SIMULTANEOUS DETERMINATION OF CORTISOL, CORTISONE, AND CORTICOSTERONE IN HUMAN PLASMA OF PARACHUTISTS IN VIEW OF PHARMACOKINETIC STUDIES

Abstract

A rapid high-performance liquid chromatographic method with UV detection (HPLC-UV) for the simultaneous quantification of cortisol, cortisone, and corticosterone was developed and validated. The method involved solid-phase extraction (SPE) followed by chromatographic separation using a Nucleosil 100 C-18, 5 µm, 250 × 4 mm i.d. chromatographic column. The mobile phase was binary mixture of acetonitrile and water (30:70 v/v) at a flow rate of 1 mL/min. The proposed method was validated for specificity, linearity, limits of detection and quantitation, precision, and accuracy. Calibrations curves prepared in human plasma extracts were linear with r > 0.9997 for all analytes. The concentration of each steroid was determined in the range from 2 to 300 ng/mL and accuracy ranged from 95.10 to 103.88% for cortisol, from 101.40 to 102.47%, and from 94.00 to 102.21% for cortisol, cortisone, and corticosterone, respectively. Good repeatabilities (RSD <9.74%) were found for all compounds in the biological matrix. The limit of quantitation (LOQ) was 2 ng/mL for cortisol, cortisone, and corticosterone, respectively. The described method was successfully applied for monitoring three endogenous steroid levels in human plasma. Studies were performed on 20 healthy volunteers of both sexes, comprising two sociably diversified groups of people, namely, 10 parachutists before and after effort and 10 patients with depression. The method proposed is simple, inexpensive, and reproducible with the capabilities of accurate quantification of steroids. The method is suitable for a wide applications in human and physiological studies, thus appearing as a fast and reliable alternative to be used for routine steroids analysis.

Keywords:

• corticosterone
• cortisol
• cortisone
• high-performance liquid chromatography method
• human plasma
• SPE extraction

Notes

1 – Healthy volunteers, Female; 2 – Healthy volunteers, Male; 3 – Depression patients, Female; 4 – Depression patients, Male; 5 – Athletes before extreme exercise, Female; 6 – Athletes before extreme exercise, Male; 7 – Athletes after extreme exercise, Female; 8 – Athletes before extreme exercise, Male.