Agenda. IVT Method Validation Conference July 28-30, 2010 Sir Francis Drake Hotel San Francisco, CA

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1 IVT Method Validation Conference July 28-30, 2010 Sir Francis Drake Hotel San Francisco, CA Analytical Instrument Qualification (AIQ) Paul St. Jean Product Marketing Manager Global Compliance Programs Waters Corporation Milford, MA USA 2010 Waters Corporation Agenda Regulatory Expectations for Qualification Terms Warning Letters USP <1058> AIQ Guidance Chapter How it came about Developing a roadmap for the AIQ Process DQ IQ OQ PQ and more Instrument Qualification Case Study Application Developing an understanding of what s involved in qualification Examining various approaches Can we be more efficient? Interactive Exercise Discuss real challenges in your lab Group Q and A 2010 Waters Corporation 2 1

2 I Regulatory Expectations for Qualification 2010 Waters Corporation 3 Data Integrity Objective Data that supports the design, development and production of government regulated products must be accurate and defensible Waters Corporation 4 2

3 Calibration Requirements CFR cgmp Equipment Automatic, Mechanical and Electronic Equipment. equipment so shall be routinely calibrated, inspected or checked according to a written program designed to assure proper performance. Written records of these calibration checks and inspections shall be maintained Waters Corporation 5 Calibration Requirements CFR General Requirements Laboratory controls shall include The calibration of instruments, apparatus gauges and recording devices at suitable intervals in accordance with an established written program containing specific directions, schedules and limits for accuracy and precision 2010 Waters Corporation 6 3

4 Validation versus Qualification VALIDATION: Refers to the total life cycle of a product from development through use and maintenance. Customers (Owners) are responsible for Validating Their Processes (personnel, equipment, methods, SOPs) to ensure compliance to CGMP/GLP regulations. QUALIFICATION: (Inspection,functional testing and documentation review) Is a part of the validation process which verifies module and system functional performance prior to being placed on-line and thereafter according to a standard operating procedure. Calibration verification is included as part of qualification. Vendors can assist customers in meeting their validation requirements but cannot validate customers processes Waters Corporation 7 Validation versus Qualification According to an FDA inspector.. QUALIFICATION: Answers the question.. Was the instrument built right? VALIDATION: Answers the question.. Was the right instrument built? Vendors are in the best position to verify proper instrument operation Only users can determine if a system is suitable for their analysis 2010 Waters Corporation 8 4

5 Validation versus Qualification According to an USP Instruments are QUALIFIED Processes are VALIDATATED It is easiest to qualify instruments using standardized tests Procedures and methods are validated for each specific analysis 2010 Waters Corporation 9 FDA Guideline Provide Documented Evidence of HPLC System Accuracy, Linearity and Precision o from: Validation of Computerized Liquid Chromatographic Systems Furman, W.B., Layloff, T.P. & Tetzalff, R.F. August, 1992 o This may be old, but it is still valid and still gets occasional mention in journals Regulations tell us what to do, but not how to do it o CFR o CFR Waters Corporation 10 5

6 Qualification Time Line Equipment used should be of Appropriate Design, Adequate Capacity and Properly Maintained. Vendor's Site Owner's Site Owner's Site Structurally Validated Products DQ Before Purchase Installation IQ Functional Testing/Verification Qualification Operational OQ Performance PQ Maintenance Calibration PQ (Initial Calibration) Before Use After Use System Suitability During Use 2010 Waters Corporation 11 Method Validation As you know, Method Validation is another area that regulated users must be concerned with but we should not confuse method validation with system qualification Methods should be validated on qualified systems Qualification shows that the module or system performs as the vendor intended Qualification is not intended as a complete re-validation of the design but rather a way to provide documented evidence of proper system performance in terms of accuracy, linearity (accuracy across a range) and precision Qualification does not demonstrate suitability for intended use Method validation shows suitability of a system to perform a specific analysis 2010 Waters Corporation 12 6

