General Guidelines for Setting Up an In Vitro LC/MS/MS Assay

In this chapter, we will discuss the choice of proper chromatographic and mass spectrometric conditions that are critical for establishing sensitive and robust in vitro and in vivo assays. Liquid chromatography combined with mass spectrometry (LC/MS) has been a primary tool for quantitation of analytes for in vitro and in vivo assays. This is due to the sensitivity and selectivity of modern LC/MS instrumentation and materials which routinely allow detection to the low ng/mL range for many analytes. The preparation of in vitro and in vivo generated samples requires that analytes are efficiently extracted with minimal chemical or enzymatic degradation both before and during LC/MS analysis. Modern chromatographic and MS ionization methods allow quantitation of analytes from small, polar organic molecules to large proteins and peptides over a wide dynamic range. Tandem MS methods via multiple reaction monitoring (MRM) has extended quantitation to low abundance analytes previously requiring radiometric detection. Quantitation software is integral to all MS operating software and will accurately determine concentrations of unknown analyte solutions based on calibration curves generated from known standard solutions.