Sample preparation and quantitative UHPLC-MS/MS analysis in an accredited high-throughput environment: Tips and tricks to improve your success ratio

Abstract

Ultra-High Performance Liquid Chromatography coupled to tandem Mass Spectrometry (UHPLC-MS/MS) is often the technique of choice for quantitative toxicological analysis. Though accessible at face-value, routine application with consistent performance requires a good framework (quality management system, QMS) including thoroughly validated methods, trained analysts and regularly serviced hardware. Even in such contained environments, unforeseen complications can occur. Therefore rapid, accurate troubleshooting to identify swift corrective actions are needed to ensure day-to-day lab continuity. Based on our personal experience, we present a practical non-limitative guide to help troubleshoot and evade common problems when performing quantitative UHPLC-MS/MS analysis. The laboratory of toxicology at Belgium's National Institute for Criminalistics and Criminology (NICC) performs large volumes of quantitative UHPLC-MS/MS analysis of illicit drugs in blood and oral fluid (> 15 000 samples/year). The laboratory and methods used are ISO 17025 accredited. The QMS requires every deviation to be described and a root cause analysis to be performed. When troubleshooting a flawed analysis, the most important aspect is identifying whether the extraction process itself or the UHPLC-MS/MS equipment is at the origin. Therefore, before the injection of any extracted sample batch, a system suitability test (SST) should be performed. We recommend a three-way SST consisting of (1) ‘no inject’ (2) reconstitution solvent and (3) a test solution. The ‘no inject’ consists of the UHPLC running the programmed gradient without injecting a sample. Unanticipated peaks at this stage originate from within the UHPLC-MS/MS system itself. Injection of the reconstitution solvent helps to identify possible contaminations or an increased baseline in the chromatogram. The test vial contains a non-extracted solution of known composition. The resulting areas, signal-to-noise ratios, peak shapes, resolutions should be tracked as a function of time for as long as the method is routinely used. Deviations in the SST point to non-compliant equipment. In general, retention shifts and unacceptable peak shapes will originate from the UHPLC. Both can be caused by normal column wear, therefore track the amount of column injections and define an in-house maximum. Sensitivity issues in the test warrant further investigation of the MS system. The regularity and thoroughness of MS maintenance/cleaning will depend on the applications and should be determined in-house. Note that not all analytes will be affected in the same way by a poorly maintained system. If the UHPLC-MS/MS equipment functions properly but the batch analysis does not meet acceptation criteria, it's likely due to an error in the extraction process. At this stage on should evaluate whether all compounds or a certain kinds is affected. Typical examples are: amphetamines lost due to volatility, esters hydrolysing and instabilities in highly organic elution solvent. Blank's containing analytes indicate contamination. Before troubleshooting a contamination: look back at the system suitability test (no inject) and evaluate the potential presence of a highly concentrated sample in the batch (carry-over and/or system contamination). Subsequently, consider which analytes are present (all, just one, or parent + metabolite) and in which vials they are present (e.g. in each vial or only a few). If a parent and it's metabolite are present as a contamination, it may be traced to a highly concentrated sample recently processed in the lab. A practical guide is presented to assist in troubleshooting when performing quantitative UHPLC-MS/MS analysis. A well thought-out SST is most valuable. Pinpointing the extraction, the LC or the MS as the source of the problem is vital. The source of error during the extraction process can be hinted at by the amount of vials and the type of compounds affected.