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National Measurement

System 1997-2000
Valid Analytical
Measurement (VAM)
Programme

Guidance on Equipment
Qualification of Analytical
Instruments: High
Performance Liquid
Chromatography (HPLC)

June 1998
LGC/VAM/1998/026.2

LGC
Setting standards
in analytical science
National Measurement
System 1997-2000 Valid
Analytical Measurement
(VAM) Programme

Guidance on Equipment
Qualification of Analytical
Instruments: High Performance
Liquid Chromatography (HPLC)

June 1998

Contact Point:
Peter Bedson
Tel: 0181 943 7392

The development of this guidance was supported


under contract with the Department of Trade
and Industry as part of the National
Measurement System Valid Analytical
Measurement (VAM) Programme

LGC/VAM/1998/026.2
© LGC (Teddington) Limited 1998
Preface
This 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 High
Performance Liquid Chromatography (HPLC) Sub-Group, have played a major role in developing this
document and their help is gratefully acknowledged. The Secretariat would also like to thank those who
commented 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 amended
text republished. Users are invited to comment on the content of the existing text and suggest additional
material in writing to:
Mr P J Bedson, Secretary, Instrumentation Working Group
LGC, 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
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 i
1. Glossary
Many of the terms relating to equipment qualification are used in different ways to
convey a variety of meanings. The following descriptions explain how these terms
should be interpreted in this guidance document.

1.1 Instrument: an entire High Performance Liquid Chromatography (HPLC) measurement


system comprising either a fully integrated system or a series of interconnected
modules.

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 performance
of a distinct component of the instrument.

1.4 Holistic testing: the process of verifying the correct functioning and performance of
entire 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 defines
the overall requirements for the instrument, the key performance characteristics of the
instrument and ranges over which the instrument is required to operate and consistently
perform, and other critical factors relating to its use.

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

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

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

1.11 Operational Qualification (OQ): the process of undertaking confirmatory checks to


verify key aspects of performance in the absence of any contributory effects which may
be introduced by the method.

LGC/VAM/1998/026.2 Page 1
1.12 Performance Qualification (PQ): is defined as the process of demonstrating that an
instrument consistently performs according to a specification appropriate for its routine
use.

1.13 Validation: is the process of evaluating the performance of a specific measuring


procedure and checking that the performance meets certain pre-set criteria. Validation
establishes and provides documented evidence that the measuring procedure is fit for a
particular purpose.

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

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

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

1.17 Traceability: The property of a result of a measurement whereby it can be related to


appropriate standards, generally national or international standards, through an
unbroken chain of comparisons.

1.18 Standard Solution: A solution or matrix containing a known concentration of an


analyte. Standard solutions are often used to check the performance of the instrument
and establish the relationship between instrumental response and analyte concentration.

Page 2 LGC/VAM/1998/026.2
2. The equipment qualification process
2.1 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 and
calibration 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 of
each stage is summarised in Figure 1.

Design
Defines the specifications of the instrument
Qualification and details the conscious decisions in the
(DQ) selection of the supplier

Establishes that the instrument is received as


Installation
designed and specified, that it is properly
Qualification installed in the selected environment, and
(IQ) that this environment is suitable for the
operation of the instrument

Operational Confirmatory checks to verify key aspects of


Qualification performance in the absence of contributory
(OQ) effects which might be introduced by the
method

Performance
The process of demonstrating that an
Qualification instrument performs according to a
(PQ) 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 the
process of purchasing a new instrument, although it may be appropriate to repeat
aspects of DQ following a major change to the instrument or its use. Whilst
qualification of the actual instrument design is for manufacturers of instruments, users
of instruments have an important role in DQ by ensuring adoption of a user requirement
specification (URS) which meets the intended use.

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

LGC/VAM/1998/026.2 Page 3
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 at
defined intervals (e.g. annually).

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

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

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

2.8 Although formal quality standards [1,2,3,4] all require various combinations of method
validation and equipment qualification, there is a lack of clear guidance on when EQ is
appropriate, what is actually required, how it should be achieved and how it should be
documented. A key objective in developing guidance [5] has been, therefore, to provide
users and suppliers of analytical instruments, as well as those responsible for the
assessment, certification and monitoring of analytical laboratories, with a clear and
consistent approach to EQ. The guidance has been prepared with the primary aim of
assisting the interpretation of formal quality standards in order to satisfy regulatory and
accreditation requirements.

