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National MeasurementSystem 1997-2000 ValidAnalytical Measurement(VAM) Programme

Guidance on EquipmentQualification of AnalyticalInstruments: High PerformanceLiquid Chromatography (HPLC)

June 1998

LGC/VAM/1998/026.2

National MeasurementSystem 1997-2000 ValidAnalytical Measurement(VAM) Programme

Guidance on EquipmentQualification of AnalyticalInstruments: High PerformanceLiquid Chromatography (HPLC)

June 1998

Contact Point:

Peter BedsonTel: 0181 943 7392

The development of this guidance was supportedunder contract with the Department of Tradeand Industry as part of the NationalMeasurement System Valid AnalyticalMeasurement (VAM) Programme

LGC/VAM/1998/026.2© LGC (Teddington) Limited 1998

PrefaceThis document has been prepared by LGC with assistance from the Instrumentation Working Group.The members of this working group, listed below, and in particular the members of the HighPerformance Liquid Chromatography (HPLC) Sub-Group, have played a major role in developing thisdocument and their help is gratefully acknowledged. The Secretariat would also like to thank those whocommented informally on this document during the drafting process.

It is intended that after a period of use, the content of this document will be reviewed, and the amendedtext republished. Users are invited to comment on the content of the existing text and suggest additionalmaterial in writing to:

Mr P J Bedson, Secretary, Instrumentation Working GroupLGC, Queens Road, Teddington, Middlesex, TW11 0LY, UK.

Instrumentation Working Group Members

Dr Mike Sargent (Chairman) LGC

Mr Colin Andrews GAMBICA

Mr Peter Bedson (Secretary) LGC

Dr Grant Cameron Anachem

Dr Lyndon Davies Lyndon Davies Associates

Mr Terence Duley Cecil Instruments

Dr Steve Ellison LGC

Dr Mike Ford VAM Working Group

Mr Ted Glancy (replaced Dr Gary Fenton) Waters

Mr John Hammond Unicam

Mr Roy Lines Coulter Electronics

Mr Lars Lis Varian

Mr Andy Martin United Kingdom Accreditation Service (UKAS)

Mr Steve Monk Department of Health GLP Monitoring Authority

Mr Nigel Moorhouse (replaced Mr Ian Vallance) Hewlett Packard

Dr Elizabeth Prichard LGC

Dr David Rudd Glaxo Wellcome Research and Development

Dr Peter Smith Hilger Analytical

Professor Peter Stockwell P S Analytical

Mr Paul Yorke Perkin Elmer

HPLC Sub-Group Members

Dr David Rudd (Chairman) Glaxo Wellcome Research and Development

Mr Peter Bedson (Secretary) LGC

Dr Grant Cameron Anachem

Mr Stuart Cline (replaced Mr Ian Vallance) Hewlett Packard

Mr Ted Glancy (replaced Dr Gary Fenton) Waters

Mr Phil Kilby Varian

Dr Mark Upton Perkin Elmer

LGC/VAM/1998/026.2 Page i

Contents

1. Glossary 1

2. The equipment qualification process 3

3. High Performance Liquid Chromatography 6

4. Design Qualification (DQ) 8

5. Installation Qualification (IQ) 14

6. Operational Qualification (OQ) 15

7. Performance Qualification (PQ) 21

8. References 24

9. Bibliography 25

LGC/VAM/1998/026.2 Page 1

1. GlossaryMany of the terms relating to equipment qualification are used in different ways toconvey a variety of meanings. The following descriptions explain how these termsshould be interpreted in this guidance document.

1.1 Instrument: an entire High Performance Liquid Chromatography (HPLC) measurementsystem comprising either a fully integrated system or a series of interconnectedmodules.

1.2 Module: a distinct component of the instrument (e.g. an injector or detector).

1.3 Modular testing: a series of checks to verify the correct functioning and performanceof a distinct component of the instrument.

1.4 Holistic testing: the process of verifying the correct functioning and performance ofentire instrument system.

1.5 User: the organisation purchasing the instrument including its management and staff.

1.6 Supplier: the instrument manufacturer, vendor, lessor or approved agent.

1.7 User Requirement Specification (URS): The user requirement specification definesthe overall requirements for the instrument, the key performance characteristics of theinstrument and ranges over which the instrument is required to operate and consistentlyperform, and other critical factors relating to its use.

1.8 Equipment Qualification (EQ): the overall process of providing evidence that aninstrument is fit for its intended purpose and that it is kept in a state of calibration andmaintenance consistent with its use.

1.9 Design Qualification (DQ): covers all procedures prior to the installation of the systemin the selected environment. DQ defines the user requirement specification and detailsthe conscious decisions in the selection of the supplier.

1.10 Installation Qualification (IQ): covers all procedures relating to the installation of theinstrument in the selected environment. IQ establishes that the instrument is received asdesigned and specified, that it is properly installed in the selected environment, and thatthis environment is suitable for the operation and use of the instrument.

1.11 Operational Qualification (OQ): the process of undertaking confirmatory checks toverify key aspects of performance in the absence of any contributory effects which maybe introduced by the method.

LGC/VAM/1998/026.2

1.12 Performance Qualification (PQ): is defined as the process of demonstrating that aninstrument consistently performs according to a specification appropriate for its routineuse.

1.13 Validation: is the process of evaluating the performance of a specific measuringprocedure and checking that the performance meets certain pre-set criteria. Validationestablishes and provides documented evidence that the measuring procedure is fit for aparticular purpose.