7 ICH International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (European Union, Japan and USA) Purpose: Harmonization of interpretation / application of technical guidelines and requirements for product registration Reduce or obviate need for duplicate testing during the research and development of new medicines Objective: Economical use of human, animal and material resources Eliminate unnecessary development delays Sections of interest related to this talk: Web site is Q7 Good Manufacturing Practice for Active Pharmaceutical Ingredients Q9 Quality Risk Management 2010 Waters Corporation Warning Letters Issued Personnel Issues (21 CFR ) Analysts not qualified Problems traced to inadequate training No training records No list of approved computer system users (21 CFR ) Analytical Method Issues (21 CFR ) Analytical methods not validated 2010 Waters Corporation 14 7

8 483 Warning Letters Issued Data Audit Trail and Software Issues No computer system validation report (211.68) Software not revision controlled (211.68) No audit trails (21 CFR Part 11) Lack of audit trails (electronic) proving retention of all raw data files ( & 21 CFR Part 11) Equipment Issues No equipment PM records (211.67) Un-qualified computer systems (211.68) Failure to calibrate no written program (211.68) Instruments not qualified/calibrated ( ) Calibration not conducted at suitable intervals ( ) 2010 Waters Corporation 15 II USP <1058> Analytical Instrument Qualification (AIQ) Guidance Document 2010 Waters Corporation 16 8

9 INTRODUCTION 2010 Waters Corporation 17 Workshop on AIQ AAPS Workshop co-sponsored with FIP and ISPE Scientific Approach to Analytical Instrument Validation, o Held in Arlington, VA, March 3-5, 2003 Proceedings are published and entered in FDA Docket Bansal SK, Layloff T, Bush ED, Hamilton M, Hankinson EA, Landy JS, Lowes S, Nasr MM, St. Jean PA, Shah VP. Qualification of Analytical Instruments for Use in the Pharmaceutical Industry: A Scientific Approach. AAPS PharmSciTech. 2004; 5(1): article 22. Proceedings form Basis of new USP Guidance <1058> Analytical Instrument Qualification o Pharmacopeial Forum Vol. 31(4) July-Aug Waters Corporation 18 9

10 Terminology Ambiguous terms: Validation or Qualification Agreed at the workshop that Processes are validated Instruments are qualified AIQ (Analytical Instrument Qualification) AIQ Is documented evidence that an instrument performs suitably for its intended purpose and that it is properly maintained and calibrated Does not include people (training), processes performed on instruments (analytical methods) Helps to justify the continued use of instrument, but it alone does not ensure quality of the data 2010 Waters Corporation 19 Components of Data Quality Quality Control Checks System Suitability Tests Analytical Methods Validation Analytical Instrument Qualification 2010 Waters Corporation 20 10

11 Component of Data Quality: Analytical Method Validation Documented evidence that an analytical method does what it purports to do and addresses the required attributes of the method Use of a validated method should instill confidence that the method can generate data of acceptable quality 2010 Waters Corporation 21 Component of Data Quality: System Suitability Tests Equipment, electronics, software, analytical operations and samples to be analyzed constitute an integral system SST verifies the system s performance according to the analyst s expectation and criteria set forth in the method H/W, S/W and analysis checks to provide assurance of system integrity before, during and following analysis of unknown samples System Precision Separation Parameters System suitability testing alone is not sufficient, Qualification is still required Waters Corporation 22 11

12 Component of Data Quality: Quality Control Checks Most analyses are performed on calibrated or standardized instruments Single or multi-point calibration performed Additional quality control check samples used in some analyses (e.g. bioanalyses) provide an in-process assurance of the test s suitable performance 2010 Waters Corporation 23 Extent of SST or QC checks Chemical analyses subject to GMP regulations, requiring tighter precision and accuracy may require more SST Bioanalyses, largely subject to GLP regulations, requires low level and broad range analysis, is performed with more QC checks during sample analysis 2010 Waters Corporation 24 12

13 Components of Data Quality Quality Control Checks System Suitability Tests Analytical Methods Validation Analytical Instrument Qualification 2010 Waters Corporation 25 III Developing a Roadmap for the AIQ Process 2010 Waters Corporation 26 13

