Validation of ACQUITY UPLC™ Methods · • Changes in Incoming Raw Material • Changes in Method • To Take Advantage of New Technology – Cost/Benefit Economic Exercise –

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


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."


The Process of Validation

Methods Method

System System

Suitability

Validation

Software

Hardware


USP Analytical Performance Characteristics

Precision

Accuracy

Limit of Detection

Limit of Quantitation

Specificity

Linearity

Robustness

Range

MethodValidation


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.


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!"


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


Smaller ParticlesThe Enabler of Productivity

Optimal velocity range


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


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


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!


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


Converting MethodsACQUITY UPLC Calculator


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


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


Converting MethodsReversed-Phase Column Selectivity Chart


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


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


Validation Example of a Redeveloped Method

USP Method for a Topical Anesthetic


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


Redevelop or Convert?

• Things to Improve:– Poor retention of Benzocaine– Poor peak shape for Tetracaine– Mobile phase uses ion pair reagent


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


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


Validation of the ACQUITY UPLC Method

Precision

Accuracy

Limit of Detection

Limit of Quantitation

Specificity

Linearity

Robustness

Range

MethodValidation


Accuracy: Definition

• The closeness of test results obtained by the method to the true value.– Established across the range


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


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


Accuracy/Recovery Results

100.3 ± 1.098.8 ± 0.8101.5 ± 0.9Spiked at 120% of Label



TetracaineButambenBenzocaine


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


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


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.


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


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


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


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


Specificity (Selectivity)

• Separation– Resolution

Determination of separation between peaks– Plate Count

Determination of a systems efficiency– Tailing Factor

Calculation referencing peak shape


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)


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


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


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


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


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


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


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 (%

)


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


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


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.


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)


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


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


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


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


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


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


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


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


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


Acknowledgements

• Katherine Hynes

• Michael D. Jones

• Mark Benvenuti

• Lauren Wood


Download Waters presentations at www.waters.com/slides

Documents

Validation of ACQUITY UPLC™ Methods · • Changes in Incoming Raw Material • Changes in Method • To Take Advantage of New Technology – Cost/Benefit Economic Exercise –