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High‐throughput determination of cortisol, cortisone, and melatonin in oral fluid by on‐line turbulent flow liquid chromatography interfaced with liquid chromatography/tandem mass spectrometry
Author(s) -
Fustii Silvia,
Polledri Elisa,
Mercadante Rosa
Publication year - 2013
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.6601
Subject(s) - chemistry , chromatography , mass spectrometry , cortisone , tandem mass spectrometry , liquid chromatography–mass spectrometry , high performance liquid chromatography , selected reaction monitoring , electrospray ionization , medicine
RATIONALE Cortisol, cortisone, and melatonin (CORTol, CORTone, and MELA, respectively) are hormones related to stress and sleep disorders. Their detection is relevant to epidemiological studies aimed at investigating the effects of circadian cycle disruption. The aim of this study was to develop and evaluate a high‐throughput assay for the detection of CORTol, CORTone, and MELA concentrations in non‐invasively collected oral fluid samples. METHODS A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method to measure levels of CORTol, CORTone, and MELA in oral fluid samples in the presence of deuterated analogs was optimized and validated. A 50 μL aliquot of oral fluid sample, obtained by centrifugation of a chewed swab, was purified using on‐line turbulent flow liquid chromatography. Analytes were then separated using C18 reversed‐phase chromatography, subjected to positive ionization using an electrospray source, then quantitated using a triple quadrupole mass detector in the selected reaction monitoring mode. RESULTS Limits of quantification and linear dynamic ranges were found to be 0.55 nmol/L, 5.5 nmol/L, and 0.004 nmol/L, and up to 28 nmol/L, 277 nmol/L, and 0.43 nmol/L for CORTol, CORTone, and MELA, respectively. Inter‐ and intra‐run precisions as relative standard deviation values were <5%, and accuracies were within 95–106% of theoretical concentrations. An evaluation of matrix effects showed that the use of deuterated analogs controlled sources of bias. Furthermore, the total analysis time per sample was 13 min, resulting in a throughput of approximately 100 samples/day. CONCLUSIONS To our knowledge, this is the first automated, high‐throughput assay for the simultaneous quantification of CORTol, CORTone, and MELA in oral fluid specimens. Copyright © 2013 John Wiley & Sons, Ltd.