14 PROCEDURES 2010 Waters Corporation 27 Performing AIQ Essential Parameters Several options were discussed, but DQ/IQ/OQ/PQ model was preferred Most widely understood parameters Need to provide AIQ specific definitions and scope for these parameters Recommended set of parameters for AIQ: o Design Qualification (DQ) o Installation Qualification (IQ) o Operational Qualification (OQ) o Performance Qualification (PQ) o Change Control 2010 Waters Corporation 28 14

15 Design Qualification Needed when designing new instrument Most suitably Performed by developer/manufacturer Not necessary for user to repeat DQ Users should ensure: The instrument is fit for their intended use Manufacturer has adopted a quality system for development, manufacturing and testing Manufacturer has adequate support for installation, service and training Procedure Depends on the intended use, complexity of instrument, acceptability of instrument in industry and prior experience Fulfilled by vendor documentation/audits, peer reviews and testing 2010 Waters Corporation 29 Installation Qualification IQ is a documented collection of activities needed to install the instrument in the environment where it will operate System Description Delivery: Did we receive what was ordered? Facility/Environment/Utilities Network and Data Storage Assembly and Installation Installation Verification IQ can be performed for new, pre-owned or existing unqualified instruments 2010 Waters Corporation 30 15

16 Operational Qualification OQ Parameters: Test operation of instrument as per specifications in the user s environment Test critical parameters to assure required performance Parameters based on manufacturer s recommendation and on user s intended use Secure data storage, backup and archive process Performance of OQ Can perform holistic or modular Modular when applicable Modular approach will allow mix and match of modules in a system Use non-method specific testing Repeat relevant OQ tests when instrument undergoes major repairs or modifications 2010 Waters Corporation 31 Performance Qualification PQ tests are performed on a periodic basis to ensure that the instrument remains in qualified state after IQ/OQ Run tests to check and verify satisfactory performance of the instrument Preventive maintenance and repairs Maintain an SOP for operation/calibration/maintenance Performance of PQ Perform at specified intervals (Same tests to generate history of instrument performance) Can perform holistic (preferred) or modular Specifications for tests can be broader than for OQ 2010 Waters Corporation 32 16

17 Timing and Applicability DQ IQ OQ PQ Prior to purchase of a new type of instrument At installation of each instrument (new, old or existing unqualified) After installation or major repair of each instrument Periodically at specified intervals for each instrument 2010 Waters Corporation 33 Qualification Activities DQ IQ OQ PQ Assurance of vendor s DQ System description Fixed parameters Preventive maintenance and repairs Assurance of adequate support availability from manufacturer Instrument Delivery SOPs operation, calibration and maintenance Instrument s fitness for use in laboratory Utilities/facility/ environment Network and data storage Secure data storage, backup and archive Assembly and installation Installation verification Instrument Function Tests Performance Checks 2010 Waters Corporation 34 17

18 Software Validation Software for analytical work classified into the following categories Firmware (low-level software for integrated chips in instruments) o Considered a component of the instrument itself o Qualified at the user site simultaneously with qualification of instrument Instrument control, data acquisition and processing software o Hardware and software are inextricably intertwined and both are necessary for the operation of the instrument o Manufacturers perform the validation and provide users a summary o User perform holistic qualification involving entire instrument and software system through AIQ process 2010 Waters Corporation 35 Software Validation (Contd.) Stand-alone software (e.g. LIMS) o An authoritative guide for validating software is available: General Principles of Software Validation, Final Guidance for Industry and FDA Staff, Jan. 11, 2002 o Software developer specifies the development model appropriate for the software o Validation takes place in a series of activities planned and executed through various stages of development cycle o User site testing is performed as an essential part of the software development cycle o User testing, though essential, is only a part of the full validation 2010 Waters Corporation 36 18

19 Change Control Changes are inevitable as new features are added and corrections are made Follow DQ/IQ/OQ/PQ classification process DQ: Review the change; adopt only useful or necessary changes IQ: Install the changes OQ/PQ: o Revise tests and specifications as necessitated by the change o Change SOP as necessary o Perform changed OQ or PQ tests 2010 Waters Corporation 37 AIQ Documentation Static Documents: Obtained during DQ, IQ and OQ phases For multiple instruments place common documents in one binder or section and instrument specific documents in separate binders or sections Dynamic Documents: Obtained during OQ and PQ phase during instrument maintenance or performance check Provide a running record of instrument performance 2010 Waters Corporation 38 19