1.14 Analytical Quality Control (AQC): The set of procedures undertaken by the user forthe continuous monitoring of operations and the results of measurements in order todecide whether results are sufficiently reliable.

1.15 System Suitability Checking (SSC): A series of tests to check the performance of ameasurement process. SSC may form part of the process of validation when applied toa particular measuring procedure. SSC establishes that the operational conditionsrequired for a specific measurement process are being achieved and can be used toprovide evidence of satisfactory instrumental performance during actual use.

1.16 Calibration: The set of operations which establish, under specified conditions, therelationship between values indicated by a measuring instrument or process and thecorresponding known values of the measurand.

1.17 Traceability: The property of a result of a measurement whereby it can be related toappropriate standards, generally national or international standards, through anunbroken chain of comparisons.

1.18 Standard Solution: A solution or matrix containing a known concentration of ananalyte. Standard solutions are often used to check the performance of the instrumentand establish the relationship between instrumental response and analyte concentration.


2. The equipment qualification process2.1 Equipment qualification (EQ) is a formal process that provides documented evidence

that an instrument is fit for its intended purpose and kept in a state of maintenance andcalibration consistent with its use.

2.2 EQ is divided into four stages: design qualification (DQ); installation qualification(IQ); operational qualification (OQ); and performance qualification (PQ). The role ofeach stage is summarised in Figure 1.

DesignQualification

(DQ)

InstallationQualification

(IQ)

OperationalQualification

(OQ)

PerformanceQualification

(PQ)

Defines the specifications of the instrumentand details the conscious decisions in the

selection of the supplier

Establishes that the instrument is received asdesigned and specified, that it is properlyinstalled in the selected environment, andthat this environment is suitable for the

operation of the instrument

Confirmatory checks to verify key aspects ofperformance in the absence of contributoryeffects which might be introduced by the

method

The process of demonstrating that aninstrument performs according to a

specification appropriate for its routine use

Figure 1 - The EQ process

2.3 DQ is the ‘planning’ part of the EQ process and is most often undertaken as part of theprocess of purchasing a new instrument, although it may be appropriate to repeataspects of DQ following a major change to the instrument or its use. Whilstqualification of the actual instrument design is for manufacturers of instruments, usersof instruments have an important role in DQ by ensuring adoption of a user requirementspecification (URS) which meets the intended use.

2.4 IQ, OQ and PQ are the ‘implementation’ stages of the EQ process and provide anassurance that the instrument is installed properly, that it operates correctly, and that itsongoing performance remains within the limits required for its actual use. IQ covers theinstallation of the instrument up to and including its response to the initial application

LGC/VAM/1998/026.2

of power. OQ should be carried out after the initial installation of the instrument (IQ)and repeated following a major event (e.g. relocation or maintenance) or periodically atdefined intervals (e.g. annually).

2.5 PQ is undertaken regularly during the routine use of the instrument. The role of PQ isto provide continued evidence that, even though the performance of the instrument maychange due to factors such as wear or contamination, its performance remains withinthe limits required for its actual use. As such, much of the evidence needed for PQ isavailable from ‘everyday’ procedures, for example, method validation, systemsuitability checking (SSC), routine calibration and analytical quality control.

2.6 The terms ‘validation’ and ‘qualification’ are used widely and often to convey the samemeaning. The approach taken in this guidance document is that validation is applicationorientated and relates to a specific measurement method or process, whereasqualification is instrument orientated and relates primarily to providing evidence ofsatisfactory performance of the instrument.

2.7 Increasingly, both analytical laboratories and regulatory authorities are acknowledgingthat, under some circumstances, both method validation and equipment qualificationare important prerequisites for obtaining reliable data. In particular, OQ provides anassurance that an instrument functions correctly independently of the applications ormethods with which it is used.

2.8 Although formal quality standards [1,2,3,4] all require various combinations of methodvalidation and equipment qualification, there is a lack of clear guidance on when EQ isappropriate, what is actually required, how it should be achieved and how it should bedocumented. A key objective in developing guidance [5] has been, therefore, to provideusers and suppliers of analytical instruments, as well as those responsible for theassessment, certification and monitoring of analytical laboratories, with a clear andconsistent approach to EQ. The guidance has been prepared with the primary aim ofassisting the interpretation of formal quality standards in order to satisfy regulatory andaccreditation requirements.

2.9 EQ must be documented (Section 4 of the general guidance [5] provides more detailedguidance on requirements for EQ documentation). EQ documentation can be preparedand provided by the user, the supplier, or both. Where it is provided by the supplier(e.g. in a qualification protocol), it should be written in such a way that it can be readilyfollowed and understood by the user.

2.10 The responsibility for equipment qualification rests with the users of analyticalinstruments. Suppliers can be expected to make available documentation, tools andservices to assist EQ and, in particular, to provide clear instructions and details of testsand checks required to demonstrate satisfactory performance. Although suchperformance testing can be carried out by the supplier or the user, it must remain underthe control of a suitably qualified user.


2.11 The user must establish the level of EQ required and what aspects of EQ (particularlyOQ) will be done in-house and what will be carried out by an external organisation.Where any aspect of EQ, and/or a performance check or test is undertaken, users mustsatisfy themselves that it has been carried out competently and correctly (evidence ofcurrent competence should be established, for example, through a valid training recordor certificate).

2.12 This document is not intended to describe an exhaustive series of compulsory tests thatmust be carried out under all circumstances. Those undertaking EQ must exercise theirprofessional judgement and common sense to decide on which tests are relevant, and onwhat test criteria and tolerance limits are appropriate. An important consideration indetermining what is checked and verified during EQ is the supplier’s track record andthe user’s experience with previously supplied equipment.

