
Inte
nsity
0
6
6
6
9x106
0.690 min194.3
Inte
nsity
0.0
2.0x106
1.118 min265.4
Inte
nsity
0.0
3.0x10 7
m/z100.00 120.00 140.00 160.00 180.00 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
Benzocaine
Butamben
Tetracaine
MS Data indicated peak purity
©2006 Waters Corporation
Linearity and Range: Definition
• Linearity– The ability of the method to elicit test results that are directly
proportional to concentration within a given range– Expressed as the variance of the slope of the regression line
• Range – Interval between upper and lower levels of analyte
demonstrated by the method– Precision and Accuracy expressed in the same units as the
test results
©2006 Waters Corporation
Linearity: Determination
• Established across the Range of the method– Dilutions– Separate Weighings
• Evaluate by Appropriate Statistical Methods (e.g. Regression)– Include Correlation Coefficient, y-Intercept, Slope, Residual Sum
of Squares, Plot Itself
• Minimum 5 Concentrations
©2006 Waters Corporation
Determination of Appropriate Range
• Minimum Specified Ranges– Assay
80-120%– Impurity Test
From QL to 120% of spec.Toxic or more potent impurities: commensurate with the controlled level
– Content Uniformity70-130% of test concentration
– Dissolution Testing+/- 20% over specified range
©2006 Waters Corporation
Linearity and Range Results
Y = 4.24e+006 X – 2.09e+0020.16%0.999883TetracaineY = 4.71e+006 X + 5.39e+0020.37%0.999897ButambenY = 2.49e+006 X + 5.47e+0031.08%0.999855Benzocaine
EquationY-Intercept %Difference
R2Name
Area
0
200000
400000
600000
800000
Amount0.000 0.032 0.064 0.096 0.128 0.160 0.192 0.224 0.256 0.288
BenzocaineAr
ea
0
60000
120000
180000
240000
Amount0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 0.040 0.045
Butamben
Tetracaine
©2006 Waters Corporation
Linearity and Range Results
• Residuals plot indicates linearity
-4
-3
-2
-1
0
1
2
3
4
1 2 3 4 5
Standard Level
Resi
dual
Dev
iatio
n (%
)
©2006 Waters Corporation
Robustness: Definition
• Robustness– Measure of the capacity to remain unaffected by small
(deliberate) variations in method parameters– Indication of reliability during normal use– Evaluate during method development– Used to set system suitability specifications
©2006 Waters Corporation
Robustness: Determination
• Consider during development of method
• Shows reliability of method with respect to deliberate changes
• If measurements are susceptible to variations in analytical procedures, these conditions should be controlled and a precautionary statement included.
• Establish System Suitability parameters to ensure the validity of the method
©2006 Waters Corporation
Robustness Parameters
2, 105Sample Prep Shake Time (min)9.0, 9.5, 10.5, 11.010.0Buffer pH
8, 9, 11, 1210Buffer Concentration (mM)50/50, 55/45, 65/35, 70/3060/40Mobile Phase Composition
0.8, 0.9, 1.1, 1.21.0Injection Volume (μL)38, 39, 41, 4240Column Temperature (°C)
0.90, 0.95, 1.05, 1.101.00Flow Rate (mL/min)225, 295, 312, and 215, 285, 302220, 290, 307Wavelength (nm)
Modified ConditionsSpecified ConditionsParameter
Only condition which caused a variation of more than 2.0% was a Column Temperature of 38 °C.