20 Instrument Categories for AIQ A single set of principles to qualify the wide variety of instruments is scientifically inappropriate Three categories of instruments are suggested Users are most qualified to establish the level of qualification needed Exact category of an instrument should be determined by the user for their specific instrument or application Modern Laboratories typically have a suite of instruments from simple to complex automated 2010 Waters Corporation 39 Instrument Categories for AIQ : Group A Conformance to user requirements determined by visual observations No independent qualification required Example instruments: o Magnetic Stirrers o Nitrogen evaporators o Ovens o Vortex mixers o Spatula o Glass pipettes o Mortar and pestle 2010 Waters Corporation 40 20

21 Instrument Categories for AIQ : Group B Conformance to user requirements is generally unambiguous and established according to instrument s SOPs. Causes of failure are discernible by simple observations. Example Instruments: o Balances o ph meters o Variable pipettes o Refrigerator/Freezers o Thermometers o Viscometers o IR spectrometers o Titrators o Melting point apparatus 2010 Waters Corporation 41 Instrument Categories for AIQ : Group C Conformance is complex, highly method specific and the conformity bounds are determined by their application. A full qualification process applies. Example Instruments: o HPLC o Mass spectrometers o GC o Diode-array detectors o Atomic absorption spectrometers o Electron Microscope o Micro-plate readers o Thermal gravimetric analyzers o X-ray fluorescence spectrometers o Elemental analyzers 2010 Waters Corporation 42 21

22 Roles and Responsibilities 2010 Waters Corporation 43 Users Users include the analysts and management Are most familiar with their analysis Ultimately responsible for AIQ Can contract out parts of AIQ, but are still responsible Must keep instrument qualified through PQ 2010 Waters Corporation 44 22

23 Quality Assurance (QA) QA role remains as in any other regulated study Ensure that AIQ meets regulatory requirements Have users attest to the scientific validity of AIQ 2010 Waters Corporation 45 Manufacturer Perform DQ when designing instrument Validate the software and hardware Provide summary of their validation work Provide results of final test on hardware Should provide critical functional test scripts for qualification of instrument/software at the user site Make available any software/hardware bugs found after release Support installation, training and service Be open to audit by users 2010 Waters Corporation 46 23

24 AIQ Conclusions Use the term Qualification instead of validation for instruments AIQ provides a level of confidence to users that the instrument is suitable for its intended use AIQ is only one component in delivering reliable and quality data Use DQ/IQ/OQ/PQ model with specific definitions and scope for each qualification stage White paper is available on FDA docket, and AAPS Pharm SciTech (AAPS website). AAPS PharmSciTech. 2004; 5(1): article Waters Corporation 47 Affect on Your Laboratory 2010 Waters Corporation 48 24

25 What does AIQ mean to You? AIQ is formalizing a long used process.but: It is an opportunity to revisit what you are doing Evaluate using a Scientific Approach What value added are you getting from your tests? Is there a way to simplify what is being done? Are you doing enough? Are you re-qualifying on a regular scheduled basis? Are you covering the high risk areas? Are you doing too much? Does it take you two years to implement a new Data System? Do you take measurements on hardware merely because you can? Are you re-qualifying more frequently than needed? Are you testing things that need not be tested as part of AIQ? Risk analysis is a good way to decide 2010 Waters Corporation 49 Questions? 2010 Waters Corporation 50 25

26 IV Qualification Case Studies 2010 Waters Corporation 51 Agenda for Module IV Determining what is important to test Some questions asked by equipment users How various instruments work and the effect on testing Various approaches in qualifying LC modules and systems Automating and streamlining the qualification process 2010 Waters Corporation 52 26

