Bioanalytical method validation of quantitative mass spectrometry based assay: experimental protocols and regulations

Liquid chromatography-mass spectrometry (LC-MS) technique has rapidly extended the spectrum of clinical chemistry methods during the past decade and it has became a standard tool for endogenous and exogenous substances in research laboratories as well as in many clinical laboratories. To date, many guidelines for bioanalytical method validation have been published, but there is still the need to have specific guidelines and experimental protocols. In particular, we need to further focus and investigate the overall clinical aspects of spectrometry method validation, for both exogenous and endogenous molecules. The aim of the present document is to suggest specific experimental regulations and protocols for bioanalytical LC-MS method validation for exogenous and endogenous molecules, inspired by actual international guidelines and integrated with practical tips to obtain better performance on methods used in routine clinical practice. Here we have introduced new concepts such as "normalized matrix effect", and protocols for method validation using "pathological samples" and "altered samples", as these are the most commonly samples encountered in laboratory routine. In conclusion, the new rules and protocols reported in this work do not intend to replace international guidelines; we believe that these rules have to be considered besides the available guidelines with the aim to help the LC-MS users by recommending a number of experimental steps to be performed during each method/kit validation.