Tanya Jenkins Andrew Aubin Dr. Michael Swartz Waters Corporation 34 Maple Street Milford, MA, 01757,USA Validation of ACQUITY UPLC Methods
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 Hardware Methods Method Validation Software System System Suitability
USP Analytical Performance Characteristics Precision Accuracy Limit of Detection Method Validation Limit of Quantitation Specificity Linearity Range Robustness
Validation Characteristics Vs. Type of Analytical Method Analytical Performance Parameter Category 1: Assays Category 2: Impurities Category 3: Quant. Limit Tests Specific Tests Category 4: I.D. Accuracy Yes Yes * * No Precision Yes Yes No Yes No Specificity Yes Yes Yes * Yes LOD No No Yes * No LOQ No Yes No * No Linearity Yes Yes No * No Range Yes Yes No * No Robustness Yes Yes No Yes No * May be required, depending on the nature of the specific test.
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 Particles The Enabler of Productivity Optimal velocity range
Same Resolution and Selectivity with Increased Speed - Constant L / dp AU AU AU AU 0.20 0.10 0.00 0.00 2.00 4.00 6.00 8.00 Minutes 0.20 0.10 0.00 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 Minutes 0.20 0.10 0.00 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 Minutes 0.20 0.10 0.00 0.00 0.20 0.40 0.60 0.80 1.00 Minutes 10.00 4.00 2.00 1.10 5 µm 150 mm F = 200 µl/min Injection = 5.0 µl Rs (2,3) = 2.28 3.5 µm 100 mm F = 300 µl/min Injection = 3.3 µl Rs (2,3) = 2.32 2.5 µm 75 mm F = 500 µl/min Injection = 2.5 µl Rs (2,3) = 2.34 1.7 µm 50 mm F = 600 µl/min Injection = 1.7 µl Rs (2,3) = 2.29 Improvement Resolution Same Speed 9X Pressure 9X Improvement Resolution Same Speed 3X Pressure 3X
HPLC vs. UPLC Speed, Sensitivity and Resolution 0.26 Absorbance at 270 nm HPLC 1 2.1 x 150 mm, 5 µm Rs (2,3) = 4.29 2 3 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 Minutes 20.00 Faster, More Sensitive Methods Faster, More Sensitive, Higher Resolution Methods 0.26 Absorbance at 270 nm UPLC 1 8X Speed 3.4X Sensitivity Same Resolution 2.1 x 50 mm, 1.7 µm Rs (2,3) = 4.28 2 3 0.26 Absorbance at 270 nm UPLC 4.5X Speed 2X Sensitivity 1.5X Resolution 1 2.1 x 100 mm, 1.7 µm Rs (2,3) = 6.38 2 3 0.00 0.40 0.80 1.20 Minutes 1.60 2.00 2.50 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 Minutes 4.50
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!
Target injection volume = Converting Methods Geometrically Scaling from HPLC to UPLC Original injection volume X Target Column Volume Original Column Volume Target Flow Rate = Original Flow Rate x d 2 Target d 2 Original Gradient Volume = Flow Rate x Time Column Volume = π x r 2 x L Gradient Duration (cv) = Gradient Volume Column Volume
Converting Methods ACQUITY UPLC Calculator
Converting Methods Choosing an ACQUITY UPLC Column ACQUITY UPLC BEH C18 USP L1 ACQUITY UPLC BEH C8 USP L7 ACQUITY UPLC BEH Shield RP18 USP L1 ACQUITY UPLC BEH Phenyl USP L11 Revisions are currently under way to formally include 1.7μm particles in the USP listings
Converting Methods Reversed-Phase Column Selectivity Chart 3.6 Waters Spherisorb S5 P Selectivity (ln [a] amitriptyline/acenaphthene) 3.3 3 2.7 2.4 2.1 1.8 1.5 1.2 0.9 0.6 0.3 0-0.3-0.6 Waters Spherisorb S5CN Nova-Pak CN HP Hypersil CPS Cyano Inertsil CN-3 Inertsil Ph-3 Hypersil Phenyl YMC-Pack Phenyl Nova-Pak Phenyl Hypersil BDS Phenyl YMC J'sphere ODS M80 YMCbasic Chromolith Nova-Pak YMC J'sphere ODS H80 XTerra TM RP-18 C18 Phenyl Nova-Pak Luna YMC-Pack ODS AQ YMC-Pack CN YMC-Pack Pro C4 C8 Phenyl Hexyl Atlantis dc18 YMC-Pack Pro C8 YMC-Pack ODS-A ACQUITY UPLC ACT Ace C18 Symmetry C8 Zorbax XDB C18 XTerra Luna BEH C8 MS C8 YMC-Pack Inertsil ODS-3 C8 (2) Pro C18 SunFire C18 XTerra Luna SunFire C8 MS C18 C18 Symmetry C18 SymmetryShield RP8 (2) XTerra RP18 Zorbax SB C18 SymmetryShield RP18 XTerra RP8 YMC-Pack PolymerC18 Retentivity (ln [k] acenaphthene) ACQUITY UPLC BEH Phenyl YMC J'sphere ODS L80 µbondapak C18 Waters Spherisorb ODS1 Resolve C18 Waters Spherisorb ODS2 Nucleosil C18 ACQUITY UPLC BEH C18-1.5-0.5 0.5 1.5 2.5 3.5 ACQUITY UPLC Shield RP18