2.13 EQ provides important evidence of an instrument’s satisfactory performance and itsfitness for the purpose for which it is used. However, EQ is just one of a range ofactivities which contribute towards achieving and demonstrating reliable and valid data.

LGC/VAM/1998/026.2

3. High Performance LiquidChromatography

3.1 A high performance liquid chromatography (HPLC) system consists of a number ofcomponents which can either be purchased as individual modules, from one or moresuppliers, or as a completely integrated system.

3.2 Whilst suppliers will be able to assist with and undertake qualification of their owninstruments, they may not be able to perform qualification of other suppliers’instruments. Where systems comprise modules from different suppliers, testing of thecomplete system (holistic testing), such as that carried out in PQ, will normally have tobe undertaken by the user.

3.3 This document provides guidance on the equipment qualification of the principlecomponents (solvent delivery system; injection system; column oven, and detector) ofan analytical HPLC system employing UV detection. However, most principles willapply to other (e.g. preparative) LC systems and alternative types of detection. Aschematic diagram illustrating the main components of a HPLC system is shown inFigure 2. Whilst the guidance does not specifically cover data handling devices(integrators or computerised system controllers) an assurance of the correct functioningof these components can be inferred from holistic performance checks such as thosecarried out in PQ.

3.4 The subsequent sections of this document provide more detailed guidance on therequirements of each stage of qualification (DQ⇒IQ⇒OQ⇒PQ) and how each stageshould be applied to the qualification of the main features and functions of an HPLCinstrument.

3.5 In OQ, the aim is to verify that the main operating parameters (e.g. injection volume,flow rate, mobile phase mixing, column thermostatting temperature, detectionwavelength) are within their specified limits for accuracy and precision. This providesconfidence that the instrument is operating correctly, to specification, and that there areno unacceptable differences between a parameter’s selected and actual values. Forexample, when the pump is set to deliver 1.0ml per minute the actual flow is withinrequired tolerances (e.g. 0.95-1.05ml per minute) and not significantly different (e.g.0.7 or 1.3 ml per minute) from the selected value.

3.6 In order to enable correct functioning and operation to be verified in the absence of anycontributory effects that might be introduced by the method, OQ performance tests aredesigned to check the performance of individual modules without involving the qualityof the fittings, solvents or the chemistry normally involved in separation science (i.e.the column). These issues which are so important during method validation and theeveryday functioning of the system to maintain data integrity are intentionallyminimised in OQ.


3.7 However, although OQ testing might provide useful evidence that the individualmodules of the instrument are operating correctly and within specification, such testsalone cannot guarantee satisfactory performance of the entire instrument system eitherfollowing initial assembly or on an ongoing basis.

3.8 The aim of PQ is to provide evidence that, following initial assembly, the entire HPLCinstrument is functioning correctly and within specification and that its performanceremains satisfactory during routine use. PQ can, therefore, be considered as having twostages: initial holistic testing to provide evidence that the complete instrumentfunctions correctly; and system suitability checking (SSC) to provide evidence offitness for purpose and satisfactory performance during actual use.

Detector Data handling device

Solventdeliverysystem

Colum

n oven

Injector

Systemcontroller

Figure 2 - Schematic diagram of HPLC system

3.9 Many quality standards and enforcement authorities stipulate that “ where possiblecalibrations should be traceable to national or international standards” in order toensure accuracy. This leads to confusion as to the need for traceable standards andcalibrated apparatus when checking an instrument’s operating parameters. Tests toverify the accuracy of critical parameters (e.g. wavelength accuracy) will necessitate theuse of traceable standards and calibrated apparatus.

LGC/VAM/1998/026.2

4. Design Qualification (DQ)4.1 Design Qualification is concerned with what the instrument is required to do and links

directly to fitness for purpose. DQ provides an opportunity for the user to demonstratethat the instrument’s fitness for purpose has been considered at an early stage and builtinto the procurement process.

(Section 5 of the general guidance [5] provides more detailed information on genericrequirements for design qualification)

4.2 DQ should, where possible, establish the intended or likely use of the instrument andshould define an appropriate user requirement specification (URS). The URS definesthe key performance characteristics of the instrument and the ranges over which theinstrument is required to operate and consistently perform along with other criticalfactors relating to its use. The URS may be a compromise between the ideal and thepracticalities of what is actually available. Whilst it is the responsibility of the user toensure that specifications exist, and that they are appropriate, they may be prepared bythe user, the supplier(s), or by discussion between the two.

4.3 In undertaking DQ, information and knowledge of existing equipment should be takeninto account. If an instrument is mature in design and has a proven track record, thismay provide a basic confidence and evidence about its suitability for use. For newtechniques or instruments DQ may require more effort.

4.4 The selection of the supplier and instrument is entirely at the discretion of the user.However, in selecting the supplier and instrument, the user should bear in mind thatregulators are likely to require evidence of: the use of rigorous design and specificationmethods; fully documented quality control and quality assurance procedures; the use, atall times of suitably qualified and experienced personnel; comprehensive, plannedtesting of the system at all levels of the system; and the application of stringent changecontrol, error reporting and corrective procedures. A suitable questionnaire, third partyaudit, or independent certification of the supplier to an approved quality scheme mayprovide the user with evidence that regulatory requirements have been met duringdesign and manufacture of the instrument. Where such evidence is not available, it isthe responsibility of the user to carry out more extensive qualification in order toprovide the necessary assurance of the instrument’s fitness for use.