2.9 EQ must be documented (Section 4 of the general guidance [5] provides more detailed
guidance on requirements for EQ documentation). EQ documentation can be prepared
and 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 readily
followed and understood by the user.

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

Page 4 LGC/VAM/1998/026.2
2.11 The user must establish the level of EQ required and what aspects of EQ (particularly
OQ) 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 must
satisfy themselves that it has been carried out competently and correctly (evidence of
current competence should be established, for example, through a valid training record
or certificate).

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

2.13 EQ provides important evidence of an instrument’s satisfactory performance and its


fitness for the purpose for which it is used. However, EQ is just one of a range of
activities which contribute towards achieving and demonstrating reliable and valid data.

LGC/VAM/1998/026.2 Page 5
3. High Performance Liquid
Chromatography
3.1 A high performance liquid chromatography (HPLC) system consists of a number of
components which can either be purchased as individual modules, from one or more
suppliers, or as a completely integrated system.

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

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

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

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, detection
wavelength) are within their specified limits for accuracy and precision. This provides
confidence that the instrument is operating correctly, to specification, and that there are
no unacceptable differences between a parameter’s selected and actual values. For
example, when the pump is set to deliver 1.0ml per minute the actual flow is within
required 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 any
contributory effects that might be introduced by the method, OQ performance tests are
designed to check the performance of individual modules without involving the quality
of 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 the
everyday functioning of the system to maintain data integrity are intentionally
minimised in OQ.

Page 6 LGC/VAM/1998/026.2
3.7 However, although OQ testing might provide useful evidence that the individual
modules of the instrument are operating correctly and within specification, such tests
alone cannot guarantee satisfactory performance of the entire instrument system either
following initial assembly or on an ongoing basis.

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

Solvent
delivery
system

Injector

System
controller
Column oven

Detector Data handling device

Figure 2 - Schematic diagram of HPLC system

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

LGC/VAM/1998/026.2 Page 7
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 demonstrate
that the instrument’s fitness for purpose has been considered at an early stage and built
into the procurement process.

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

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

4.3 In undertaking DQ, information and knowledge of existing equipment should be taken
into account. If an instrument is mature in design and has a proven track record, this
may provide a basic confidence and evidence about its suitability for use. For new
techniques 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 that
regulators are likely to require evidence of: the use of rigorous design and specification
methods; fully documented quality control and quality assurance procedures; the use, at
all times of suitably qualified and experienced personnel; comprehensive, planned
testing of the system at all levels of the system; and the application of stringent change
control, error reporting and corrective procedures. A suitable questionnaire, third party
audit, or independent certification of the supplier to an approved quality scheme may
provide the user with evidence that regulatory requirements have been met during
design and manufacture of the instrument. Where such evidence is not available, it is
the responsibility of the user to carry out more extensive qualification in order to
provide the necessary assurance of the instrument’s fitness for use.

4.5 Where instruments are intended to be used to make measurements supporting


regulatory studies, the user may also need to seek confirmation that the manufacturer is
prepared, if required, to allow regulatory authorities access to detailed information and
records relating to the instrument’s manufacture and development, for example: source
codes; instrument development records and procedures; calibration and qualification
documentation; batch test records and reports; hardware and software qualification
documentation; and credentials of staff involved with the development of the
instrument.

Page 8 LGC/VAM/1998/026.2
4.6 Detailed listings of criteria to be considered when selecting and purchasing HPLC
instrumentation have been published by the Analytical Methods Committee of the
Royal Society of Chemistry [6]. Tables 1-5 summarise key features which might be
considered 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 of
issue.
Size, power & utility requirements Limitations on, requirements for and expected consumption of
services, 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 and
calibration 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 of
any training necessary and courses provided by the supplier.
Environmental conditions Environmental conditions within which, or range over which, the
instrument must work.
Health and Safety Health and safety and environmental issues and/or requirements

LGC/VAM/1998/026.2 Page 9
Table 2 - Design Qualification of HPLC Instruments - Injection Systems

