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

Balázs Magda, Zoltán Dobi, Katalin Mészáros, Éva Szabó, Zoltán Márta, T. Imre, P. Szabó

Research output: Contribution to journalArticle

2 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)223-231
Number of pages9
JournalJournal of Pharmaceutical and Biomedical Analysis
Publication statusPublished - Jun 5 2017



  • 2-Hydrazino-1-methylpyridine
  • Column-switching
  • Cortisol
  • Cortisone
  • LC–MS/MS

ASJC Scopus subject areas

  • Analytical Chemistry
  • Pharmaceutical Science
  • Drug Discovery
  • Spectroscopy
  • Clinical Biochemistry

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