4.5 Where instruments are intended to be used to make measurements supportingregulatory studies, the user may also need to seek confirmation that the manufacturer isprepared, if required, to allow regulatory authorities access to detailed information andrecords relating to the instrument’s manufacture and development, for example: sourcecodes; instrument development records and procedures; calibration and qualificationdocumentation; batch test records and reports; hardware and software qualificationdocumentation; and credentials of staff involved with the development of theinstrument.


4.6 Detailed listings of criteria to be considered when selecting and purchasing HPLCinstrumentation have been published by the Analytical Methods Committee of theRoyal Society of Chemistry [6]. Tables 1-5 summarise key features which might beconsidered during the development of the URS.

Table 1 - Design Qualification of HPLC Instruments - General Considerations

Feature Consideration

Instrument set-up & control Modular or integrated system.

Compatibility between modules and with existing equipment.

Ease of changing modules (e.g. to change detector).

Software control of operating conditions and parameters.

Data acquisition, processing and presentation needs.

In-built diagnostic facilities.

Sample throughput & introduction Sample preparation / clean-up / pre-treatment needs.

Sample throughput, presentation and introduction needs.

Manual or automated system.

Materials of construction Resistance to corrosion, contamination.

Inert to solvent and sample.

Documentation Clarity and ease of use of documentation (e.g. operating manuals,qualification protocols, model SOPs).

Unique document identification by version number and date ofissue.

Size, power & utility requirements Limitations on, requirements for and expected consumption ofservices, utilities, and consumables (e.g. electricity, compressed air,helium).

Maintenance & support Ease of user maintenance and cleaning.

Cost and availability of spares and parts.

Cost and availability of service contracts and technical support.

Suggested intervals between and procedures for maintenance andcalibration of the instrument.

The period for which support (qualification, maintenance, parts etc.)for the instrument can be guaranteed.

Training requirements The level of skill required to operate the instrument and details ofany training necessary and courses provided by the supplier.

Environmental conditions Environmental conditions within which, or range over which, theinstrument must work.

Health and Safety Health and safety and environmental issues and/or requirements

LGC/VAM/1998/026.2

Table 2 - Design Qualification of HPLC Instruments - Injection Systems

Feature Consideration Specification

System control andcommunication

Ability fully to select, control, store andretrieve injection parameters locally withininjector.

Stand-alone programmabilityrequirements.

Ability to send / accept signals (e.g. throughcontact closures) and to communicate (e.g.RS232) with other devices.

Requirements for communicatingwith other devices

Ability fully to control injector from a remotePC or system controller.

Integrated system controlrequirements.

Ability to store and retrieve injectionparameters as part of the method in a PC orremote system controller.

Manual or automaticinjection

Ability to carry out unattended injections. Manual or automatic injection.

Autosampler capacity Ability to carry out unattended analysis ofmultiple samples.

Autosampler capacity.

Autosamplerthermostatting

Ability to thermostatically control sampletemperature.

Temperature range, accuracy andstability.

Injector temperaturecontrol

Ability to minimise temperature effects andchromatographic distortion through injection ofsample at same temperature as mobile phase.

Sample loop/valve pre-heat rangeand limits.

Injection volume Ability to make injections of fixed or variablesample volumes.

Injection volume range.

Ability to make variable volume injectionswithout changing loops.

Constant or variable volume loop.

Ability / ease of changing injection loops. Range of loop sizes required.

Ability to make accurate and repeatableinjections across the operating range.

Injection volume linearity.

Injection volume precision.

Sample capacity Ability to make injections from small samplevolumes.

Minimum volume of samplerequired to make an injection.

Carryover Minimising carryover improves accuracy andreproducibility of results.

Limits of carryover.

Needle wash Ability to flush or wash injector / needle tominimise cross-contamination of samples andreduce carryover.

Needle / injector washrequirements.

Operating pressure Ability to make injection at required systemoperating pressure.

Operating pressure range andmaximum operating pressure.

Liquid transfer &dilution

Ability to perform liquid transfer and dilution.

Sample preparation Ability to carry out sample pre-treatment andconcentration.

LGC/VAM/1998/026.2 Page 11

Table 3 - Design Qualification of HPLC Instruments - Solvent Delivery Systems



Ability fully to select, control, store and retrieveoperational conditions (mobile phase flow rate,composition, and gradient programmes) locallywithin SDS.




Ability fully to control SDS from a remote PC orsystem controller.


Ability to store and retrieve SDS parameters aspart of the method in a PC or remote systemcontroller.

Mobile phaseproportioning/mixing

Ability to perform analysis under isocratic orgradient conditions.

Number of solvent channels

Mobile phaseproportioning/mixingaccuracy & precision

Ability to mix solvents accurately andreproducibly over the required gradient range.

Limits for proportioning accuracyand precision over the requiredrange.

Mobile phasedegassing andfiltration

Ability to degass and filter solvents in situ. Degassing & filtration requirements- inlet pressure limits for Helium

Eluent / columnswitching

Ability to switch between eluents and/orcolumns.

Mobile phaserecycling

Ability to re-use mobile phase.

Flow range Ability to deliver a wide range of flow rates ofsolvents required.

Flow range and incremental steprequired.

Flow rate accuracy,stability and precision

Ability to deliver accurate flow rates,reproducibly, over a long period of time.

Accuracy, precision and stability offlow rate over the specified range.