27 Considerations As we proceed lets Consider the importance of the parameters we test o What value added does the test supply Evaluate to what level they need to be tested o Time to test versus benefit or risk mitigation Consider the various approaches we might use to test these Gain an understanding how each test and each module under test functions Remember that there is no one right way to qualify that an instrument or a system is functioning as intended 2010 Waters Corporation 53 Considerations (2) Remember, in the end, the user must be prepared to answer any questions an auditor may have Understanding how the equipment functions and how it has been qualified leaves you better prepared to support the process in an audit situation Qualification testing indicates that a module or system performs as intended Qualification testing does not indicate suitability for a particular application 2010 Waters Corporation 54 27

28 What about Part 11 Requirements? Electronic Records and Signatures Recent Changes Risk Based Approach Predicate Rules Apply Laboratory Data Equivalency to paper based Systems Can qualification results be made part 11 compliant? If the results are treated as chromatography data, they can 2010 Waters Corporation 55 Determining what is important to test (continued) On LC, we are generally concerned with Accuracy Linearity Precision There are multiple ways to get this information There is no one right way The FDA does not dictate the method to use 2010 Waters Corporation 56 28

29 Manual SOPs for Qualification Traditional/Manual SOPs (Qualification Workbooks) are designed to test key product specifications Generally, on Liquid Chromatographs, we verify Accuracy, Linearity and Precision where possible (per FDA guideline) Workbook tests are derived from factory tests for individual modules adapted for use in the field The workbook tests also allow for typical testing error Errors in class a volumetric flasks Errors in tests performed by NIST on the test solutions Errors in meters and temperature probes Test errors are additive with the product error Workbooks provide a high degree of assurance that product specifications are met They test the module as a module not as it will be used in the system so system error is not be taken into account The main disadvantages of this form of testing are the time required systems not tested similar to the way they will be used 2010 Waters Corporation 57 Exploring Ways to Qualify LC Systems A new streamlined approach is needed A scientific look is taken at the modular tests Redesigning existing modular tests can simplify the process Using the power of the Chromatography Data Software (CDS), the system itself can be used to verify calibration and provide qualification data Calculations can be performed internally avoiding the need to deal with external spreadsheets Automatic reporting and calculations reduces the risk of human error and interference Results can be kept on-line and be part 11 compliant The concept of Advanced Qualification Technology (AQT) and the Waters Systems Qualification Tool (SystemsQT) is created 2010 Waters Corporation 58 29

30 Design Criteria A design requirement of the SystemsQT was that it could perform all the tests normally performed using a traditional qualification workbook The system was to be tested the way it will be used Workbooks were initially developed using tests off the Waters manufacturing floor SystemsQT was developed with a clean sheet of paper All procedures were reviewed and new methodologies developed that combined tests wherever possible New test solutions were incorporated with the aim of minimizing the various chemical kits used 2010 Waters Corporation 59 Development Original factory tests and specifications were developed to test a specific module and only that module Interference from other modules, solvents, columns and other factors were minimized or eliminated when testing at the modular level The SystemsQT is designed to test the chromatograph as a system the way it will be used, not as individual modules New pass/fail criteria had to be developed based on all the factors that could affect the outcome A number of qualified, characterized systems were used to build tolerance windows around procedures Pass/fail criteria were empirically derived from qualified systems 2010 Waters Corporation 60 30

31 UV Detector Qualification User question: I have a UV detector (Variable λ or Photo Diode Array) I want it qualified How are all the wavelengths tested for accuracy? Do we need to test all the wavelengths? Why or why not? What happens if I change a lamp? What if an inspector asks how I know λ is right? Don t know? A better answer Did you document it? 2010 Waters Corporation 61 UV Detector Qualification 2010 Waters Corporation 62 31

32 Wavelength Accuracy (1) Example Specification Wavelength accuracy is one of the most important test we run and inspectors frequently ask about this It is NOT necessary to test every wavelength in a detector s range to provide a high degree of assurance that an absorbance detector is working properly in terms of wavelength accuracy Not enough suitable standards are available It would take lots of time Replicate gratings are highly reproducible Design of the wavelength algorithms are validated as part of the instrument design qualification (DQ) 2010 Waters Corporation 63 Wavelength Accuracy (2) For an example, if we are qualifying a Waters Tunable UV detector, the unit has on-board diagnostics that can be used to verify proper wavelength accuracy at every power-up The user is alerted if a wavelength is found out of specification The cause could be due to something in the cell The detector uses wavelengths from both the lamp and from an internal filter that is inserted into the light path. A number of choices are available: Zero Order diffraction grating energy spike (tunable detectors) o reflects all wavelengths (used in some older detectors) 257 nm erbium nitrate filter absorbance maxima 379 nm erbium nitrate filter absorbance maxima 486 nm deuterium energy line (used as anchor for 500nm top of range) 487nm erbium nitrate filter (generally not used because of 486nm deuterium line) 521 nm erbium nitrate filter absorbance maxima 656 nm deuterium energy line (anchor wavelength peak) 2010 Waters Corporation 64 32

