Determination of hydrocortisone and cortisone in artificial saliva by spray assisted fine droplet formation liquid phase microextraction coupled to LC-MS/MS

Abstract

Hydrocortisone and cortisone are clinically informative steroids whose joint measurement at low concentrations is essential for ratio-reliable interpretation. A spraying-assisted fine droplet formation liquid-phase microextraction (SA-FDF-LPME) was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for simultaneous quantitation in saliva-type matrices as part of a method-development study. Method parameters were optimized to secure chromatographic resolution, stable ion ratios, and high sensitivity. Under direct injection, LOD/LOQ values were 1.05/3.50 & micro;g kg-1 for cortisone and 0.80/2.68 & micro;g kg-1 for hydrocortisone, with dynamic ranges of 3.65-150.77 and 2.59-268.45 & micro;g kg-1, respectively. With SA-FDF-LPME coupled to LC-MS/MS, LOD/LOQ were 0.0164/0.0547 & micro;g kg-1 for cortisone and 0.0734/0.245 & micro;g kg-1 for hydrocortisone, and dynamic ranges were 0.049-4.983 and 0.210-5.50 & micro;g kg-1, respectively. Accuracy was established by pre-extraction spiking at different concentrations in two artificial saliva formulations. Using external-standard calibration, recoveries were 105.3-129.4%/110.4-141.1% for cortisone and 110.0-134.9%/94.1-142.8% for hydrocortisone; with matrix-matched calibration, recoveries were 80.3-103.2%/98.1-117.8% for cortisone and 79.8-105.7%/97.3-114.0% for hydrocortisone. The developed SA-FDF-LPME-LC-MS/MS method enables sensitive, accurate, and simultaneous quantification of hydrocortisone and cortisone at saliva-relevant concentrations and offers a practical platform for selective bioanalysis of related small molecules.