Feature Consideration Specification


System control and Ability fully to select, control, store and Stand-alone programmability
communication retrieve injection parameters locally within requirements.
injector.
Ability to send / accept signals (e.g. through Requirements for communicating
contact closures) and to communicate (e.g. with other devices
RS232) with other devices.
Ability fully to control injector from a remote Integrated system control
PC or system controller. requirements.
Ability to store and retrieve injection
parameters as part of the method in a PC or
remote system controller.
Manual or automatic Ability to carry out unattended injections. Manual or automatic injection.
injection
Autosampler capacity Ability to carry out unattended analysis of Autosampler capacity.
multiple samples.
Autosampler Ability to thermostatically control sample Temperature range, accuracy and
thermostatting temperature. stability.
Injector temperature Ability to minimise temperature effects and Sample loop/valve pre-heat range
control chromatographic distortion through injection of and limits.
sample at same temperature as mobile phase.
Injection volume Ability to make injections of fixed or variable Injection volume range.
sample volumes.
Ability to make variable volume injections Constant or variable volume loop.
without changing loops.
Ability / ease of changing injection loops. Range of loop sizes required.
Ability to make accurate and repeatable Injection volume linearity.
injections across the operating range.
Injection volume precision.
Sample capacity Ability to make injections from small sample Minimum volume of sample
volumes. required to make an injection.
Carryover Minimising carryover improves accuracy and Limits of carryover.
reproducibility of results.
Needle wash Ability to flush or wash injector / needle to Needle / injector wash
minimise cross-contamination of samples and requirements.
reduce carryover.
Operating pressure Ability to make injection at required system Operating pressure range and
operating pressure. maximum operating pressure.
Liquid transfer & Ability to perform liquid transfer and dilution.
dilution
Sample preparation Ability to carry out sample pre-treatment and
concentration.

Page 10 LGC/VAM/1998/026.2
Table 3 - Design Qualification of HPLC Instruments - Solvent Delivery Systems

Feature Consideration Specification


System control and Ability fully to select, control, store and retrieve Stand-alone programmability
communication operational conditions (mobile phase flow rate, requirements.
composition, and gradient programmes) locally
within SDS.
Ability to send / accept signals (e.g. through Requirements for communicating
contact closures) and to communicate (e.g. with other devices
RS232) with other devices.
Ability fully to control SDS from a remote PC or Integrated system control
system controller. requirements.
Ability to store and retrieve SDS parameters as
part of the method in a PC or remote system
controller.
Mobile phase Ability to perform analysis under isocratic or Number of solvent channels
proportioning/mixing gradient conditions.
Mobile phase Ability to mix solvents accurately and Limits for proportioning accuracy
proportioning/mixing reproducibly over the required gradient range. and precision over the required
accuracy & precision range.
Mobile phase Ability to degass and filter solvents in situ. Degassing & filtration requirements
degassing and - inlet pressure limits for Helium
filtration
Eluent / column Ability to switch between eluents and/or
switching columns.
Mobile phase Ability to re-use mobile phase.
recycling
Flow range Ability to deliver a wide range of flow rates of Flow range and incremental step
solvents required. required.
Flow rate accuracy, Ability to deliver accurate flow rates, Accuracy, precision and stability of
stability and precision reproducibly, over a long period of time. flow rate over the specified range.
Solvent storage Ability to perform long unattended runs using Number and capacity of solvent
mobile phases which comprise one or more reservoirs
solvent mixtures.
Spillage containment Ability to detect leaks and spillage Automatic shut down
Gradient delay volume Ability to deliver changes in mobile phase Dead volume
composition with minimal delay.

LGC/VAM/1998/026.2 Page 11
Table 4 - Design Qualification of HPLC Instruments - UV/VIS Detectors

