Quantifying endogenous androgens, estrogens, pregnenolone and progesterone metabolites in human urine by gas chromatography tandem mass spectrometry.

A method for the quantitation of 22 urinary steroids (androgens, estrogens and the main pregnenolone and progesterone metabolites) by means of gas chromatography tandem mass spectrometry using a triple quadrupole analyzer has been developed. Two different enzymatic hydrolysis protocols were investigated; one capable of releasing steroids present as both sulfates and glucuronides (total fraction), and another with ß-glucuronidase activity only. After selecting adequate internal standards and choosing the optimal instrumental parameters, i.e. chromatographic separation and ion transition conditions, the method was fully validated using both hydrolysis protocols. The method was shown to be linear (r >0.99) in the range of endogenous concentrations for all studied steroids with extraction recoveries higher than 80%. The use of labeled internal standards allowed for both a correct quantification and the evaluation of the rate of deconjugation for sulfates and glucuronides in every sample. In general, the sensitivity of the method was suitable for the detection of the endogenous levels, with limits of quantification ranging from 0.1 to 20ng/mL. Accuracies ranging from 80% to 120%, and relative standard deviations below 25% in intra- and inter- assay experiments were found for most of the analytes. The applicability of the validated method was tested by quantifying twenty-two metabolites in 24-h urine samples collected from healthy individuals. The ranges for the excretion of steroids in the total and glucuronide fractions obtained with the new method were compared with those available in the literature. By comparing the figures in both fractions, an estimation of the percentage that the sulfation represents for each steroid was also calculated. The presence of side enzymatic activities and the utility of the method for clinical studies as well as for doping control analysis is discussed.

Targeting tryptophan and tyrosine metabolism by liquid chromatography tandem mass spectrometry.

An imbalance in tryptophan (Trp) and tyrosine (Tyr) metabolites is associated with neurological and inflammatory disorders. The accurate and precise measurement of these compounds in biological specimens is a powerful tool to understand the biochemical state in several diseases. In this study, a rapid, accurate and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the targeted analysis of the metabolism of Trp and Tyr has been developed and validated. The method allows for the adequate quantification of Trp, Tyr and, eight Trp metabolites, three Tyr metabolites, together with four competitive large neutral amino acids. Serotonin, 5-hydroxyindoleacetic acid, kynurenine, kynurenic acid, dopamine, and homovanilic acid were among the targeted compounds. Sample preparation, chromatographic separation and mass spectrometric detection were optimized in human urine, human plasma and mice prefrontal cortex extracts. The method was shown to be linear (r>0.98) in the range of endogenous concentrations for all studied metabolites. In general, the limits of detection were suitable for the detection of the endogenous levels. Intra- and inter-assay precisions below 25% and accuracies ranging from 80 to 120% were found for most of the analytes. The use of labeled internal standards corrected the moderate matrix effect observed for some compounds. The applicability of the method was confirmed by analyzing urine samples collected from 13 healthy volunteers and comparing the results with previously established normal ranges. In addition, urine samples from two patients and a heterozygous carrier of a family with disturbed monoamine metabolism due to a loss of function mutation in the MAOA gene (X-linked) were analyzed and compared with samples from controls. All data together show the potential of the developed approach for targeted metabolomic studies.

Urinary cysteinyl progestogens: Occurrence and origin.

The presence of two cysteinyl progestogens, 16-cysteinyl-progesterone (16-Cys-Prog) and 16-cysteinyl-pregnenolone (16-Cys-Preg), in human urine is described for the first time. Their occurrence was unequivocally confirmed by comparison with synthesized material by using mass spectrometric detectors. Several experiments were performed in order to clarify their origin. The adrenal origin of both 16-Cys-Prog and 16-Cys-Preg can be inferred from the increase in their concentrations after ACTH stimulatory test, together with their circadian variation similar to the one observed for cortisol. Moreover, the notable increase in excretions of 16-Cys-Prog during the luteal phase of the menstrual cycle points towards an ovarian production for this progestogen. However, the analysis of samples during the course of two pregnancies revealed that, in spite of the large amounts of progesterone produced during gestation, the human placenta lacks the capacity to make 16-Cys-Prog. The adrenal and ovarian origin has been further indicated by the absence of both metabolites in samples collected from a subject with bilateral adrenalectomy and hypogonadotrophyic hypogonadism. Regarding liver action, in vitro studies with hepatocytes and progesterone indicate that, although the liver is able to metabolize progesterone to 6-dehydroprogesterone, it has not the enzymatic machinery for the generation of 16-dehydroprogesterone. Taken together, these results open the possibility for a noninvasive test for the simultaneous evaluation of progesterone biosynthesis in different organs.

Investigation of endogenous corticosteroids profiles in human urine based on liquid chromatography tandem mass spectrometry.

The accurate and precise measurement of endogenous corticosteroids in urine is a powerful tool to understand the biochemical state in several diseases. In this study, a rapid, accurate, and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of 67 endogenous gluco- and mineralo-corticosteroids and progestins has been developed and validated. Sample preparation, chromatographic separation, and mass spectrometric detection were optimized. Urine samples (0.5 mL) were hydrolyzed with ß-glucuronidase and the released analytes were extracted by liquid-liquid extraction. The chromatographic separation was performed in 20 min after redisolution of the extract. MS behavior of endogenous corticosteroids was evaluated in order to select the most specific precursor ion ([M+H](+), [M+NH4](+), or [M+H-nH2O](+)) for the detection. MS/MS determination was performed under selected reaction monitoring mode using electrospray ionization in positive mode. The method was shown to be linear (r>0.99) in the range of endogenous concentrations for all studied metabolites. Limits of detection (LOD) below 1 ng mL(-1) were typically obtained for analytes with a 3-oxo-4-ene structure whereas LODs below 15 ng mL(-1) were common for the rest of analytes. Recoveries were higher than 80% and intra-assay precisions below 20%, evaluated at three concentration levels, were found for most steroids. No significant or moderate matrix effect, ranging from 54 to 155%, was observed for most of the analytes. The applicability of the method was confirmed by analyzing 24h urine samples collected from twenty healthy volunteers and comparing the results with previously established normal ranges. The wide coverage of corticosteroid metabolism, together with short analysis time, low sample volume, simple sample preparation, and satisfactory quantitative results make this method useful for clinical purposes.