Converting Methods Reversed-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
Redeveloping Methods Taking Advantage of New Column Chemistries New Hybrid Technology allows for high ph retention of bases Retention Factor (k) 40 35 30 25 20 15 10 Reversed-Phase Retention Map Note: Retention of neutral analytes not affected by ph Acid Increased acid retention Neutral Increased, robust base retention Note: Column Particle, Temperature and % Organic Held Constant 5 0 Base 0 2 4 6 8 10 12 ph Silica ph Range Hybrid Particle ph Range
Validation of an ACQUITY UPLC Method
Validation Example of a Redeveloped Method USP Method for a Topical Anesthetic
HPLC Method Conditions Conditions System: Alliance XC System 2487 UV/Vis Detector Empower CDS μbondapak C18, 3.9 x 300 mm, 10 μm Topical Anesthetic 500:500:20 Water:Methanol:0.25M 1-heptanesulfonate 2.0 ml/min Column: Sample: Mobile Phase: Flow Rate: Injection Volume: 10 µl Needlewash: 5:1:1 Acetonitrile:Water:Isopropanol Temperature: 25 C Detection: 313 nm Data Rate: 1 Hz Filter 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 1.00 0.75 Benzocaine - 0.293 40 minutes by HPLC ~ 27X Faster 1.5 minutes by UPLC AU 0.50 0.25 Butamben - 0.654 Tetracaine - 1.045 0.00 0.15 0.30 0.45 0.60 0.75 0.90 1.05 1.20 1.35 1.50 Minutes
Conditions System: Column: Sample: Mobile Phase: Flow Rate: Injection Volume: Weak Wash: Strong Wash: Temperature: 40 C Detection: Data Rate: Filter Constant: UPLC Method Conditions ACQUITY UPLC System Tunable UV Detector (TUV) Empower CDS ACQUITY UPLC BEH C 18, 2.1 x 50 mm, 1.7 μm 0.20 mg/ml Benzocaine 0.030 mg/ml Butamben and Tetracaine HCl 60/40 10 mm Ammonium Bicarbonate ph 10 / Acetonitrile 1.0 ml/min 1 µl PLUNO with 5 μl Loop 60/40 Water/Acetonitrile 1200 μl 10/90 Water/Acetonitrile 400 μl 0.0 0.5 min 220 nm 0.5 0.9 min 290 nm 0.9 1.5 min 307 nm 20 Hz 0.1 sec
Validation of the ACQUITY UPLC Method Precision Accuracy Limit of Detection Method Validation Limit of Quantitation Specificity Linearity Range Robustness
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 Benzocaine Butamben Tetracaine Spiked at 80% of Label 100.4 ± 1.2 100.2 ± 1.2 98.8 ± 1.1 Spiked at 100% of Label 100.5 ± 0.6 100.5 ± 0.5 98.8 ± 0.8 Spiked at 120% of Label 101.5 ± 0.9 98.8 ± 0.8 100.3 ± 1.0
Precision: Definition Precision The measure of the degree of agreement among test results when the method is applied repeatedly to multiple samplings of a homogeneous 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), or Minimum 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 Benzocaine Butamben Tetracaine Mean 0.101 0.0152 0.0150 50% Std. Dev. %RSD 0.0003 0.34 0.0001 0.561 0.0001 0.485 Mean 0.204 0.0306 0.0305 100% Std. Dev. %RSD 0.0001 0.54 0.0002 0.62 0.0002 0.62 Mean 0.302 0.0457 0.0452 150% Std. Dev. %RSD 0.0001 0.36 0.0001 0.28 0.0001 0.30
Intermediate Precision Results Benzocaine %Active Butamben %Active Tetracaine %Active Analyst 1 Analyst 2 Analyst 1 Analyst 2 Analyst 1 Analyst 2 Mean 13.9 14.0 1.99 1.96 1.96 1.97 Std. Dev. 0.05 0.03 0.007 0.004 0.005 0.004 %RSD 0.33 0.80 0.36 0.02 0.26 0.22 % Diff. 0.70 1.50 0.05
Reproducibility Results Benzocaine %Active Butamben %Active Tetracaine %Active Lab 1 Lab 2 Lab 1 Lab 2 Lab 1 Lab 2 Mean 14.0 13.8 1.98 1.95 2.02 2.00 Std. Dev. 0.07 0.14 0.012 0.021 0.026 0.027 %RSD 0.51 1.04 0.59 1.08 1.30 1.36 % Diff. 1.43 1.51 1.00
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 Ingredients Excipients Impurities Degradation Products Placebo 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 Results for 6 Replicate Injections Peak Retention Time (min) Resolution Purity Angle Purity Threshold Unknown 1 0.14 --- 3.611 ± 8.9% 0.498 ± 5.0% Benzocaine 0.30 11.3 ± 1.8 0.225 ± 11.5% 0.604 ± 2.1% Unknown 2 0.37 4.5 ± 1.4 2.117 ± 15.5% 2.612 ± 8.4% Butamben 0.66 12.9 ± 0.9 0.252 ± 9.0% 0.419 ± 7.7% Tetracaine 1.05 11.7 ± 0.5 0.437 ± 8.9% 0.603 ± 8.9% Purity Angle was below the Purity Threshold for all major peaks indicating purity. Blank injections demonstrated no co-elutions