Feature Consideration Specification


System control and Ability fully to select, control, store and retrieve Stand-alone programmability
communication operational conditions (wavelength, bandwidth) requirements.
locally within detector.
Ability to send / accept signals (e.g. through Requirements for communicating
contact closures) and to communicate (e.g. with other devices
RS232) with other devices.
Ability fully to control detector from a remote Integrated system control
PC or system controller. requirements.
Ability to store and retrieve operational
conditions as part of the method in a PC or
remote system controller.
Detector type Ability to monitor a single, multiple or variable Detector type: single wavelength;
wavelengths and/or full spectral characteristics. continuously variable wavelength; or
diode array.
Flow cells Ability easily to remove and clean flow cells, to Requirements for removal and
inject liquid standards, and to connect detectors cleaning of flow cells, injection of
in series without band broadening or loss of liquid standards. Dead volume
chromatographic efficiency. limits. Sensitivity to back pressure.
Ability to change flow cell for other uses, e.g.
microbore / preparative applications.
Wavelength accuracy Ability accurately and reproducibly to select Accuracy and precision of
wavelengths monitored. wavelength selection.
Wavelength range Ability to select and monitor a wide range of Wavelength range. Ease of lamp
wavelengths, with or without changing source. changing.
Linear dynamic range - Ability for accurate quantitation over a large Linear dynamic range
stray light concentration range
Noise Low noise facilitates improved sensitivity and Signal to noise ratio
lower detection limits
Drift Low drift facilitates improved sensitivity and Flow cell thermostatting and
lower detection limits sensitivity to back pressure
Spillage containment Ability to detect leaks and spillage Automatic shut down

Page 12 LGC/VAM/1998/026.2
Table 5 - Design Qualification of HPLC Instruments - Column Ovens

Feature Consideration Specification


System control and Ability fully to select, control, store and retrieve Stand-alone programmability
communication operational conditions (wavelength, bandwidth) requirements.
locally within detector.
Ability to send / accept signals (e.g. through Requirements for communicating
contact closures) and to communicate (e.g. with other devices
RS232) with other devices.
Ability fully to control detector from a remote Integrated system control
PC or system controller. requirements.
Ability to store and retrieve operational
conditions as part of the method in a PC or
remote system controller.
Size Ability to accommodate a range of columns Number and size of columns that
(including guard columns) of different sizes. can be accommodated.
Ability easily to access, install or replace
columns.
Temperature control Ability to reach thermostatting temperatures Equilibration time.
quickly and to maintain accurate and stable
temperatures over a range of external Accuracy, precision and stability
conditions, flow rates and solvents. over required temperature range.

Spillage containment Ability to detect leaks and spillage Automatic shut down

LGC/VAM/1998/026.2 Page 13
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 is
properly installed in the selected environment.

5.2 IQ may be carried out either by the supplier and/or the user. However, it should be
noted that, in some cases, the complexity of the instrument alone may preclude the user
performing IQ and, in others, the unpacking of the equipment by the user may
invalidate 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 recorded
and, where these have been carried out by the supplier, the results of these tests must be
communicated to the user.

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

Has the instrument been checked and verified as undamaged? ü

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

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

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

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

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

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

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

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

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

Page 14 LGC/VAM/1998/026.2
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 the
method.

6.2 Instrumental operating conditions (e.g. injection volume, mobile phase flow rate,
gradient composition, column thermostatting, detection wavelength) are normally
specified as part of an HPLC method. Whilst many methods might be robust to small
differences between the selected and actual value of an operating condition, significant
differences may impact on the validity of the method and the data generated by it. The
role of OQ can, therefore, be considered as the process of checking that key operating
conditions 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) stages
throughout the life of the instrument.

6.4 Whilst suppliers should provide advice on recommended intervals and events after
which OQ testing should be repeated, the responsibility for defining and setting the
frequency and extent of OQ testing rests with users of instruments. This is because the
need and frequency of OQ testing will depend on a variety of factors, not least the level
and 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 usually
linked to and carried out following planned events such as routine maintenance or
replacement of consumable parts (e.g. replacement of piston seals in a pump). This may
be the only planned or defined interval at which OQ testing is carried out to verify
some 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 less
stable aspects of performance.

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

• the manufacturer’s recommended intervals;

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

• the level of use of the instrument (higher workloads might accelerate


deterioration in overall performance and therefore necessitate more frequent OQ
testing);

LGC/VAM/1998/026.2 Page 15
• the nature of use (aggressive solvents may cause faster deterioration of pump
performance; some sample matrices may cause faster deterioration of injector
performance);

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

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

6.7 For event-driven OQ, the extent to which OQ is repeated will depend on the impact that
the event has on the performance of the instrument. For example, whilst the
replacement of a flow cell is likely to affect the performance of the detector, it is
unlikely to impact on the performance of the injection system. Therefore, although it
will be necessary to repeat OQ to verify the performance of the detector, it should not
be 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 Table
7. It is important to emphasise that this is not intended to be an exhaustive list of
checks and tests that must be carried out under all circumstances. Users must exercise
their professional judgement as to the checks which are relevant and extent of testing
required.