Solvent storage Ability to perform long unattended runs usingmobile phases which comprise one or moresolvent mixtures.

Number and capacity of solventreservoirs

Spillage containment Ability to detect leaks and spillage Automatic shut down

Gradient delay volume Ability to deliver changes in mobile phasecomposition with minimal delay.

Dead volume

LGC/VAM/1998/026.2

Table 4 - Design Qualification of HPLC Instruments - UV/VIS Detectors



Ability fully to select, control, store and retrieveoperational conditions (wavelength, bandwidth)locally within detector.




Ability fully to control detector from a remotePC or system controller.


Ability to store and retrieve operationalconditions as part of the method in a PC orremote system controller.

Detector type Ability to monitor a single, multiple or variablewavelengths and/or full spectral characteristics.

Detector type: single wavelength;continuously variable wavelength; ordiode array.

Flow cells Ability easily to remove and clean flow cells, toinject liquid standards, and to connect detectorsin series without band broadening or loss ofchromatographic efficiency.

Ability to change flow cell for other uses, e.g.microbore / preparative applications.

Requirements for removal andcleaning of flow cells, injection ofliquid standards. Dead volumelimits. Sensitivity to back pressure.

Wavelength accuracy Ability accurately and reproducibly to selectwavelengths monitored.

Accuracy and precision ofwavelength selection.

Wavelength range Ability to select and monitor a wide range ofwavelengths, with or without changing source.

Wavelength range. Ease of lampchanging.

Linear dynamic range -stray light

Ability for accurate quantitation over a largeconcentration range

Linear dynamic range

Noise Low noise facilitates improved sensitivity andlower detection limits

Signal to noise ratio

Drift Low drift facilitates improved sensitivity andlower detection limits

Flow cell thermostatting andsensitivity to back pressure


LGC/VAM/1998/026.2 Page 13

Table 5 - Design Qualification of HPLC Instruments - Column Ovens



Ability fully to select, control, store and retrieveoperational conditions (wavelength, bandwidth)locally within detector.




Ability fully to control detector from a remotePC or system controller.


Ability to store and retrieve operationalconditions as part of the method in a PC orremote system controller.

Size Ability to accommodate a range of columns(including guard columns) of different sizes.

Number and size of columns thatcan be accommodated.

Ability easily to access, install or replacecolumns.

Temperature control Ability to reach thermostatting temperaturesquickly and to maintain accurate and stabletemperatures over a range of externalconditions, flow rates and solvents.

Equilibration time.

Accuracy, precision and stabilityover required temperature range.


LGC/VAM/1998/026.2

5. Installation Qualification (IQ)5.1 IQ covers the installation of the instrument up to and including its response to the

initial application of power. IQ involves formal checks to confirm that the instrument,its modules and accessories have been supplied as ordered and that the instrument isproperly installed in the selected environment.

5.2 IQ may be carried out either by the supplier and/or the user. However, it should benoted that, in some cases, the complexity of the instrument alone may preclude the userperforming IQ and, in others, the unpacking of the equipment by the user mayinvalidate the warranty.

5.3 IQ must be undertaken in accordance with the supplier’s instructions and procedures.The success or failure of each of the IQ checks performed should be formally recordedand, where these have been carried out by the supplier, the results of these tests must becommunicated to the user.

5.4 The principles relating to IQ are primarily generic in nature and more detailedinformation on the requirements for IQ is provided in Section 6 of the general guidance[5] published previously. For convenience, a checklist covering the main requirementsfor IQ is provided in Table 6.

Table 6 - Installation Qualification Checklist

Has the instrument been delivered as ordered, e.g. according to the URS or purchase order? üü

Has the instrument been checked and verified as undamaged? üü

Has the required documentation been supplied, is it of correct issue and uniquely identifiedby version number and date?

üü

Have details of all services and utilities required to operate the instrument been provided? üü

Have details of recommended service and calibration intervals been provided? üü

Have methods and instructions for user-maintenance been provided along with contactpoints for service and spare parts?

üü

Has the correct hardware, firmware and software been supplied and is it of correct issue? üü

Has information on consumables required during the normal operation of the instrumentsystem, particularly during start-up or shut-down procedures, been provided?

üü

Is the selected environment for the instrument system suitable, with adequate room forinstallation, operation and servicing, and have appropriate services and utilities (electricity,helium etc.) been provided?

üü

Has health and safety and environmental information relating to the operation of theinstrument been provided?

üü

Is the response of the instrument to the initial application of power as expected and haveany deviations been recorded?

üü

LGC/VAM/1998/026.2 Page 15

6. Operational Qualification (OQ)6.1 The purpose of Operational Qualification (OQ) is to verify key aspects of instrumental

performance in the absence of any contributory effects which may be introduced by themethod.

6.2 Instrumental operating conditions (e.g. injection volume, mobile phase flow rate,gradient composition, column thermostatting, detection wavelength) are normallyspecified as part of an HPLC method. Whilst many methods might be robust to smalldifferences between the selected and actual value of an operating condition, significantdifferences may impact on the validity of the method and the data generated by it. Therole of OQ can, therefore, be considered as the process of checking that key operatingconditions are within any specified limits.

6.3 OQ should be carried out after the initial installation of the instrument. Following this,OQ testing will need to be repeated at regular (but not necessarily frequent) stagesthroughout the life of the instrument.

