This article discusses how laboratory testing should be performed in a quality regulated environment and will describe the controls and procedures which should be followed to assure the quality, reliability, trustworthiness and validity of analytical data.

Analytical Test Methods

All analytical testing should be carried out according to written, validated and approved procedures, variously called: analytical methods, testing procedures, analytical testing methods. The procedure should be written in such a way that a trained but uninformed person can read it and carry out the analysis without having to refer to the originator for clarification.

Written analytical procedures should contain the following elements:

Introduction: should describe the purpose and provide a brief description of the method

Scope: should state what samples the method applies to and include any limitations for use, such as phase of clinical study

Equipment: should state instrument requirements, including any special requirements, such as specific instrument or any special configuration, should also include chromatographic column specifications

Reagents: List reagents required together with reagent grade specifications

Preparation of Solvents and Test Reagents: Provide instructions for the preparation of mobile phase for HPLC, extraction solvents for preparing sample solutions, dissolution media etc.

Standard Preparation: Provide instructions for the preparation of the reference standard solutions

System Suitability Requirements: List all required systemsuitability requirements

Sample Preparation: Provide instructions for the preparation of the sample solutions

Calculations: Provide clear instructions on how the results should be calculated from the instrument output to obtain the final results providing a clear equation

Reporting Requirements: Provide instructions on reporting the results, such as number of decimal places or significant figures

Instructions within a method should be presented in a numbered list format, such as:

Sample Preparation

1. Prepare samples in duplicate

2. Transfer ten 25 mg AAA-122 tablets to a labelled 100 ml volumetric flask

3. Add approximately 60 ml of extraction solvent

4. Sonicate for 15 mins and then swirl to mix the contents

5. Sonicate for a further 15 mins and then swirl again to mix the contents

6. Sonicate for a further 15 mins and then swirl again to mix the contents

7. Allow to cool to ambient

8. Make up to 100 ml and mix well

9. Decant a portion of the suspension in to a stoppered labelled centrifuge tube and centrifuge at 3000 rpm in a centrifuge with a radius of 300 mm for 10 mins

10. Pipette 10 ml of the supernatant in to a labelled 100 ml volumetric flask

11. Make up to 100ml and mix well

12. These are the sample solutions

This format is much easier to follow and is less prone to having steps missed, compared to the paragraph format, shown below:

Prepare samples in duplicate. Transfer ten 25 mg AAA-122 tablets to a labelled 100 ml volumetric flask and add approximately 60 ml of extraction solvent. Sonicate for 15 mins and then swirl to mix the contents, sonicate for a further 15 mins and then swirl again to mix the contents, sonicate for a
further 15 mins and then swirl again to mix the contents.

Allow to cool to ambient and make up to 100 ml and mix well. Decant a portion of the suspension in to a stoppered labelled centrifuge tube and centrifuge at 3000 rpm in a centrifuge with a radius of 300 mm, for 10 mins. Pipette 10 ml of the supernatant in to a labelled 100 ml volumetric flask, make up to 100ml and mix well. These are the sample solutions.

Assuring Standard Accuracy

Most analytical methods rely on comparing the instrument output, such as a peak area, from a sample solution to the instrument output from a standard solution of known concentration. The accuracy of the standard concentration is therefore fundamental to the accuracy of the final result. It is therefore necessary to provide assurance that the standard concentration(s) are accurate.

If the results are expected to fall within a ±25 % range, this can be achieved by independently preparing standard solutions in duplicate. A recovery of one standard verses the other standard can be performed for example:

A standard recovery of between 98.0 and 102.0 % is usually considered sufficient assurance of standard accuracy.

If the results are expected to fall outside a ±25 % range, then a series of standards should be independently prepared, that bracket the expected sample concentration range, and a correlation between the instrument output and the standard concentrations should be obtained. A correlation 2 coefficient (r ) value of ≥ 0.999 is usually considered acceptable.

System Suitability Tests

System Suitability tests are an integral part of analytical methods intended to provide assurance that the instrument is performing to appropriate specifications at the start of, and throughout an entire analytical run.

At a minimum system suitability should consist of:

• Providing assurance against contamination interfering with analysis; before making any standard or sample measurements, make one or more blanks measurements, and determine that no interfering outputs are obtained
• Determining the repeatability of a set of five or six standards measurements at the start of an analytical run. A relative standard deviation ≤ 2.0 % is a usual acceptance criteria
• Making standard recovery measurements at appropriate intervals throughout, typically every five or six samples, and at the end of the analytical run. A recovery of between 98.0 to 102.0 % is a usual acceptance criteria

In addition, for chromatographic methods, system suitability criteria based on typical chromatographic parameters should be established, such as those shown in Table 4.

It is good practice to establish that all system suitability and standard recovery requirement are met prior to preparing any samples. This will avoid wasting time preparing samples, which could then not be analysed or running samples which must subsequently be rejected because standard recovery and system suitability requirements are not met.

Controlling Chromatographic Peak Integration

The quantitation of the output from chromatographic methods entails determining the area of the respective peak. All chromatographic data processing packages have the ability to perform this task automatically. To do this it is necessary to select appropriate values for the integration parameters that control this process. These parameters have various names, depending on the data processing package used. Some of the most common are slope sensitivity, peak width, noise threshold, area threshold and bunching factor.

The general process is to manipulate the relevant integration parameters, until accurate integration is achieved. From a quality and data reliability perspective, this presents a couple of issues:

• What constitutes accurate integration?
• The potential to manipulate the final results by tweaking the peak
  integration to obtain a passing result

This is an area which is currently attracting much regulatory scrutiny. Obviously manipulating the peak area to obtain a within specification result, that would otherwise be out of specification is unacceptable. The regulatory expectation is that selecting the most appropriate integration parameters is an activity that should be controlled by a SOP, and should follow a predefined, scientifically sound procedure. Once a method has been validated the same integration parameters should be used for all subsequent analysis.

Any re-integration should be explained and justified. Performing multiple re-integrations of chromatograms without explanation are unacceptable.

Extract taken from “An Easy to Understand Guide to Good Laboratory Practices”