Page 16 LGC/VAM/1998/026.2
Table 7 - Operational Qualification of HPLC Instruments

Module Parameter Reason Procedure / Comments Typical


Tolerance
Injector Injection volume Important for system repeatability Can be determined from the relative standard deviation (RSD) of weights of a sample of known <1% RSD
precision and precision of results. density drawn from a vial (repeatedly carrying out the injection volume accuracy test and
determining the RSD).
Injection volume precision can be affected by a variety of factors (e.g. sample viscosity) and the
value of such testing at the modular level is questionable. In general, it is recommended that
holistic testing (initial PQ) be used to verify that the injector precision is within specification and
that SSC be used to provide continued evidence of precision during routine use. However, where
PQ/SSC indicates problem with precision, it might be necessary to carry out modular testing to
identify the cause of the problem.
Injection volume Important for analyses requiring Can be determined by measuring the weight of a sample of known density drawn from a vial, but not
accuracy accurate injections or liquid this does not necessarily guarantee that this volume will be injected onto the column. normally
handling (e.g. dilution) prior to specified
analysis; mostly not the case. Whilst modular testing can provide evidence that injection volume accuracy is within
specification, constant volumes of sample and standard are normally injected and, therefore,
injection volume precision is more important.
Injection volume Important for analyses requiring Can be determined by measuring the weight of a sample of known density drawn from a vial (over not
linearity accurate injections or liquid the operational or required injection volume range), but this does not necessarily guarantee that normally
handling (e.g. dilution) prior to this volume will be injected onto the column. specified
analysis; mostly not the case.
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 volume
precision is more important.
Autosampler Thermostatting Important for comparability when Can be determined by measuring and comparing the selected temperature with the actual ±2ºC (for
accuracy transferring methods between temperature inside the autosampler, for example, using a calibrated temperature probe. systems with
systems. temperature
Can be repeated at different temperatures to determine the autosampler thermostatting linearity - control)
the temperature accuracy over the operational or required thermostatting range.
Thermostatting Important for system repeatability Can be determined by measuring the temperature inside the autosampler, for example, using a ±2ºC (for
precision and comparability between/during calibrated temperature probe, over a defined period of time. systems with
analyses. temperature
control)

LGC/VAM/1998/026.2 Page 17
Table 7 - Operational Qualification of HPLC Instruments (continued)

Module Parameter Reason Procedure / Comments Typical


Tolerance
Solvent Flow rate Important for comparability when Can be determined by attaching a capillary to generate back pressure and measuring the volume of ±3% at up
delivery accuracy transferring methods between mobile phase delivered over a set period of time. to 5000psi
system systems. (~345bar)
Can also be used to check flow rate linearity - i.e. accuracy over the operational or required flow
rate range.
Although flow rate accuracy can be verified at a modular level, it may be more convenient to
measure and verify flow rate during holistic (initial PQ) testing of the instrument.
Flow rate Important for repeatability of Can be determined at a modular level by attaching a capillary to generate a back pressure and ±3% at up
precision retention times and peak volumetrically measuring the flow rate at several intervals over time. to 5000psi
area/height. (~345bar)
Although flow rate precision can be verified at a modular level, it may be more convenient to
measure and verify flow rate precision during holistic (initial PQ) testing of the instrument.
Proportioning Important for comparability when Can be determined at the modular level by running an additive through one of the solvent channels
accuracy transferring methods between and independently measuring the concentration of the additive in the mobile phase following
systems. stepped or constant percentage increases or decreases in the solvent channel.
Although proportioning accuracy can be verified at a modular level, it may be more conveniently
verified using a holistic test, for example, by running an additive through one of the solvent
channels and measuring the relative response of the detector to stepped or constant percentage
increases or decreases in the solvent channel.
Proportioning Important for repeatability of Can be determined by running an additive through one of the solvent channels and measuring the
precision retention times and peak resultant concentration of the additive in the mobile phase following stepped increases and
area/height. decreases in solvent composition over time.
Although proportioning precision can be verified at a modular level, it may be more conveniently
verified using a holistic test, for example, by running an additive through one of the solvent
channels and measuring the relative response of the detector to stepped or constant percentage
increases or decreases in the solvent channel.

