The methyl-5 alpha-dihydrotestosterones mesterolone and drostanolone; gas chromatographic/mass spectrometric characterization of the urinary metabolites.

Before including the detection of the methyl-5 alpha-dihydrotestosterones mesterolone (1 alpha-methyl-17 beta-hydroxy-5 alpha-androstan-3-one) and drostanolone (2 alpha-methyl-17 beta-hydroxy-5 alpha-androstan-3-one) in doping control procedures, their urinary metabolites were characterized by gas chromatography/mass spectrometry. Several metabolites were found after enzymatic hydrolysis and conversion of the respective metabolites to their trimethylsilyl-enol-trimethylsilyl ether derivatives. The major metabolites of mesterolone and drostanolone were identified as 1 alpha-methyl-androsterone and 2 alpha-methyl-androsterone, respectively. The parent compounds and the intermediate 3 alpha,17 beta-dihydroxysteroid metabolites were detected as well. The reduction into the corresponding 3 beta-hydroxysteroids was a minor metabolic pathway. All metabolites were found to be conjugated to glucuronic acid.

The detection of danazol and its significance in doping analysis.

The use of anabolic steroids and related compounds in sport is forbidden by the International Olympic Committee (IOC). Because danazol (17 alpha-pregna-2,4-dien-20-yno[2,3-D] isoxazol-17 beta-ol) is structurally related to the anabolic steroid stanozolol, its use should be questioned. Therefore, the detection and the significance of danazol in doping analysis are discussed. A urine specimen suspected of containing danazol metabolites was analyzed in order to characterize the metabolites. After isolation and conversion into three different derivatives, the metabolites were subjected to gas chromatography/mass spectrometry (GC/MS) in the electron impact (EI) mode. The structure assignment was based on the molecular ions, fragmentation patterns observed for the three different derivatives, and the possible metabolite structures given in the literature. Ethisterone was identified as a nonconjugated metabolite. 2-Hydroxymethylethisterone was observed in two stereoisomeric forms. One stereoisomer was found mainly in the nonconjugated steroid fraction and the other in the conjugated fraction. The results were confirmed by analyzing urine specimens of a volunteer who was known to have taken danazol. Derivatization methods and GC/MS data are given to implement danazol detection in routine screening and confirmation procedures.

Doping control of testosterone and human chorionic gonadotrophin: a case study.

Doping control for testosterone and human Chorionic Gonadotrophin (hCG) requires special attention as a difference must be made between the endogenous and exogenous origin of both substances. The detection of exogenous testosterone is based on the ratio of testosterone- to epitestosterone-glucuronide (T/E) in urine. The problems with this ratio are discussed. For hCG analysis in urine the utilization of sandwich-type hCG specific assays instead of hCG/hCG beta competitive assays is recommended. A case study in which an athlete self-administered testosterone and hCG before a competition is described. The T/E ratio and hCG concentration in urine were followed during this period of self-administration. The results demonstrate the relevance of the T/E ratio and of the selected hCG assay. The ratio of testosterone to human Luteinizing Hormone (T/hLH) in serum also indicated the use of hormones. Although the athlete's urine was negative for exogenous testosterone directly after competition, he would have been found positive for hCG.

DNA content and Ks8.12 binding of the psoriatic lesion during treatment with the vitamin D3 analogue MC903 and betamethasone.

Twenty patients with psoriasis were treated with the vitamin D3 analogue MC903 and betamethasone ointment in a double-blind trial with a left-right comparison. In addition to the clinical severity scores, Ks8.12 binding which detects keratin 16 expression and the DNA synthesis were quantified using flow cytometry. Both markers decreased significantly with treatment, but remained above the normal range even in those who had total clearance of the lesions. Treatment with MC903 with regard to Ks8.12 binding was significantly better than with betamethasone.

Mass spectrometric characterization of different norandrosterone derivatives by low-cost mass spectrometric detectors using electron ionization and chemical ionization.

