Liquid chromatography linked to tandem mass spectrometry (LC–MS/MS) is the ‘go to’ method for pharmacokinetics and metabolism studies across drug development with recent advances in separation, throughput and methods for analysing protein biotherapeutics.
But not all LC-MS/MS assays are created equal. There are many pitfalls that await even experienced scientists developing and validating, for example, a new bioassay. However, with experience and knowing what pitfalls to look out for, these issues don’t need to stand in the way of producing a robust bioassay and achieving analysis goals for your compound in good time.
Choosing the right method approach
When selecting your LC-MS/MS method approach it is worth doing your homework. Pick an assay format that is best suited to your compound and consider factors such as matrix type, analyte structure, choice and availability of reagents, and the sensitivity and specificity you need.
What about sample preparation? Will protein precipitation or solid phase extraction be enough or should you consider enrichment, for example using affinity capture steps? Use the simplest approach available to the required selectivity/specificity, sensitivity, accuracy, and precision in the intended matrix and species.
Accuracy, precision, reliability, throughput and sensitivity all make for a good assay. But what are the common pitfalls you should be on top of when commissioning a new LC-MS/MS assay?
The increase in sensitivity of recent LC-MS/MS instruments and the use of wide calibration ranges can make carry over and contamination an issue, but these can be avoided if detected and addressed during validation.
The retention factor of the analyte needs to be optimised in order to allow for the analyte to have sufficient time to interact with the stationary phase on the column. This will allow for the best sensitivity to be achieved and for the analyte to elute from the column before any other matrix interferences in the sample.
LC-MS/MS has a well-deserved reputation for excellent selectivity but interference from the sample matrix (matrix effect) or metabolites can catch you out. Careful validation is key to optimising methods to eliminate or minimise these issues¹.
Best recommendation? Find a partner with expertise in a wide range of LC-MS/MS methodologies
LGC has extensive experience developing LC-MS/MS bioassays for many different compounds and applies an intelligent, streamlined process to new method development that highlights issues at an early stage and creates a solid basis for future troubleshooting. Read our poster ‘A step-by-step guide to developing a robust assay in bioanalysis using LC-MS/MS’ to learn about LGC’s proven approach to developing industry-leading LC-MS/MS bioassays.
1. Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC− MS/MS. Analytical chemistry. 2003 Jul 1;75(13):3019-30.