Overcoming ionization effects through chromatography: A case study for the ESI-LC-MS/MS quantitation of a hydrophobic therapeutic agent in human serum using a stable-label internal standard

Significant electrospray ionization effects were observed for a hydrophobic analyte and its stable isotopically labeled internal standard (SIL IS) in human plasma extracts. Analyte and SIL IS were slightly offset in retention within the suppression region resulting in a differential suppression that biased calculated concentrations. Six abundant endogenous phospholipids were identified as potential contributors to ionization suppression. Chromatographic conditions were optimized using pH (relative to the log D of the analytes), to better resolve nearby phospholipids from the analyte and SIL IS and minimize ionization suppression. The successful validation of this method demonstrates the value of investigating minor chromatographic changes to remediate detrimental ionization effects without further altering extraction procedures.