33 Wavelength Accuracy (3) The OQ is used as a means of qualifying not only the detector but also the internal diagnostics Caffeine can be used for qualification purposes and has peaks at 205nm and 273nm for 1nm Slit Band Width Testing 205nm with Caffeine is about as far as we can get from the 656nm anchor wavelength provided by the lamp and used in the start up calibration If a wavelength 452nm away from the calibration wavelength is accurate those closer should also be accurate Using the diagnostics and the caffeine solution, we have verified 6 different wavelengths, 205, 257, 273, 379, 521 and 656nm Using the power up diagnostics between qualifications allows us to check 257, 379, 521 and 656nm It is good practice to power down and back up weekly 2010 Waters Corporation 65 Wavelength Accuracy (4) Absorbance detector test procedures are based on ASTM E * *Standard Practice for Testing Variable-Wavelength Photometric Detectors Used in Liquid Chromatography Qualification of wavelength accuracy in this manner is common practice been audited successfully on many occasions Knowing about the onboard diagnostics in this case and documenting their use allows you to explain to an inspector how you know the wavelengths are accurate after a lamp change between qualifications 2010 Waters Corporation 66 33

34 Wavelength Accuracy (5) The test solutions used were tested by NIST with an accuracy of + or- 0.3nm Wavelength is fixed by the physics of the test solution Lambda max can change with detector slit bandwidth depending upon peak shape of each peak in the solution Concentration of the accuracy test solutions is not critical The product specification of 1nm is added to the NIST 0.3nm error and rounded up to a pass/fail criteria of +/- 2nm because errors are additive and because the detector bins the results to the nearest nanometer The pass/fail criteria in this case becomes +/- 2nm 2010 Waters Corporation 67 Wavelength Accuracy (5) One way of automating this test is by having the Chromatography Data System make successive injections of the caffeine solution, incrementing the wavelength on each injection and determining the lambda max for the two caffeine peaks Since there are two detector channels, both 205 and 273nm can be tested at once Alternating between 2 wavelengths also verifies λ precision 2010 Waters Corporation 68 34

35 Wavelength Accuracy (6) λ Max For Caffeine nm 1.20 Absorbance nm Wavelength (nm) 2010 Waters Corporation 69 Absorbance Linearity (1) Tested by injecting different concentrations of a solution Caffeine is also a suitable solution for this test Peaks are symmetrical Different cell path lengths require different concentration test solutions to test the linear range of the detector Beer s law The shorter the path length, the more concentrated the solution must be to absorb the same amount of light A passing detector is generally defined as one where the drop from a theoretical straight line is 5% at the top end of it s specified linear range (per ASTM 1657) 2010 Waters Corporation 70 35

36 Absorbance Linearity (2) A line can be drawn from the different injection areas and the straightness of that line or the linearity can be determined by the R 2 of the curve Acceptable pass/fail values are determined by the linearity specification of the detector and the test solutions used It is impractical to test linearity at every wavelength Linearity will very at every wavelength due to lamp energy Linearity will vary with every mobile phase, diluent and sample Qualification provides assurance that the instrument is functioning as intended Method validation should be used to test specific samples o Suitability for intended use 2010 Waters Corporation 71 Detector Linearity and Sensitivity Sensitivity is %RSD Of Peak Heights / Amounts Linearity is R 2 of curve Peak Height (AU) % RSD = 3.96 R 2 = Peak AU/Amount Amount Injected 2010 Waters Corporation 72 36

