System Suitability Failures

The purpose of system suitability testing is summarised by the USP as follows: "System suitability tests are an integral part of gas and liquid chromatographic methods. They are used to verify that the chromatographic system is adequate for the intended analysis. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such."[1] In practice the testing consists of measurements performed on the chromatograms obtained for particular injections during the analysis which provide an indication of whether the HPLC method and system is performing as would be expected. Typical system suitability parameters, as defined by the FDA[2], are summarised in Table 1, with definition of terms for the parameters provided in Figure 1. Acceptance criteria based on recommendations provided by the FDA[2] are provided in Table 2, these are often implemented as a ‘generic’ set of conditions when new methods are developed. Additional requirements may be added as required, e.g. a measure of detector sensitivity may be assessed for a low level impurity method.

Table 1



Figure 1


Table 2

When a system suitability test fails, an investigation into the cause is required. Data generated from the analysis cannot be used. Ideally analytical results should not be generated until the system suitability test results have been shown to pass. In practice however, the processing for an HPLC analysis is usually automated and thus the results are often generated before the results of the system suitability test have been evaluated. In this situation it is very important that the documentation accompanying the analysis is very clear about why the results have been rejected. The available regulatory guidance[3,4] for the investigation of out of specification (OOS) and out of trend (OOT) results involves inspection of the system suitability conditions for the analysis, even though it should already have been assessed prior to accepting the analysis as valid. This may seem redundant but it is important to review all available data during this type of laboratory investigation. It is also possible that the system suitability testing for the method is inadequate and may need to be reviewed and updated.

It is desirable to minimise the occurrence of system suitability failures since each failure represents wasted time spent on analysis and additional time spent conducting an investigation into the failure. The consequences of the failure are not severe: the cause is documented and the analysis repeated. However, regular failures are of concern and may indicate a more serious root cause. The test measures the operation of an HPLC method in a holistic fashion and the reasons for failure of the test are varied. The most common reasons are related to:
1. Degradation of the HPLC column,
2. Competence of the analyst, and
3. Maintenance of the HPLC system.

Degradation of the HPLC column
The column has been singled out since it is a consumable item and it is expected that over time it will degrade and in the process have an effect on the chromatographic performance of the method. Resolution and efficiency are likely to decrease and tailing to increase over time. Under ideal conditions the primary purpose of the system suitability test would be to indicate when the column was no longer performing adequately for the method and should be replaced. In many laboratories the expected lifetime of a HPLC column for a particular method is not investigated and thus there is a risk that using a generic set of acceptance criteria for system suitability testing may result in a failure of the test when in fact the column is still performing adequately, or potentially (though less likely) a pass when the column has actually degraded past the point where acceptable results can be obtained.

Typically the number of injections that can be obtained for a column depends on the combination of the mobile phase, the system conditions, and the sample being analysed. Columns which are subjected to method conditions which include inorganic buffers, high pH or high temperature may result in a reduced number of injections, as will columns which are used for ‘dirty’ samples. An evaluation of the aging of the column for a method is advisable to set appropriate system suitability criteria. This may be carried out as a dedicated study during method development, or a column may be monitored in normal use of the method. Either way the data gained will provide a sound scientific basis for a set of relevant system suitability criteria. In addition, warning limits may be set which alert the operator that the column needs to be replaced soon, thus eliminating any system suitability failure due to the aging column.

Competence of the analyst
The analyst performing HPLC analysis must not only be competent in the preparation of mobile phase and test solutions but also in setting up the HPLC system. Errors in mobile phase preparation can affect the capacity factor and thus the retention time of analytes. If the analyst introduces contamination in the mobile phase this may introduce extra (unwanted) peaks, decrease resolution and degrade the overall chromatography. Errors in preparation of test solutions can affect the accuracy and precision of the analytical results. Examples of problems due to incorrect set up of the HPLC system include unsteady and noisy baselines if the pump is not adequately primed, peak broadening effects (decreased efficiency) due to unsatisfactory connections and drifting baselines and retention times if the temperature is not controlled.

If the operator has a good understanding of the method, this will help to ensure that important method parameters are controlled, e.g. in some HPLC methods the retention times of the analytes are very sensitive to the pH of the buffer in the mobile phase and thus strict control of the pH is critical. If the analyst is aware of this requirement they will be more careful during the pH adjustment task. Suitable training is essential to ensure that system suitability failures are not due to the competence of the analyst.

Maintenance of the HPLC System
If not correctly maintained the HPLC instrumentation can contribute to system suitability testing failures, e.g. worn injector parts may result in repeatability failure, an aged lamp in a UV detector can cause baseline noise and have a detrimental effect on the quantification of low level analytes. The system suitability test is not a substitute for the performance qualification of the HPLC instrument since it is method based rather than instrument based. Although it provides assurance that the method may be used satisfactorily at that time it does not test that the instrument is functioning correctly, e.g. accuracy of flow rate, wavelength, injection volume, temperature, etc. Regular maintenance and performance verification should be scheduled to ensure that the HPLC system does not contribute to system suitability testing failures.

In summary, system suitability testing failures can be reduced by a combination of three measures:

1. Set system suitability criteria which relate specifically to the method in use. A column degradation study will identify the parameters of resolution, tailing and efficiency which indicate that a new column should be used.

2. Ensure that HPLC operators have received suitable training and are familiar with the method in use, and in particular, the robustness issues relating to the method.

3. Implement a regular maintenance and performance verification procedure for HPLC systems.

References:
1. United States Pharmacopeia (USP), Chromatography <621>
2. Reviewer Guidance: Validation of Chromatographic Methods, US Food and Drug Administration, 1994
3. Guidance for Industry: Investigating Out-of-Specification (OOS) Test Results fror Pharmaceutical Production US Food and Drug Administration, 2006
4. Out of Specification Investigations, MHRA