Sample preparation techniques for mass spectrometry in the clinical laboratory

The principles and optimization of sample preparation protocols commonly used in clinical LC-MS/MS are described and compared in this chapter. Although the practices reviewed here are more broadly applicable, in this chapter “LC-MS/MS” refers to reverse-phase LC, UPLC, or UHPLC used with electrospray (ESI), atmospheric pressure chemical ionization (APCI), and triple quadrupole (QqQ) quantitative mass analysis. In general, those protocols that are more labor intensive, complex and/or expensive are also more effective at concentrating analytes and removing matrix than simpler and cheaper protocols. Matrix removal is beneficial because injecting less matrix/sample typically yields more robust methods and longer maintenance-free intervals for the LC-MS/MS instrument. Goals for method performance, such as precision and quantitation limit, are well defined in clinical chemistry, but there is less agreement on what constitutes method robustness. The challenge for clinical laboratories using LC-MS/MS is to select sample preparation protocols that deliver not only acceptable method performance but also the desired cost/reportable test, practicality and robustness. Strategies for evaluating robustness during validation—at best an incomplete proxy for robustness in production—are presented and metrics for quantifying robustness in production are proposed.