37 Injection Accuracy, Linearity and Precision Injection Accuracy Linearity and Precision are affected by many factors including: Mobile phase used Degassed state of mobile phase Sample diluent used Viscosity of the sample System dead volume Sample loop volume Syringe size Syringe draw rate Number of motor steps per microliter (varies with syringe size) Vial septa used Injection mode used 2010 Waters Corporation 73 Injection Accuracy While precision is most important, injection accuracy allows for easier movement of an analysis from one system to the next One way to test is by weighing the aspirated amount of the sample and translating that to volume Using water for the sample makes this easier at room temps Enough weight must be removed from a vial such that the weighing error is not a significant part of the measurement 2010 Waters Corporation 74 37

38 Injection Accuracy An example injection accuracy specification: The accuracy specification is at 50uL with specific solvents Why was the specification written this way? Six injections are made to remove enough weight (300uL) The injector is deemed to meet specification if it passes the test Apparent performance at different injection volumes, with different solvents, sample loops, syringes, mobile phases columns and samples will vary It is not practical to test at all volumes o Method Validation and System Suitability should be used to demonstrate suitability for intended use 2010 Waters Corporation 75 Injection Accuracy Another way to test, one that can be automated, is by performing actual injections at different volumes to determine the linearity and then using the x intercept as a means to determine the accuracy error While this may be less metrologic, it demonstrates the injector in the same way that it will be used and provides a high degree of assurance that the injector is operating as intended The qualification test assures that the syringe drives, needle, valves and injection algorithms are all functioning as intended Method Validation and System Suitability should be used to demonstrate suitability for the specific intended use 2010 Waters Corporation 76 38

39 Injector Linearity and Accuracy Linearity = R 2 of curve Accuracy = X Intercept area (uv*sec) y = 25000x X-intercept = ul volume injected (ul) 2010 Waters Corporation 77 Injection Linearity Linearity o Linearity, for this example, is specified from 1 to 100uL o Linearity above 100uL is not specified o Injections above 150uL will provide best results with the 2500uL syringe so they can be made in one syringe draw o Making injections at 5uL using a large size syringe and large volume sample loop is not the best practice because a large loop means more air in the system that will stretch before the actual sample is pulled in o But, a 250uL sample loop is not large enough for accurate 200uL injections. At least a 330uL loop needed for 400uL of total sample loop volume o One system configuration is not the best for all injection volume sizes (just like columns!) o Think about how you will be using your system before choosing the configuration o Use method validation and system suitability to demonstrate suitability for intended use 2010 Waters Corporation 78 39

40 Injection Linearity Injection linearity testing in the OQ is used as a means of determining if the injector is functioning as expected across the typically used analytical range The algorithms that determine motor drive functionality and hence linearity are validated as part of the design of the product It is always best to perform linearity tests with specific compounds, mobile phases, wavelengths, sensitivities, sample loops, syringes and columns to be used Method Validation and System Suitability Demonstrates suitability for intended use 2010 Waters Corporation 79 Injection Linearity A user Question: An HPLC injector has been qualified using at 5, 10, 20 and 50uL Is it still linear at 200uL? The answer is it depends what you mean By design, the motor will step the proper amount of steps and the syringe will move the specified amount so in that sense, it will be linear but: Physics will get in the way to make the actual results less linear o Any injection volume much over one half the sample loop volume will start to disperse out of the loop o A 200uL injection will require a larger sample loop and that will allow more air in the system. That air stretches when aspirating an injection. This will particularly affect small injection volumes o A 200uL injection with a standard syringe will require multiple syringe draws and valve movements this will affect accuracy and linearity o Mobile phase and degassing used will affect accuracy and linearity o The column used will affect accuracy and linearity 2010 Waters Corporation 80 40

41 Column Effects A rule of thumb for columns Do not inject more than 5% of the column void volume To inject 200uL into a column, the column should have a 4mL void volume Otherwise the sample diluent becomes the mobile phase Injecting any more than that alters the chemistry and will make the analysis non-linear A 4.6 x 150mm column has a void volume of only 2.7 mls Using a 4mL void volume column will mean waiting about an hour for the peaks to come out or a much higher flow rate If you are using a system in a non-standard configuration, you can qualify using the standard column and loop, then perform PQ and/or System Suitability with the column and loop to be used for the specific analysis 2010 Waters Corporation 81 UPLC Flow Rate Linearity Linearity = R 2 of curve 1/void tim e y = x X-intercept = ml/min flow rate (ml/min) 2010 Waters Corporation 82 41

