Qualification of High Performance Liquid Chromatography

Equipment qualification is a formal process that provides documented evidence that an instrument is fit for its intended use and kept in a state of maintenance and calibration consistent with its use. The entire qualification process consists of four parts:

1. Design qualification (DQ)
2. Installation qualification (IQ)
3. Operational qualification (OQ)
4. Performance qualification (PQ)

Individual steps and frequency of qualifications depend on the type of equipment and application. This article describes steps required for the qualification of HPLC systems.

Design Qualification

Design qualification (DQ) describes the user requirements and defines the functional and operational specifications of the instrument. It should ensure that the instruments to be purchased have the necessary functions and performance that will enable them be suitable for the intended applications. 

Later on the DQ document will be used as a basis for tests in the OQ phase. Table-1 lists elements with examples that should be included in the design qualification for the HPLC systems in the selected QA/QC laboratory. Please note that all instruments are required to have gradient pumps, thermostatted column compartments and diode-array detectors to ensure that they all can be used for all applications.

Design elements	Examples
Intended use	Analysis of drug compounds and impurities.
User requirement specification for the HPLC analysis	Up to 100 samples / day.
	Automated over-night analysis.
	Limit of quantitation: 0.1%.
	Automated confirmation of peak identity and purity with diode-array detection.
	Automated compound quantitation and printing of report.
Functional specifications	Pump: Binary or higher gradient.
	Detector: UV/Vis Diode-array, 190-900 nm.
	Autosampler: 100 samples, 0.5 µl to 5 ml sample volume.
	Column compartment: 15 to 60 Deg C, Peltier controlled.
	Computer: System control, data acquisition for signals and spectra, peak integration and quantitation, spectral evaluation for peak purity and compound confirmation. Electronically save all chromatograms generated by the system.
Operational specifications	Detector: Baseline noise: <5 x 10-5 AU Sampler: Precision inj. volume: <0.5% RSD, sample carry over: <0.5% Pump: precision of retention time: <0.5% RSD
User instructions	Operational manual on paper. Computer-based tutorial.
Validation/qualification	Vendor must provide procedures and services for IQ and OQ.
Maintenance	Vendor must deliver maintenance procedure and recommend schedule. Instrument must include early maintenance feedback for timely exchange.

Table 1. Design qualification elements

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Installation Qualification

Installation qualification 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. Table 2 lists the steps as recommended before and during installation. 

IQ should include an analysis of a test sample. A successful run of such a sample verifies the correct installation of all modules and electrical and fluid connections. 

Before installation

• Obtain the manufacturer's recommendations for the installation site requirements.
• Check the site for the fulfillment of the manufacturer’s recommendations (utilities such as electricity, and environmental conditions such as humidity and temperature).
• Allow sufficient shelf space for the equipment, SOPs, operating manuals and software.

During installation

• Compare equipment, as received, with purchase order (including software, accessories, spare parts)
• Check documentation for completeness (operating manuals, maintenance instructions, standard operating procedures for testing, safety and validation certificates)
• Check equipment for any damage
• Install hardware (computer, equipment, fittings and tubings for fluid connections, columns in HPLC and GC, power cables, data flow and instrument control cables)
• Switch on the instruments and ensure that all modules power up and perform an electronic self-test.

• Identify and make a list with a description of all hardware, include drawings where appropriate.
• Run test sample and compare chromatogram print-out with reference chromatogram.
• List equipment manuals and SOPs
• Prepare an installation report

Operational Qualification

Operational qualification (OQ) is the process of demonstrating that an instrument will function according to its operational specification in the selected environment“ It verifies that the HPLC system complies with key functional and operational requirements as specified in the design qualification.

Parameter	Procedure	User Limit
Leak testing	Flow test by volume or weight/time	± 5 %
Baseline drift	ASTM Method E19.09, 20 min	<2 x 10-3 AU
Baseline noise	ASTM Method E19.09, 20 x 1 min	<5 x 10-5 AU
Precision of injection volume	6 x injection of caffeine standard, RSD of peak areas	0.3 % RSD
Detector linearity	inject 5 standards	>1.5 AU, 5%
Wavelength accuracy	holmium oxide filter	± 1 nm
Temperature accuracy	comparison with external measuring device	± 1 °C
Temperature precision	monitoring temperature over 20 min	± 0.25 °C
Autosampler carry over	Injection of blank solvent after large concentration	< 0.5 %
Mobile phase composition accuracy	Step gradients from 4 to 7 % B, step heights relative to 100%, with acetone tracer	± 1 %

Table 2. Test parameters and acceptance criteria for the case study.

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In modular HPLC systems it is recommended to perform system tests (holistic testing), rather than performing tests module by module (modular testing). Individual module tests should be performed as part of the diagnosis if the system fails.

Performance Qualification

Performance Qualification (PQ) is the process of demonstrating that an instrument consistently performs according to a specification appropriate for its routine use. Important here is the word ‘consistently’. The test frequency is much higher than for OQ.

Another difference is that PQ should always be performed under conditions that are similar to routine sample analysis. For a chromatograph, this means using the same column, the same analysis conditions and the same or similar test compounds.

PQ should be performed on a daily basis or whenever the instrument is used. The test frequency not only depends on the stability of the equipment but on everything in the system that may contribute to the analysis results. For a liquid chromatograph, this may be the chromatographic column or a detector’s lamp. The test criteria and frequency should be determined during the development and validation of the analytical method.

In practice, PQ can mean system suitability testing, where critical key system performance characteristics are measured and compared with documented, preset limits. For example, a well-characterized standard can be injected 5 or 6 times and the standard deviation of amounts are then compared with a predefined value. 

PQ tests are applications specific. If the limits of detection and/or quantitation are critical, the lamp’s intensity profile or the baseline noise should be tested. They should use the same column and chemicals as used for the real samples. Test should include,

1. The precision of the amounts
2. The precision of retention times
3. Resolution between two peaks
4. Peak width at half height or
5. Peak tailing
6. Baseline noise
7. Wavelength accuracy of the UV/Visible wavelength detector, preferably using built-in holmium-oxide filters

Documentation

On completion of equipment qualification, documentation should be available that consists of:

• Design qualification document
• IQ document (includes a description of hardware and software)
• Procedures for OQ testing
• OQ test reports
• PQ test procedures and representative results

OQ test reports should include test parameters, acceptance criteria and actual results.

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