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

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 µg kg –1 for cortisone and 0.80/2.68 µg kg –1 for hydrocortisone, with dynamic ranges of 3.65–150.77 and 2.59–268.45 µg kg –1 , respectively. With SA-FDF-LPME coupled to LC–MS/MS, LOD/LOQ were 0.0164 / 0.0547 µg kg –1 for cortisone and 0.0734/0.245 µg kg –1 for hydrocortisone, and dynamic ranges were 0.049–4.983 and 0.210–5.50 µg 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.