A sharp, robust, and quantitative method by liquid chromatography tandem mass spectrometry for the measurement of EAD for acute radiation syndrome and its application.

17-Ethinyl-3,17-dihydroxyandrost-5-ene (EAD) is an agent designed for the treatment of acute radiation syndrome (ARS). Given its vital role played in the prevention and mitigation of ARS, the development of a sharp, sensitive and robust liquid chromatography tandem mass spectrometry (LC-MS/MS) method to monitor the metabolism of EAD in vivo was crucial. A new method was constructed and validated for the determination of EAD with the internal standard of androst-5-ene-3ß,17ß-diol (5-AED). The blood samples were precipitated with methanol, centrifuged, from which the supernatant was separated on UPLC with C18 column and eluted in gradient with acetonitrile and Milli-Q water both containing 0.1% formic acid (FA). Quantification was performed by a triple quadrupole mass spectrometer with electro spray ionization (ESI) in multiple reactive monitoring (MRM) positive mode. A good linearity was obtained with R>0.99 for EAD within its calibration range from 5 to 1000ngmL-1 with a lowest limit of quantification (LLOQ) of 5ngmL-1. Inter- and intra-day accuracy and precision of three levels of quality control (QC) samples were within the range of 15%, while the LLOQ was within 20%. Samples were stable under the circumstances of the experiments. The method was simple, accurate and robust applied to determine the concentrations of EAD in Wistar rat after a single administration of EAD orally at the dose of 100mgkg-1.

Gas chromatography-mass spectrometry method for the analysis of 19-nor-4-androstenediol and metabolites in human plasma: application to pharmacokinetic studies after oral administration of a prohormone supplement.

19-Nor-4-androstenediol is a prohormone of nandrolone. Both substances are included in the WADA list of prohibited classes of substances. The aim of this study is to determine the plasma levels of 19-nor-4-androstenediol and its metabolites after oral administration of a nutritional supplement containing the drug. Two capsules of Norandrodiol Select 300 were orally administered to six healthy male volunteers. Plasma samples were collected up to 24h. Samples were extracted to obtain free and glucuronoconjugated metabolic fractions. Trimethylsilyl derivatives of both fractions were analyzed by gas chromatography coupled to mass spectrometry (GC-MS). The method was validated to determine linearity, extraction recovery, limit of detection and quantification, intra- and inter-day precision and accuracy. After administration of 19-nor-4-androstenediol, the main metabolites detected were norandrosterone and noretiocholanolone, mainly in the glucuronide fraction. Nandrolone, norandrostenedione and 19-nor-4-androstenediol were also detected at lower concentrations.

Quantitative determination of metabolic products of 19-norandrostenediol in human plasma using gas chromatography/mass spectrometry.

Prohormones such as 19-norandrostenediol (estr-4-ene-3beta,17beta-diol) have been added to the list of prohibited substances of the World Anti-Doping Agency because they are metabolized to the common nandrolone metabolites norandrosterone and noretiocholanolone. So far, no studies on the metabolism and in vivo conversion of 19-norandrostenediol after oral or sublingual administration have been reported nor have had quantified data on resulting plasma nandrolone levels. In the present study, an open-label crossover trial with eight healthy male volunteers was conducted. After application of capsules or sublingual tablets of 19-norandrostenediol plasma concentrations of 19-norandrostenediol, nandrolone as well as major metabolites (19-norandrosterone and 19-noretiocholanolone) were determined using a validated assay based on gas chromatography/mass spectrometry. The administration of 100-mg capsules of 19-norandrostenediol yielded maximum plasma total concentrations (i.e., conjugated plus unconjugated compounds) of 1.1 ng/ml (+/-0.7) for 19-norandrostenediol, 4.0 ng/ml (+/-2.6) for nandrolone, 154.8 ng/ml (+/-130.8) for 19-norandrosterone, and 37.7 ng/ml (+/-6.9) for 19-noretiocholanolone. The use of 25-mg sublingual tablets resulted in 3.3 ng/ml (+/-1.0) for 19-norandrostenediol, 11.0 ng/ml (+/-6.4) for nandrolone, 106.3 ng/ml (+/-40.1) for 19-norandrosterone, and 28.5 ng/ml (+/-20.8) for 19-noretiocholanolone. Most interestingly, the pharmacologically active unconjugated nandrolone was determined after administration of sublingual tablets (up to 5.7 ng/ml) in contrast to capsule applications. These results demonstrate the importance of prohibiting prohormones such as 19-norandrostenediol, in particular, since plasma concentrations of nandrolone between 0.3 to 1.2 ng/ml have been reported to influence endocrinological parameters.

Quantification and profiling of 19-norandrosterone and 19-noretiocholanolone in human urine after consumption of a nutritional supplement and norsteroids.

Nandrolone is one of the synthetic anabolic steroids banned in sports and has been a popular substance abused by athletes in recent years. One of its major metabolites, 19-norandrosterone (19-NA), has been used as a determinant for drug violations in sports. Current reports regarding nandrolone-positive cases have been related to intake of some nandrolone-free nutritional supplements. The aim of this study was to learn whether if a nutritional supplement sold by over-the-counter (OTC) nutritional stores could yield the same metabolic products as that of nandrolone. If so, what is (are) the substance(s) that contributed to the nandrolone metabolites? To determine the content of an OTC nutritional supplement, a tablet was dissolved in methanol, followed by N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA)-trimethyliodosilane (TMIS) derivatization prior to gas chromatography-mass spectrometry (GC-MS) analysis. The collected urine samples underwent extraction, enzymatic hydrolysis, and derivatization before the analyses of GC-MS. The results showed that seven anabolic steroids were found as contaminants in the nutritional supplement, in addition to six that were listed in the ingredients by the manufacturer. We confirmed previous reports that administration of the OTC supplement could produce a positive urine test for nandrolone metabolites. Furthermore, the results from excretion studies showed that 19-NA and 19-noretiocholanolone (19-NE) were present in urine after consuming the nutritional supplement, nandrolone, 19-nor-4-androsten-3,17-dione, 19-nor-4-androsten-3beta,17beta-diol, and 19-nor-5-androsten-3beta,17beta-diol. The 19-NA concentrations in urine were generally higher than that of 19-NE (19-NA/19-NE ratio > 1.0) especially during the early stage of excretion, that is, before 6 h post-administration. After this period of time, the concentrations of 19-NA and 19-NE fluctuated and might even have reversed (19-NA/19-NE ratio < 1.0) in their ratio, that is, higher yield in 19-NE than that in 19-NA. On the basis of this study, we postulate that some doping violations of nandrolone could be attributed by indiscriminate administration of the OTC nutritional supplements that contained 19-norsteroids.

The metabolism of orally ingested 19-nor-4-androstene-3,17-dione and 19-nor-4-androstene-3,17-diol in healthy, resistance-trained men.

The purpose of this investigation was to determine the metabolism of 2 over-the-counter steroids (Nortesten, which contains 36 mg of 19-nor-4-androstene-3,17-dione and 36 mg of 19-nor-4-androstene-3,17-diol) in healthy, resistance-trained men. Subjects were administered either low (72 mg) or high doses (144 mg) of Nortesten twice daily for 10 days. All subjects tested positive via urinalysis for the presence of nortestosterone at days 3, 5, 7, and 10. There was no change in the urine testosterone-epitestosterone ratio at any day. Furthermore, as determined by serum chemistry tests, there was no effect on renal, hepatic, hematological, or bone marrow function. Thus, short-term ingestion of 19-nor-4-androstene-3,17-dione and 19-nor-4-androstene-3,17-diol may result in a positive drug test result without any harmful side effects.