6.4 Whilst suppliers should provide advice on recommended intervals and events afterwhich OQ testing should be repeated, the responsibility for defining and setting thefrequency and extent of OQ testing rests with users of instruments. This is because theneed and frequency of OQ testing will depend on a variety of factors, not least the leveland nature of use of the instrument, and this is likely to vary between laboratories.

6.5 OQ testing is usually carried out either periodically (at defined intervals, e.g. annually)or following an event which affects the performance of the instrument(e.g. maintenance or repair). However, for convenience, periodic OQ testing is usuallylinked to and carried out following planned events such as routine maintenance orreplacement of consumable parts (e.g. replacement of piston seals in a pump). This maybe the only planned or defined interval at which OQ testing is carried out to verifysome of the more stable aspects of performance (e.g. pump flow rate accuracy).However, it may also be necessary to carry out more frequent OQ testing to verify lessstable aspects of performance.

6.6 The frequency at which periodic OQ testing is undertaken will depend on a variety offactors including:

• the manufacturer’s recommended intervals;

• the required performance (accuracy, precision) of the instrument;

• the level of use of the instrument (higher workloads might acceleratedeterioration in overall performance and therefore necessitate more frequent OQtesting);

LGC/VAM/1998/026.2

• the nature of use (aggressive solvents may cause faster deterioration of pumpperformance; some sample matrices may cause faster deterioration of injectorperformance);

• the environment in which the instrument is used (it is likely that a UV/VISdetector in a mobile laboratory will require more frequent OQ testing to verifywavelength accuracy than the same detector would require if housed in apermanent location); and

• the time that the instrument has been found to remain within requiredperformance limits under the conditions used.

6.7 For event-driven OQ, the extent to which OQ is repeated will depend on the impact thatthe event has on the performance of the instrument. For example, whilst thereplacement of a flow cell is likely to affect the performance of the detector, it isunlikely to impact on the performance of the injection system. Therefore, although itwill be necessary to repeat OQ to verify the performance of the detector, it should notbe necessary to repeat OQ testing to verify the performance of the injector.

6.8 Examples of events that may necessitate repeating OQ might include:

• routine maintenance, servicing and replacement of parts;

• movement or relocation;

• interruption to services and/or utilities;

• modification or upgrades; and

• as part of troubleshooting / fault finding following PQ failure.

6.9 A list of the checks and tests that might be carried out during OQ is provided in Table7. It is important to emphasise that this is not intended to be an exhaustive list ofchecks and tests that must be carried out under all circumstances. Users must exercisetheir professional judgement as to the checks which are relevant and extent of testingrequired.

LGC/VAM/1998/026.2 Page 17

Table 7 - Operational Qualification of HPLC Instruments

Module Parameter Reason Procedure / Comments TypicalTolerance

Injector Injection volumeprecision

Important for system repeatabilityand precision of results.

Can be determined from the relative standard deviation (RSD) of weights of a sample of knowndensity drawn from a vial (repeatedly carrying out the injection volume accuracy test anddetermining the RSD).

Injection volume precision can be affected by a variety of factors (e.g. sample viscosity) and thevalue of such testing at the modular level is questionable. In general, it is recommended thatholistic testing (initial PQ) be used to verify that the injector precision is within specification andthat SSC be used to provide continued evidence of precision during routine use. However, wherePQ/SSC indicates problem with precision, it might be necessary to carry out modular testing toidentify the cause of the problem.

<1% RSD

Injection volumeaccuracy

Important for analyses requiringaccurate injections or liquidhandling (e.g. dilution) prior toanalysis; mostly not the case.

Can be determined by measuring the weight of a sample of known density drawn from a vial, butthis does not necessarily guarantee that this volume will be injected onto the column.

Whilst modular testing can provide evidence that injection volume accuracy is withinspecification, constant volumes of sample and standard are normally injected and, therefore,injection volume precision is more important.

notnormallyspecified

Injection volumelinearity

Important for analyses requiringaccurate injections or liquidhandling (e.g. dilution) prior toanalysis; mostly not the case.

Can be determined by measuring the weight of a sample of known density drawn from a vial (overthe operational or required injection volume range), but this does not necessarily guarantee thatthis volume will be injected onto the column.

Whilst modular testing can provide evidence that injection volume linearity is within specification,constant volumes of sample and standard are normally injected and, therefore, injection volumeprecision is more important.

notnormallyspecified

Autosampler Thermostattingaccuracy

Important for comparability whentransferring methods betweensystems.

Can be determined by measuring and comparing the selected temperature with the actualtemperature inside the autosampler, for example, using a calibrated temperature probe.

Can be repeated at different temperatures to determine the autosampler thermostatting linearity -the temperature accuracy over the operational or required thermostatting range.

±2ºC (forsystems withtemperature

control)

Thermostattingprecision

Important for system repeatabilityand comparability between/duringanalyses.

Can be determined by measuring the temperature inside the autosampler, for example, using acalibrated temperature probe, over a defined period of time.

±2ºC (forsystems withtemperature

control)

LGC/VAM/1998/026.2 Page 18

Table 7 - Operational Qualification of HPLC Instruments (continued)


Solventdeliverysystem

Flow rateaccuracy


Can be determined by attaching a capillary to generate back pressure and measuring the volume ofmobile phase delivered over a set period of time.

Can also be used to check flow rate linearity - i.e. accuracy over the operational or required flowrate range.

Although flow rate accuracy can be verified at a modular level, it may be more convenient tomeasure and verify flow rate during holistic (initial PQ) testing of the instrument.

±3% at upto 5000psi(~345bar)

Flow rateprecision

Important for repeatability ofretention times and peakarea/height.

