Abstract
Objective: To develop and validate a simple and rapid assay for simultaneous measurement of cortisol and cortisone in human saliva by ultra-performance liquid chromatography-tandem mass spectrometry. Methods: Chromatographic analysis was performed on an Atlantis dC18 column (2.1 x 100 mm, 3 µm) using a mobile phase consisting of acetonitrile and 2 mmol ammonium-acetate (50:50, v; v) that was delivered at a flow rate of 0.3 ml/min. The eluents were monitored using electrospray ionization in the positive ion mode set at transition set of mass-to-charge (m/z): 363.11 → 121.00, 361.18 → 163.11, and 367.19 → 121.24 for cortisol, cortisone and internal standard (IS), respectively the method was validated for linearity, accuracy, precision, and recovery, according to international guidelines. Results: The retention times of cortisol, cortisone and internal were about 1.38, 1.43 and 1.38 min, respectively. Cortisol level and cortisone level relationship to the ratio of their respective peak-area to IS’s peak-area was linear (range of 0.5-100 ng/ml). Coefficients of variation and inaccuracy were, ≤9.9% and-0.3 to 6.9 for cortisol and ≤8.4 and-1.5 to 4.8 for cortisone, respectively. Extraction recoveries for cortisol, cortisone, and the IS were 90%, 94%, and 98%, respectively. Cortisol and cortisone stability was evaluated in processed saliva samples (stored at room temperature for 24 h) and unprocessed saliva samples (stored at room temperature for 24 h or at-20 °C for 10 w) and after 3 freeze-thaw cycles was ≥ 86%. Conclusion: The proposed method is simple, precise, and accurate for the rapid simultaneous measurement of cortisol and cortisone levels in saliva. The assay was successfully applied to determine levels of cortisol and cortisone in human saliva samples obtained from healthy volunteers.
Highlights
Cortisol, a steroid hormone secreted by the adrenal cortex, has an important function in metabolism, electrolyte balance, and the immune response, among others [1, 2]
Estimation of cortisol and cortisone in the saliva is used in evaluating the adrenal axis as it reflects that biologically active cortisol and sample assortment is not related to stress [3, 4]
Ultra-performance liquid chromatographytandem mass spectrometry (UPLC-MS/MS) method for simultaneous measurement of cortisol and cortisone levels in saliva, utilizing liquid-liquid extraction and cortisol-d4 as an internal standard (IS)
Summary
A steroid hormone secreted by the adrenal cortex, has an important function in metabolism, electrolyte balance, and the immune response, among others [1, 2]. Estimation of cortisol and cortisone in the saliva is used in evaluating the adrenal axis as it reflects that biologically active cortisol and sample assortment is not related to stress [3, 4]. Since cortisol is metabolized to cortisone (biologically inactive) by the salivary glands, via 11-ßhydroxysteroid dehydrogenase type-2 (11ß-HSD2), an assay that can simultaneously measure cortisol and cortisone would allow better assessment of the adrenal axis and is essential for studying the salivary gland 11ß-HSD2 activity [5, 6]. High-performance-liquidchromatography (HPLC) equipped with ultraviolet [9] or fluorescence [10] detector or tandem-mass-spectrometry (LCMS/MS) [11,12,13,14,15] were used for simultaneous measurement of cortisol and cortisone levels in human plasma [11], serum [12] urine [13] and saliva [14, 15]. For Bioanalysis, often derivatizers [14] or expensive solid-phase extraction cartridges [13, 15] are utilized in sample preparation
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