How do I generate authentic Chromatographic results?

Is my data authentic ?

As an analytical chemist your primary concern is to generate authentic data which is universally accepted and reproducible under identical conditions in any other laboratory. Even the use of most sophisticated instruments will not serve the purpose if globally accepted norms are overlooked. System suitability is the key to generation of valid data in chemical analysis.

Chromatography, specially, liquid chromatography is the most popular technique in laboratories engaged in pharmaceuticals, foods, forensics, polymers, life sciences and environmental studies. Thousands of HPLC’s are working in research and development and quality control laboratories in pharmaceuticals sector alone across the world. Immense amount of data is generated daily which contributes in developing safe and efficacious drugs for human consumption. Given the seriousness of such activity it is of utmost significance that the HPLC systems should satisfy the requirements of system suitability before decisions are taken on basis of such data.

What is System Suitability?

System suitability tests constitute significant part of chromatographic analysis. They help us decide that the detection sensitivity, resolution, and reproducibility are adequate for the required analysis. The United States Pharmacopoeia (USP) is an authoritative source for guidelines on chromatography of the drug substances. Section 621 USP states that system suitability tests are an integral part of gas and liquid chromatography analysis. The tests are based upon the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such. This means that no sample analysis is acceptable unless the requirements of system suitability have been met.

Factors contributing to system suitability failures in HPLC analysis

• Auto sampler – Poor precision due to inability to deliver precisely the set volumes of samples and standards.
• Columns – ageing columns or choked columns can result in poor performance due to reduction in theoretical plate count.
• Pumps – failure to deliver constant flow rate of mobile phase due to wear and tear of the operating parts
• Mobile phase- errors in making prescribed mixtures and incomplete degassing
• Sample and reference preparation – inaccuracies in preparation or non-filtration before injection

How often is system suitability testing required?

As per regulatory bodies guidelines system suitability should qualify the entire chromatographic system and not just individual modules. Whenever there is a significant change in equipment or in a critical reagent system suitability testing should be done before injection of the samples. No sample analysis is acceptable unless the requirements of system suitability have been met. Sample analysis results obtained while system is failing requirements of system suitability are of no significance.

System suitability must be performed before and throughout all regulated assays. It is not sufficient to apply suitability tests at the beginning of the chromatographic run and assume that the system will perform properly during the run. Test mixtures containing main components and expected impurities can be injected at the beginning and interspersed during testing.

On facing system suitability failure the analyst should stop the analysis immediately. After diagnosing the problem and taking remedial measures the suitability testing should be redone. Analysis of actual samples should be resumed only after all system suitability criteria are met and not only the failed criteria.