Can be determined at a modular level by attaching a capillary to generate a back pressure andvolumetrically measuring the flow rate at several intervals over time.

Although flow rate precision can be verified at a modular level, it may be more convenient tomeasure and verify flow rate precision during holistic (initial PQ) testing of the instrument.

±3% at upto 5000psi(~345bar)

Proportioningaccuracy


Can be determined at the modular level by running an additive through one of the solvent channelsand independently measuring the concentration of the additive in the mobile phase followingstepped or constant percentage increases or decreases in the solvent channel.

Although proportioning accuracy can be verified at a modular level, it may be more convenientlyverified using a holistic test, for example, by running an additive through one of the solventchannels and measuring the relative response of the detector to stepped or constant percentageincreases or decreases in the solvent channel.

Proportioningprecision


Can be determined by running an additive through one of the solvent channels and measuring theresultant concentration of the additive in the mobile phase following stepped increases anddecreases in solvent composition over time.

Although proportioning precision can be verified at a modular level, it may be more convenientlyverified using a holistic test, for example, by running an additive through one of the solventchannels and measuring the relative response of the detector to stepped or constant percentageincreases or decreases in the solvent channel.

LGC/VAM/1998/026.2 Page 19



Column Oven Thermostattingaccuracy


Can be determined by measuring the temperature inside the column oven (e.g. using a calibratedtemperature probe).

Can also be used to determine the column oven thermostatting linearity - i.e. accuracy oftemperature over the operational or required thermostatting range.

±1ºC

Thermostattingprecision


Can be determined by measuring the temperature inside the column oven (e.g. using a calibratedtemperature probe) over a defined period of time.

±1ºC

Detector Wavelengthaccuracy

Important for accuracy of resultsand comparability whentransferring methods betweensystems.

Can be determined by comparing the measured absorbance maxima with the absorbance maximaof a reference material (e.g. holmium perchlorate solution or holmium oxide filter).

Although diagnostics which facilitate wavelength calibration are built into many detectors,independent checks (using traceable reference materials) can provide valuable evidence that anycalibration adjustments are correct and the wavelength accuracy is within specification.

±2 nm

Linearity ofdetectorresponse

Important for accuracy of results. Can be determined by assessing the linearity between the detector’s output voltage and theconcentration of a material.

Although assessing the linearity of detector response at the modular level may demonstrate thatthe detector is within specification and also provide information on the boundaries ofperformance, it provides little assurance of guaranteed linearity for different compoundsencountered in actual use.

Noise Important for sensitivity and limitof detection.

Can be determined from the amplitude of random variations in the detector’s signal over time.

Drift Important for sensitivity and limitof detection.

Can be determined from the slope of the amplitude of random variations in the detector’s signalover time.

Both short-term noise, long-term noise and drift can be determined under static (no flow) and dynamic (flow) conditions. Althoughmodular testing under static conditions and holistic testing under dynamic conditions may provide evidence that the detector is withinspecification, it provides little indication or assurance of sensitivity of the detector during actual use (i.e. under a particular or changingmobile phase composition).

LGC/VAM/1998/026.2 Page 20



DataHandling

Accuracy andprecision

Important for accurate and precisemeasurement of chromatographicpeaks, including partially resolved,broad or asymmetric peaks.

Can be determined and verified using software packages or peak output simulators.

Although possible to qualify at a modular level, the value of such testing is questionable. Ingeneral, satisfactory performance would be inferred during PQ/SSC testing of the entireinstrument.

LGC/VAM/1998/026.2 Page 21

7. Performance Qualification (PQ)7.1 The purpose of PQ is to provide evidence that the entire HPLC instrument system

functions correctly and to ensure continued satisfactory performance during routine use.

7.2 For convenience, PQ can be considered as having two stages:

Initial PQ - performance testing following OQ to provide evidence that the completeHPLC instrument system functions correctly (some suppliers may include this type ofholistic testing, for example, to verify the absence of carryover, as part of OQ); and

Ongoing PQ - system suitability checking (SSC) to ensure fitness for purpose andcontinued satisfactory performance during actual use.

7.3 Following OQ, a supplier would normally be expected to carry out a holisticperformance test to verify the correct functioning and performance of the entireinstrument system. This “ Initial PQ” usually involves analysing a ‘test mix’ using a‘test column’ under defined operating conditions and thus enables the instrument to beevaluated using a well-characterised test procedure and its performance to be comparedwith that obtained previously or in the future. It also enables the performance of theinstrument to be compared with that of other instruments, either in the same laboratoryor elsewhere. As such, this provides evidence that the instrument is functioning notonly correctly, but that its performance is also predictable, comparable and withinspecification. Typical parameters verified during PQ are summarised in Table 8.

7.4 However, whilst this type of holistic testing provides valuable evidence of satisfactoryperformance under one particular set of conditions, the actual conditions or range ofconditions under which an instrument is normally used may be significantly different.During normal routine use it is also highly likely that the performance of an HPLCinstrument will change over time. Gradual deterioration in performance may result fromcontamination and normal wear of parts (e.g. contamination of a flow cell, wear to apump’s piston seals, or loss of intensity from a detector source). There may also bemore sudden changes in performance due to failure of the instrument or one of itscomponents.

7.5 The user must, therefore, carry out further checks and tests to demonstrate and providecontinued evidence of system suitability and satisfactory instrumental performanceduring actual use. The user should establish appropriate procedures to monitor keyperformance characteristics and set warning and action thresholds outside which theinstrument’s performance is deemed to be no longer acceptable for its actual use (e.g.when the retention time or the response to a standard solution is not as expected). Thesechecks and tests need not be burdensome and can be built into system suitabilitychecking and analytical quality control (AQC).

