Effervescent‐assisted Liquid‐phase Microextraction Employing Switchable Hydrophilicity Solvent for the Determination of Cortisol and Testosterone in Oral Fluid by High‐Performance Liquid Chromatography‐Diode Array Detection

Cortisol and testosterone are important biomarkers for diagnosing complex disorders, including polycystic ovary syndrome and Cushing's syndrome, where symptomatology usually overlaps with other prevalent disorders. This work proposes, for the first time, an analytical method based on a switchable hydrophilicity solvent as an extraction phase for the determination of cortisol and testosterone in oral fluid (OF) by high-performance liquid chromatography with diode-array detection. The optimized extraction conditions consisted of 1000 µL of OF, 100 µL of decanoic acid solution (65 mg/mL), 170 µL of Na₂CO₃, 900 µL of H₂SO₄ and 150 µL of acetonitrile for dilution. The method was validated, and coefficients of determination higher than 0.9926, the limit of detection of 4.55 ng/mL and the limit of quantification of 15.00 ng/mL were obtained. Intra-day precision varied from 5.6% to 11.9%, inter-day precision ranged from 6.1% to 13.5%, and relative recoveries ranged from 98.9% to 104.6% for cortisol, and 89.1% to 103.9% for testosterone. This methodology was successfully applied to five OF samples from volunteers. Moreover, the greenness of this methodology was evaluated based on the sample preparation metric of sustainability achieving a global score of 7.37 which can be considered sustainable.