Specificity Results 9x10 6 0.314 min 166.2 Benzocaine Intensity 6 6 6 2.0x10 0 6 0.690 min 194.3 Butamben Intensity 3.0x10 0.0 7 1.118 min 265.4 Tetracaine Intensity 0.0 100.00 120.00 140.00 160.00 180.00 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 m/z 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 Uniformity 70-130% of test concentration Dissolution Testing +/- 20% over specified range
Linearity and Range Results Name R 2 Y-Intercept %Difference Equation Benzocaine 0.999855 1.08% Y = 2.49e+006 X + 5.47e+003 Butamben 0.999897 0.37% Y = 4.71e+006 X + 5.39e+002 Tetracaine 0.999883 0.16% Y = 4.24e+006 X 2.09e+002 800000 240000 Area 600000 400000 Benzocaine Area 180000 120000 Butamben Tetracaine 200000 60000 0 0.000 0.032 0.064 0.096 0.128 0.160 0.192 0.224 0.256 0.288 Amount 0 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 0.040 0.045 Amount
Linearity and Range Results Residuals plot indicates linearity 4 3 Residual Deviation (%) 2 1 0-1 -2-3 -4 1 2 3 4 5 Standard Level
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 Parameter Wavelength (nm) Flow Rate (ml/min) Column Temperature ( C) Injection Volume (μl) Mobile Phase Composition Buffer Concentration (mm) Buffer ph Sample Prep Shake Time (min) Specified Conditions 220, 290, 307 1.00 40 1.0 60/40 10 10.0 5 Modified Conditions 225, 295, 312, and 215, 285, 302 0.90, 0.95, 1.05, 1.10 38, 39, 41, 42 0.8, 0.9, 1.1, 1.2 50/50, 55/45, 65/35, 70/30 8, 9, 11, 12 9.0, 9.5, 10.5, 11.0 2, 10 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 replicates For %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 Reagents System 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 Tailing factor T </= 2 Theoretical Plates In general N > 2000 Resolution Rs >/= 2 (Major peak and closest eluting)
System Suitability Results 1.00 0.75 Benzocaine - 0.293 Benzocaine Capacity Factor = 1.10 Repeatability = 0.54% Resolution = 10.8 Tailing Factor = 1.18 Theoretical Plates = 5,800 System Suitability Requirements Capacity Factor > 1.00 Repeatability (n=6) < 1.00% Resolution > 3.0 Tailing Factor < 1.30 Theoretical Plates > 5,000 AU 0.50 0.25 Butamben - 0.654 Butamben Capacity Factor = 3.67 Repeatability = 0.62% Resolution = 12.7 Tailing Factor = 1.08 Theoretical Plates = 9,800 Tetracaine - 1.045 Tetracaine Capacity Factor = 6.47 Repeatability = 0.62% Resolution = 11.6 Tailing Factor = 1.05 Theoretical Plates = 10,300 0.00 0.15 0.30 0.45 0.60 0.75 0.90 1.05 1.20 1.35 1.50 Minutes
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 Create Sample Sequence Data Acquisition & Processing Calculation Statistical Results Reports Compiled Prepare Standards & Samples Corporate Method Validation SOP Time consuming, repetitive tasks consisting of several sequential steps Data Management
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 3 rd applications Additional validation requirements for 3 rd applications
Analytical Method Validation Process with Method Validation Manager Create Sample Sequence Data Acquisition & Processing Calculation Statistical Results Reports Compiled Prepare Standards & Samples Corporate Method Method Validation Validation Manager SOP Time consuming, repetitive tasks consisting Faster and Easier Method Validation of several sequential steps Data Management
Benefits of Method Validation Manager 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 software No transcription error / No need to check data transfer No need to validate spreadsheet functions Multi-component analysis and batch processing of validation results Report templates can be used to standardize the report format Automatic report generation Ease 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 3 rd 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
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
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