LGC/VAM/1998/026.2

7.6 It is strongly recommended that SSC is undertaken before and during analysis toconfirm that the overall measurement system (chromatographic method and instrument)is performing satisfactorily during actual use. SSC not only provides the user withconfidence that the chromatographic conditions (e.g. specificity, resolution, detectionlimits) are satisfactory, but when carried out in conjunction with appropriate calibrationand AQC procedures SSC also provides evidence that the instrument’s performance(accuracy, precision and linearity) continues to be fit for purpose.

7.7 As a bare minimum users must demonstrate that the precision and linearity of theinstrument are satisfactory prior to actual use. Linearity should be assessed bycalibrating the instrument with a series of standard solutions which covers the range ofanticipated results, plus a safety margin. This type of calibration should be performedbefore sample analysis or at an interval specified in a standard operating procedure, thefrequency of which should, as a minimum, be based on the period over which theinstrument has previously been found to remain within calibration. The precisionshould be determined from the relative standard deviation (RSD) of responses toreplicate injections of a standard solution. Acceptable precision is often defined inmethods as part of system suitability requirements but, as a rule of thumb, RSDsgreater than 1% are generally unacceptable. During use, a control solution should beanalysed at regular intervals to confirm that the instrument remains within calibration.(Note: Furman et al [7] recommend that: the entire instrument system should becalibrated for linearity by injecting at least four standard solutions; that precisionshould be determined from the RSD of responses to at least six replicate injections of asingle standard solution; and that a standard solution should be run at regular intervals(every 2 hours or every 5 samples) to confirm that the instrument remains withincalibration.)

7.8 SSC can also provide an indication of which parts of the measurement system are notperforming satisfactorily. For example, if the response for a standard solution is low orvariable, then this may indicate problems with the detector or injection system - thedetector source may have deteriorated or failed, or the injector become contaminated orblocked. Similarly, variable retention times may infer problems with solvent delivery ormixing.

LGC/VAM/1998/026.2 Page 23

Table 8 - Performance Qualification of HPLC Instruments

Parameter Reason Procedure TypicalTolerance

Injection volume precision Important for accuracy and precision of results andrepeatability of peak area/height.

Can be determined from the relative standard deviation (RSD) in detectorresponse (peak area/height) to repeated injections of the same volume of astandard solution.

<1% RSD

Injection volume linearity Important where variable volumes are injected -mostly not the case.

Can be determined from the linearity between the detector response (peakarea/height) and the volume of standard solution injected.

Injection carryover Important for accuracy and precision of results andrepeatability of peak area/height.

Can be determined by measuring the detector response to a blank immediatelyfollowing a standard solution.

Note - not to be confused with “ghosting” under gradient analysis.

method specific

Flow rate precision Important for repeatability of retention times andpeak area/height.

Can be determined from repeatability of retention times of a defined “test peak”. <0.5% RSD RT

Proportioning precision Important for repeatability of retention times andpeak area/height.


Column oven thermostattingprecision

Important for repeatability of retention times andpeak area/height.


Linearity of detector response Important for accuracy of results. Can be determined from the linearity between detector response (peakarea/height) and standard solution concentration.

method specific

Signal to noise ratio Important for sensitivity and limit of detection. Can be determined from the response of a detector to a dilute standard solutionand a blank.

method specific

LGC/VAM/1998/026.2

8. References

1. Quality Systems - Model for quality assurance in design, development, production,installation and servicing. BS EN ISO 9001:1994.

2. The Good Laboratory Practice Regulations 1997. Statutory Instrument No.654. TheStationery Office.

3. Good Laboratory Practice for Non-clinical Laboratory Studies. Food and DrugAdministration (FDA); 21 CFR Ch.1 Part 58.

4. General requirements for the competence of calibration and testing laboratories.ISO/IEC Guide 25, 3rd Ed., 1990. (note new version in draft stage)

5. The development and application of guidance on equipment qualification of analyticalinstruments.P Bedson and M Sargent, Accred. Qual. Assur. (1996)1:265-274.

6. Evaluation of analytical instrumentation: Part IX Instrumentation for High PerformanceLiquid Chromatography.Analytical Methods Committee, Analyst (1997)122:387-392.

7. Validation of computerised liquid chromatographic systems.W Furman, T Layoff & R Tetzlaff, J. AOAC International (1994)77(5):1314-1318.

LGC/VAM/1998/026.2 Page 25

9. Bibliography

1. Requirements and tests for HPLC Apparatus and Methods in Pharmaceutical QualityControl. G Maldener, Chromatographia (1997)28:85-88.

2. Quality assurance and instrumentation.L Huber, Accred. Qual. Assur. (1996)1:24-34.

3. Applying the validation timeline to HPLC system validation.W Maxwell & J Sweeney, LC:GC International (1994)12(9):678-682.

4. Functional control of HPLC instruments according to quality standards.W Beinhart, LC:GC International

5. Standard practice for testing fixed-wavelength photometric detectors used in liquidchromatography, ASTM E685, 1983 Annual Book of ASTM Standards Part 14.01,American Society for Testing and Materials.

6. Position paper on the qualification of analytical equipment.M Freeman, M Leng, D Morrison and R P Munden,Pharm. Tech. Eur. (1995):7(10):40-46

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