42 HPLC Flow Linearity and Accuracy 2010 Waters Corporation 83 Flow Rate Accuracy HPLC specification example: A column is in place when using the SystemsQT Because of this, flow rates are limited to the capabilities of the column For HPLC, flow rates are tested at 0.5, 0.75, 1 and 1.5, the most common range used by users with analytical columns Flow rates across the entire range are validated as part of the design qualification (DQ) for the product If the HPLC passes at the flow rates tested, there is a high degree of assurance that it will work within specification at all flow rates (i.e. very low risk of failure) For the most part, flow error is fixed by the check valve size and design The fixed error of a properly functioning check valve shows up as a higher percentage of total flow at lower flow rates 2010 Waters Corporation 84 42

43 System Precision %RSD of 6 Replicate Injections 2010 Waters Corporation 85 Gradient Proportioning and Compositional Accuracy If you use two high pressure pumps (or a binary pump) to create gradients and they use a structurally validated gradient algorithm, so long as the flow rates on both pumps test accurately at multiple flow rates (flow rates are linear), gradients will be accurate and precise. If you use low pressure mixing on a system that has a structurally validated gradient algorithm, generally any gradient inaccuracy is caused by improper operation or sticking of the Gradient Proportioning Valves (GPVs) so focusing on testing these is one way to assure proper gradient performance If the valves remain unused for a long time (usually the C or D valves), they can become sluggish or sticky Exercising them before use or qualification in such cases is advised 2010 Waters Corporation 86 43

44 Gradient Performance SD of Retention Time from each of 7 Peaks over Six Injections SD must be < or = 1.0 sec Note: In this UPLC case, there are two high pressure pumps Waters Corporation 87 Compositional Accuracy Note: In this case, we are testing one pump with low pressure mixing capability of 4 solvents 2010 Waters Corporation 88 44

45 Demonstrating Fit for Intended Use A PQ based on specific user and method requirements Performed by the user AFTER the vendor supplied IQ,OQ and vendor PQ (system level OQ). Demonstrates that the instrument system can operate reliably under routine, minimum, and maximum operating conditions. Fit for intended use. Recommended combination of user requirements: Method specific system suitability o Precision of quantity of analite injected o Peak resolution o Peak tailing o Column efficiency Method specific concentration standards Calibration check 2010 Waters Corporation 89 FDA Requirements Vendor Supplied IQ, OQ and vendor PQ (system level test) Works according to manufacturer s specifications PQ based on User Requirements o Demonstrates Fit for Intended Use Ongoing system calibration (qualification) and documented scheduled maintenance 2010 Waters Corporation 90 45

46 Advantages of CDS Managed Qualification Accurate Qualification Testing and Analysis Less opportunity for human error Measures peak areas, peak heights and retention times accurately and consistently Custom field calculations and regression analysis Testing consistent from system to system Reduces time that system is off-line by about half Qualifies software and systems in analytical configuration 2010 Waters Corporation 91 Advantages of CDS Managed Qualification (cont) Secure and Auditable Qualification Data 21 CFR Part 11 Compliant Qualification Data On-Line Qualification Documentation for Easy Inspection Easy tracking and trending of qualification results Audit trails and change control part of the data system No need for external spreadsheets or third party software Secure data environment 2010 Waters Corporation 92 46

47 Summary There are many ways to qualify LC systems Advanced technology can save time in your laboratory SystemsQT is the Next Generation in automated Instrument Qualification It enhances qualification testing and data management in an Empower Chromatography Data System environment It offers a whole system test approach for a more accurate indication of overall system-level compliance Operates on a simple wizard built into Empower using Run Samples Built into Empower no separate software to install and uninstall Projects to qualify new modules/systems can be easily added Document review is substantially reduced 2010 Waters Corporation 93 Questions? 2010 Waters Corporation 94 47