LGC/VAM/1998/026.2 Page 18
Table 7 - Operational Qualification of HPLC Instruments (continued)

Module Parameter Reason Procedure / Comments Typical


Tolerance
Column Oven Thermostatting Important for comparability when Can be determined by measuring the temperature inside the column oven (e.g. using a calibrated ±1ºC
accuracy transferring methods between temperature probe).
systems.
Can also be used to determine the column oven thermostatting linearity - i.e. accuracy of
temperature over the operational or required thermostatting range.
Thermostatting Important for repeatability of Can be determined by measuring the temperature inside the column oven (e.g. using a calibrated ±1ºC
precision retention times and peak temperature probe) over a defined period of time.
area/height.
Detector Wavelength Important for accuracy of results Can be determined by comparing the measured absorbance maxima with the absorbance maxima ±2 nm
accuracy and comparability when of a reference material (e.g. holmium perchlorate solution or holmium oxide filter).
transferring methods between
systems. Although diagnostics which facilitate wavelength calibration are built into many detectors,
independent checks (using traceable reference materials) can provide valuable evidence that any
calibration adjustments are correct and the wavelength accuracy is within specification.
Linearity of Important for accuracy of results. Can be determined by assessing the linearity between the detector’s output voltage and the
detector concentration of a material.
response
Although assessing the linearity of detector response at the modular level may demonstrate that
the detector is within specification and also provide information on the boundaries of
performance, it provides little assurance of guaranteed linearity for different compounds
encountered in actual use.
Noise Important for sensitivity and limit Can be determined from the amplitude of random variations in the detector’s signal over time.
of detection.
Drift Important for sensitivity and limit Can be determined from the slope of the amplitude of random variations in the detector’s signal
of detection. over time.
Both short-term noise, long-term noise and drift can be determined under static (no flow) and dynamic (flow) conditions. Although
modular testing under static conditions and holistic testing under dynamic conditions may provide evidence that the detector is within
specification, it provides little indication or assurance of sensitivity of the detector during actual use (i.e. under a particular or changing
mobile phase composition).

LGC/VAM/1998/026.2 Page 19
Table 7 - Operational Qualification of HPLC Instruments (continued)

Module Parameter Reason Procedure / Comments Typical


Tolerance
Data Accuracy and Important for accurate and precise Can be determined and verified using software packages or peak output simulators.
Handling precision measurement of chromatographic
peaks, including partially resolved, Although possible to qualify at a modular level, the value of such testing is questionable. In
broad or asymmetric peaks. general, satisfactory performance would be inferred during PQ/SSC testing of the entire
instrument.

LGC/VAM/1998/026.2 Page 20
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 complete


HPLC instrument system functions correctly (some suppliers may include this type of
holistic 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 and
continued satisfactory performance during actual use.

7.3 Following OQ, a supplier would normally be expected to carry out a holistic
performance test to verify the correct functioning and performance of the entire
instrument system. This “ Initial PQ” usually involves analysing a ‘test mix’ using a
‘test column’ under defined operating conditions and thus enables the instrument to be
evaluated using a well-characterised test procedure and its performance to be compared
with that obtained previously or in the future. It also enables the performance of the
instrument to be compared with that of other instruments, either in the same laboratory
or elsewhere. As such, this provides evidence that the instrument is functioning not
only correctly, but that its performance is also predictable, comparable and within
specification. Typical parameters verified during PQ are summarised in Table 8.

7.4 However, whilst this type of holistic testing provides valuable evidence of satisfactory
performance under one particular set of conditions, the actual conditions or range of
conditions 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 HPLC
instrument will change over time. Gradual deterioration in performance may result from
contamination and normal wear of parts (e.g. contamination of a flow cell, wear to a
pump’s piston seals, or loss of intensity from a detector source). There may also be
more sudden changes in performance due to failure of the instrument or one of its
components.

