Simultaneous Determination of Free Cortisol,Cortisone and their Tetrahydrometabolites in Urine by Single Solvent Extraction and Liquid Chromatography–Tandem Mass Spectrometry

Abstract

A fast, efficient and low-cost high performance liquid chromatography–tandem mass spectrometry methodology was developed and validated for the simultaneous determination of free urinary cortisone, cortisol and their tetrahydro-metabolites. The developed method comprises a simple liquid-liquid extraction with CH₂Cl_2, followed by reversed-phase liquid chromatography–tandem mass spectrometry (LC–MS/MS) with electrospray ionization (ESI) in positive mode. The baseline chromatographic separation of the analytes, including the stereoisomers tetrahydrocortisol (THF) and allo-THF, was achieved on a Hypersil Gold C₁₈ column with a mobile phase consisting of 0.05%v/v formic acid in water—acetonitrile, using a gradient elution program. The influence of the mobile phase composition and the ESI parameters on the sensitivity of the method was extensively studied. Sample preparation was also optimized, testing two techniques: solid phase extraction (SPE) and liquid-liquid extraction (LLE). Recoveries ranged from 74.7% (a-THF) to 93.5% (cortisol) and the method limits of detection (MLD) ranged from 0.34?ng mL^?1 (cortisol) to 1.37?ng mL^?1 (THF). Intra- and inter-day coefficient of variation of the assay varied from1.5% (allo-THF) to 13% (tetrahydrocortisone) and from 3.6% (allo-THF) to 14.9% (tetrahydrocortisone), respectively. The method was applied for the analysis of urine samples from 53 healthy individuals with a mean age of 13.96?years in order to estimate the concentration of the five corticosteroids and the ratio of the metabolites. Associations between urinary cortisol/cortisone and serum cortisol/cortisone values were also characterized.     

Chemiluminometric behaviour of erythromycin and related compounds

This paper reports the chemiluminescence generated from erythromycin and other macrolide antibiotics by the action of cerium(IV) in perchloric acid or potassium permanganate in sulphuric acid or polyphosphoric acid with limits of detection (3 × noise) for erythromycin in the range 0.15–0.31 μg ml⁻¹. Permanganate in polyphosphoric acid gives the greatest sensitivity. A preliminary investigation of the reaction mechanism is also presented.     

Effects of Sideritis euboea (Lamiaceae) aqueous extract on IL-6, OPG and RANKL secretion by osteoblasts

The water extract obtained from the aerial parts of Sideritis euboea (Lamiaceae), which is known in Greece as 'mountain tea', was investigated by determining the in vitro effect of this extract on the IL-6, OPG and RANKL secretion by osteoblasts, three important molecules in osteoblast-osteoclast interplay. The results showed that this extract reduced significantly the secretion of IL-6 by KS-483 osteoblasts, while it also suppressed RANKL secretion, with both effects being dose-dependent and more potent at the higher concentrations tested (50, 100 microg/mL). We further determined the chemical profile of the extract by applying an analytical U-HPLC-DAD-ESI-MS/MS method using the high resolution hybrid LTQ-Orbitrap Discovery spectrometer. An ESI source in negative mode was employed. The analysis indicated that the water extract of S. euboea is rich in flavonoid glycosides, and phenylpropanoid glycosides.