©2006 Waters Corporation
System Suitability
• System Suitability– The checking of a system, before or during analysis of unknowns, to
insure system performance.“No sample analysis is acceptable unless the requirements for system suitability have been met.” (USP Chapter 621)
– Plate Count, Tailing, Resolution– Determination of reproducibility (%RSD)
For %RSD < 2.0%, Five replicatesFor %RSD > 2.0%, Six replicates
• System Suitability "Sample"– A mixture of main components and expected by-products utilized to
determine system suitability
• “Whenever There is a Significant change in Equipment or ReagentsSystem Suitability Testing Should be Performed” (USP Chapter 621)
©2006 Waters Corporation
Recommendations From FDA 1994 Guideline: System Suitability
• Capacity factor – k' > 2
• Precision/Injection repeatability– RSD </= 1%, n >/= 5
• Resolution– Rs >/= 2 (Major peak and
closest eluting)
• Tailing factor– T </= 2
• Theoretical Plates– In general N > 2000
©2006 Waters Corporation
System Suitability Results
Ben
zoca
ine
- 0.2
93
Buta
mbe
n - 0
.654
Tetra
cain
e - 1
.045
AU
0.00
0.25
0.50
0.75
1.00
Minutes0.15 0.30 0.45 0.60 0.75 0.90 1.05 1.20 1.35 1.50
System Suitability RequirementsCapacity Factor > 1.00Repeatability (n=6) < 1.00%Resolution > 3.0Tailing Factor < 1.30Theoretical Plates > 5,000
BenzocaineCapacity Factor = 1.10 Repeatability = 0.54%Resolution = 10.8Tailing Factor = 1.18Theoretical Plates = 5,800
ButambenCapacity Factor = 3.67Repeatability = 0.62%Resolution = 12.7Tailing Factor = 1.08Theoretical Plates = 9,800
TetracaineCapacity Factor = 6.47Repeatability = 0.62%Resolution = 11.6Tailing Factor = 1.05Theoretical Plates = 10,300
Method Validation Manager
©2006 Waters Corporation
With Method Validation Manager you can automatically…
• Manage method validation workflow in one comprehensive, automated application
• Clearly display the status of on-going validation studies – enabling you to see at what step each individual validation parameter is in the method validation process
• Perform all results and statistical calculations in Empower 2, eliminating time-consuming data transfer to spreadsheets and the associated problems of transcription errors and security concerns
• Perform multi-component analyses and batch processing of method validation results
• Generate reports with standardized templates
©2006 Waters Corporation
Analytical Method Validation Process
Corporate Method Validation
SOP
PrepareStandards & Samples
Data Acquisition& Processing
Data Management
Create SampleSequence
Calculation Statistical Results
Reports Compiled
Time consuming, repetitive tasks consisting of several sequential steps
©2006 Waters Corporation
Issues with Existing Process
• Constant referral of SOP to determine next step
• No way to determine test status
• Possibilities of collected duplicate data
• No traceability of statistical results back to chromatographic data
• Manual and error prone process
• Multiple data transfer steps to multiple 3rd applications
• Additional validation requirements for 3rd
applications
©2006 Waters Corporation
Analytical Method Validation Processwith Method Validation Manager
Corporate Method Validation
SOP
PrepareStandards & Samples
Data Acquisition& Processing
Data Management
Create SampleSequence
Calculation Statistical Results
Reports Compiled
Time consuming, repetitive tasks consisting of several sequential steps
MethodValidationManager
Faster and Easier Method Validation
©2006 Waters Corporation
Save Time / Entire Process Less Error Prone– Data management is handled by Empower, not by user– Automatic data checks performed at each step of the workflow– Data approvals can be configured at each step of the workflow– Calculations done in Empower
No transfer to spreadsheets or other softwareNo transcription error / No need to check data transferNo need to validate spreadsheet functionsMulti-component analysis and batch processing of validation results
– Report templates can be used to standardize the report format Automatic report generationEase of Review
Benefits of Method Validation Manager
©2006 Waters Corporation
Assure Regulatory Compliance (A huge concern with current validation practices)– No spreadsheets or data transfer and checking required– No concern regarding security of spreadsheets– No 3rd party statistical software required – Privileges control user activities– Structurally validated calculations– Results are secure in the Empower database– Audit trails of user activity– Data management performed by Empower, not by the user
Data mining is easy
Benefits of Method Validation Manager
©2006 Waters Corporation
Validating ACQUITY UPLC Methods
• Methods can easily be validated using the ACQUITY UPLC System
• Significant return on investment can be realized by adopting UPLC and validating new and existing methods on the ACQUITY UPLC System
• Method Validation Manager easily manages work flow in one comprehensive automated application
©2006 Waters Corporation
Acknowledgements
• Katherine Hynes
• Michael D. Jones
• Mark Benvenuti
• Lauren Wood
©2006 Waters Corporation
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