7.5 The user must, therefore, carry out further checks and tests to demonstrate and provide
continued evidence of system suitability and satisfactory instrumental performance
during actual use. The user should establish appropriate procedures to monitor key
performance characteristics and set warning and action thresholds outside which the
instrument’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). These
checks and tests need not be burdensome and can be built into system suitability
checking and analytical quality control (AQC).

LGC/VAM/1998/026.2 Page 21
7.6 It is strongly recommended that SSC is undertaken before and during analysis to
confirm that the overall measurement system (chromatographic method and instrument)
is performing satisfactorily during actual use. SSC not only provides the user with
confidence that the chromatographic conditions (e.g. specificity, resolution, detection
limits) are satisfactory, but when carried out in conjunction with appropriate calibration
and 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 the
instrument are satisfactory prior to actual use. Linearity should be assessed by
calibrating the instrument with a series of standard solutions which covers the range of
anticipated results, plus a safety margin. This type of calibration should be performed
before sample analysis or at an interval specified in a standard operating procedure, the
frequency of which should, as a minimum, be based on the period over which the
instrument has previously been found to remain within calibration. The precision
should be determined from the relative standard deviation (RSD) of responses to
replicate injections of a standard solution. Acceptable precision is often defined in
methods as part of system suitability requirements but, as a rule of thumb, RSDs
greater than 1% are generally unacceptable. During use, a control solution should be
analysed at regular intervals to confirm that the instrument remains within calibration.
(Note: Furman et al [7] recommend that: the entire instrument system should be
calibrated for linearity by injecting at least four standard solutions; that precision
should be determined from the RSD of responses to at least six replicate injections of a
single 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 within
calibration.)

7.8 SSC can also provide an indication of which parts of the measurement system are not
performing satisfactorily. For example, if the response for a standard solution is low or
variable, then this may indicate problems with the detector or injection system - the
detector source may have deteriorated or failed, or the injector become contaminated or
blocked. Similarly, variable retention times may infer problems with solvent delivery or
mixing.

Page 22 LGC/VAM/1998/026.2
Table 8 - Performance Qualification of HPLC Instruments

Parameter Reason Procedure Typical


Tolerance
Injection volume precision Important for accuracy and precision of results and Can be determined from the relative standard deviation (RSD) in detector <1% RSD
repeatability of peak area/height. response (peak area/height) to repeated injections of the same volume of a
standard solution.
Injection volume linearity Important where variable volumes are injected - Can be determined from the linearity between the detector response (peak
mostly not the case. area/height) and the volume of standard solution injected.
Injection carryover Important for accuracy and precision of results and Can be determined by measuring the detector response to a blank immediately method specific
repeatability of peak area/height. following a standard solution.
Note - not to be confused with “ghosting” under gradient analysis.
Flow rate precision Important for repeatability of retention times and Can be determined from repeatability of retention times of a defined “test peak”. <0.5% RSD RT
peak area/height.
Proportioning precision Important for repeatability of retention times and Can be determined from repeatability of retention times of a defined “test peak”. <0.5% RSD RT
peak area/height.
Column oven thermostatting Important for repeatability of retention times and Can be determined from repeatability of retention times of a defined “test peak”. <0.5% RSD RT
precision peak area/height.
Linearity of detector response Important for accuracy of results. Can be determined from the linearity between detector response (peak method specific
area/height) and standard solution concentration.
Signal to noise ratio Important for sensitivity and limit of detection. Can be determined from the response of a detector to a dilute standard solution method specific
and a blank.

LGC/VAM/1998/026.2 Page 23
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. The
Stationery Office.

3. Good Laboratory Practice for Non-clinical Laboratory Studies. Food and Drug
Administration (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 analytical


instruments.
P Bedson and M Sargent, Accred. Qual. Assur. (1996)1:265-274.

6. Evaluation of analytical instrumentation: Part IX Instrumentation for High Performance


Liquid 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.

Page 24 LGC/VAM/1998/026.2
9. Bibliography
1. Requirements and tests for HPLC Apparatus and Methods in Pharmaceutical Quality
Control. 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 liquid


chromatography, 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

LGC/VAM/1998/026.2 Page 25