Qualification and quantification of seventeen natural steroids in plasma by GC-Q-MS and GC-IT-MS/MS.

Studying the plasma steroid profile offers information about the possible existence of endocrinological alterations. This study describes the development and validation of gas chromatographic-mass spectrometric and gas tandem mass spectrometric methods for the simultaneous identification of 17 steroid hormones in human plasma using five different solvents. The n-hexane/ethyl acetate solvent mixture, in a proportion of 70/30 (v/v) provided the best results. The extracts were derivatized with N-methyl-N-trimethylsilyl-trifluoroacetamide. The obtained limits of detection were below 1 ng/mL in the majority of the studied steroids and the limits of quantification were below 5 ng/mL; the method obtained good linearity, reproducibility, repeatability, accuracy and recoveries above 95% in most cases.

Variations in urine excretion of steroid hormones after an acute session and after a 4-week programme of strength training.

Performing strength exercise, whether acutely or in a training programme, leads to alterations at the hypothalamic-pituitary-testicular and hypothalamic-pituitary-adrenal axes. One way to evaluate these changes is by analysis of the excretion of steroid hormones in the urine. The present study determined the variations in the urine profile of glucuroconjugated steroids after a single session of strength exercise and after a 4-week programme of strength training. The subjects were a group (n = 20) of non-sportsman male university students who worked out 3 days a week [Monday (M), Wednesday (W) and Friday (F)], performing the exercises at 70-75% of one repetition maximum strength (1-RM). Four urine samples were collected per subject: (A) before and (B) after a standard session prior to initiating the training programme, and (C) before and (D) after the same standard session at the end of the study, and they were assayed by gas chromatography coupled to mass spectrometry. The concentrations of the different hormones were determined relatively to the urine creatinine level (ng steroid/mg creatinine) to correct for diuresis. After the exercise sessions, both before and after the training programme, there was a fall in the urine excretion of androgens and estrogens, but no statistically significant changes in the excretion of tetrahydrocortisol (THF) and tetrahydrocortisone (THE). The anabolic/catabolic hormones ratio also decreased after the acute session, although only androstenodione + dehydroepiandrosterone (DHEA)/THE + THF ratio had a significant decrease (P < 0.05). After the training programme, there was a significant (P < 0.01) improvement in the strength of the muscle groups studied, and an increased urinary excretion of all the androgens with respect to the initial state of repose, with the difference being significant in the case of epitestosterone (Epit) (P < 0.05). The androsterone (A) + etiocholanolone (E)/THE + THF ratio increased significantly (P < 0.05) concerning the initial state. We therefore conclude that subjects suffer variations of the urine profile with regard to the steroid hormones before and after the acute strength sessions and after the training period. The alteration after the training programme seems to be due to the subjects' hypothalamic-hypophysis-testicular and hypothalamic-pituitary-adrenal axes adaptations, which enable them to increase physical strength.

Determination of natural corticosteroids in urine samples from sportsmen.

A method for the determination of natural corticosteroids (cortisone, cortisol, 5beta-dihydrocortisone, 5beta-dihydrocortisol, tetrahydrocortisone and tetrahydrocortisol) found in the urine of sportsmen, was developed using a capillary gas chromatography-mass spectrometry ion trap system. 17alpha-Methyltestosterone was used as an internal standard. The different corticosteroids were determined from the peak area ratios of the [M]; [M-90] and [M-90-90] fragment ions of their methoxime-trimethylsiyl derivatives. Sensitivity (15 ppb), specificity, accuracy (96%) and reproducibility (RSD=4-10%) of the method were demonstrated to be satisfactory for measuring the urinary concentrations of the selected natural corticosteroids.