Charged derivatization and on-line solid phase extraction to measure extremely low cortisol and cortisone levels in human saliva with liquid chromatography–tandem mass spectrometry

Abstract The aim of this study was to develop a sensitive, reliable and high-throughput liquid chromatography – electrospray ionization – mass spectrometric (LC-ESI–MS/MS) method for the simultaneous quantitation of cortisol and cortisone in human saliva. Derivatization with 2-hydrazino-1-methylpyridine (HMP) was one of the most challenging aspects of the method development. The reagent was reacting with cortisol and cortisone at 60 °C within 1 h, giving mono- and bis-hydrazone derivatives. Investigation of derivatization reaction and sample preparation was detailed and discussed. Improvement of method sensitivity was achieved with charged derivatization and use of on-line solid phase extraction (on-line SPE). The lower limit of quantitation (LLOQ) was 5 and 10 pg/ml for cortisol and cortisone, respectively. The developed method was subsequently applied to clinical laboratory measurement of cortisol and cortisone in human saliva.