The abuse of nortestosterone in sport is an important problem in doping-control analysis. In order to detect the main urinary metabolite of nortestosterone, norandrosterone (NA), sensitive and specific methodology is necessary. In this context the use of a low-cost mass spectrometric detector such as the Finnigan MAT ion-trap detector (ITD) was studied. The electron ionization (EI) and positive-ion chemical ionization (PICI) mass spectra of the methoxime-trimethylsilyl, trimethylsilyl-enol trimethylsilyl ether and pentafluoropropionic ester derivatives of NA are described. The limits of detection of these derivatives are compared with those obtained by the Hewlett-Packard mass selective detector (MSD), another low-cost mass spectrometric detector and operating only in the EI mode. For the derivatives of the reference standard of NA the ITD has in the EI mode the same limit of detection, in the range of 0.5 to 1 ng injected on the column, as the MSD. However, under these conditions the ITD provides more spectrometric information, because it gives full scan data. Moreover, with the same or even improved limits of detection the ITD can operate in the PICI mode. On the other hand, for the analysis of NA isolated from urine samples, the performance of the MSD was better than that of the ITD. The ion trapping technique is probably limited when the chemical background is high.

Comparison of the GC/MS ion trapping technique with GC-FTIR for the identification of stimulants in drug testing.

The identification of banned stimulants in the urine of athletes is typically performed by gas chromatography with electron impact mass spectrometry (GC-EIMS). Derivatization is usually used to improve the chromatographic properties and to produce unique mass spectra for closely related compounds. Both gas chromatography with chemical ionization mass spectrometry (GC-CIMS) and gas chromatography with Fourier transform infrared spectroscopy (GC-FTIR) were compared for three test mixtures of stimulants. Both techniques were capable of supplying the necessary information for conclusive confirmation. However, the CI option on the ion trap mass detector (ITD) offers the most sensitive and fastest way for the confirmation of positive samples with detection limits of 1-10 ng. The GC-FTIR is from a chemometrics point of view more specific but needs 25-75 ng for a reliable spectrum.

Improved screening method for beta-blockers in urine using solid-phase extraction and capillary gas chromatography-mass spectrometry.

An improved screening method for beta-blockers in urine is proposed, involving enzymatic hydrolysis, solid-phase extraction and capillary gas chromatography-mass spectrometry. Several extraction methods for beta-blockers, such as conventional liquid-liquid and solid-phase extraction procedures, have been evaluated for at least eight beta-blockers. Additionally, the gas chromatographic properties and mass fragmentation of the trimethylsilyl-trifluoroacetyl, trifluoroacetyl and cyclic n-butylboronate derivatives of beta-blockers have been compared and evaluated with respect to their efficiency for screening urine. The resulting screening method proved to be a specific and sensitive procedure, enabling these analytes to be detected and identified up to 48 h after the administration of a dosage, usually encountered in doping cases.

The determination of non-steroidal anti-inflammatory drugs by GC-MS-MS in equine urine.

Results are given for a more sensitive screening procedure for non-steroidal anti-inflammatory drugs using GC-MS-MS. By monitoring a selected characteristic reaction for each drug very low detection limits are reached even in a difficult biological matrix such as equine urine. Detection down to 5 ng ml-1 for ibuprofen, ibufenac, alclofenac, fenoprofen, ketoprofen, naproxen and diclofenac is possible in contrast to the 0.5 microgram ml-1 limit for normal GC-MS detection. Examples are given of real positive cases for diclofenac and ibuprofen.

Why do doping control labs need a tandem mass spectrometer?

The International Olympic Committee requires a confirmation by gas chromatography/mass spectrometry for all positive doping cases. Because of the severe consequences involved they should be more specific about the required information and determine the differences allowed. The use of tandem mass spectrometry in dope control should be required before the Olympic Games in Barcelona in 1992. This article shows the power of tandem mass spectrometry in the definite identification of a drug.

The structure of the m/z 101 ion in the spectra of permethylated saccharides.

In this paper the structure of the most important fragment ion ([C5H9O2]+, m/z 101) in the spectra of permethylated saccharides is investigated. The participation of the C-1 carbon atom in the formation of this ion has been quantitatively determined from the spectra of 13C labelled permethylated glucofuranosides. Investigation of the metastable ion and collisional activation spectra of m/z 101 yielded evidence for an acylic ion structure.

The use of collisional activation spectra in the discrimination of stereoisomeric permethylated disaccharides.

Collisional activation spectra of quasimolecular ions generated by chemical ionization can be used for the discrimination of stereoisomeric permethylated disaccharides. The best results have been obtained by using ammonia and trimethylamine as reagent gases. In the case of trimethylamine the presence of water seems to play an important role in order to obtain distinctive collisional activation spectra. The structural information obtained with the chemical ionization gases mentioned is complementary. A flow chart is presented for the identification of an unknown permethylated disaccharide.