M. C. Dumasia

Screening and confirmatory analysis of β-agonists, β-antagonists and their metabolites in horse urine by capillary gas chromatography—mass spectrometry

A method for the screening and confirmatory analysis of β-agonists and -antagonists in equine urine is described. Following initial enzymic hydrolysis, the basic drugs and metabolites are extracted using Clean Screen® DAU or Bond Elut Certify™ cartridges, and analysed as their trimethylsilyl ether or 2-(dimethyl) silamorpholine derivatives by capillary gas chromatography—mass spectrometry. The method proved to be very sensitive and selective for basic drugs. After administration of therapeutic doses of propranolol, metoprolol, timolol, isoxsuprine and clenbuterol to thoroughbred horses, the parent compound/metabolites could be detected in urine for upto 14–120 h depending on the drug.

Development of a gas chromatographic—mass spectrometric method using multiple analytes for the confirmatory analysis of anabolic steroid residues in horse urine : II. Detection of administration of 19-nortestosterone phenylpropionate to equine male castrates and fillies

Esters of 19-nortestosterone form an important group of anabolic preparations used in veterinary practice. Based upon results from detailed metabolic studies for 19-nortestosterone in the horse, a method to confirm the administration of anabolic preparations of this steroid to castrated male horses and fillies is described; the method is based upon the use of multiple analytes. Following administration of the anabolic preparations, solid-phase extraction of urinary conjugates and the separation of the conjugate groups prior to hydrolysis allow for the determination of specific metabolites conjugated with either glucuronic acid or sulphate. Following hydrolysis of the conjugates, purification of the free neutral steroids on thin-layer chromatography, derivatisation and gas chromatographic-mass spectrometric analysis, the presence of the major metabolites, estrane-3,17 alpha-diol in the glucuronic acid fraction and 19-nortestosterone and two isomers of estrane-3,17-diol in the sulphate fraction, could be confirmed for 17-18 days after administration of Nandrolin (19-nortestosterone phenylpropionate).

Improved capillary gas-chromatographic-mass spectrometric method for the determination of anabolic steroid and corticosteroid metabolites in horse urine using on-column injection with high-boiling solvents

When analysing large numbers of samples containing high-boiling components by capillary column gas chromatography, the analysis time using hexane as solvent in the splitless mode is prohibitive. In order to reduce the analysis time for the steroid TMS and MO-TMS derivatives but yet maintain a solvent effect, the use of solvents with high boiling-points has been investigated. The use of solvents with high boiling-points has now been extended to on-column injection without any loss in resolution or adverse effects on column lifetime.

Development of a gas chromatographic—mass spectrometric method using multiple analytes for the confirmatory analysis of anabolic steroids in horse urine : I. Detection of testosterone phenylpropionate administrations to equine male castrates

A gas chromatographic-mass spectrometric (GC-MS) method using three analytes to detect and confirm the administration to equine male castrates of veterinary pro-drugs based upon esters of testosterone is described. The method involves extraction of steroid conjugates from horse urine by C18-bonded cartridges and fractionation into glucuronic acid and sulpho-conjugates by Sephadex LH-20 column chromatography. After deconjugation, the free neutral steroids were partially purified by thin-layer chromatography and following derivatization (methyloxime-trimethylsilyl ether) were analysed by capillary GC-MS in the selected-ion or full-scan mode. Of the three analytes, 5 alpha-androstane-3 beta, 17 alpha-diol could be detected in the glucuronic acid fraction for about ten days and 5 alpha-androstane-3 beta,17 beta-diol and testosterone could be detected in the sulpho-conjugate fraction for up to nineteen days after administration of a single therapeutic dose (50 mg) of testosterone phenylpropionate to cross-bred and thoroughbred castrated male horses. The reasons for development of such a method, its validation and its potential for the detection of neutral metabolites of other veterinary anabolic steroids in horse urine are discussed.

Comparison of the use of mass spectrometry and methylene unit values in the determination of the stereochemistry of estranediol, the major urinary metabolite of 19-nortestosterone in the horse.

The stereochemistry of an isomer of 5-estrane-3,17 alpha-diol, the major metabolite of 19-nortestosterone in horse urine has been established by the use of methylene unit (MU) values. The empirical MU values of the bis-trimethylsilyl (TMS) derivatives of the eight available isomers of 5-androstane-3,17-diol and four isomers of 5-estrane-3,17 beta-diol were determined by capillary gas chromatography using three different columns. From this data the theoretical MU values for the bis-TMS derivatives of the four 5-estrane-3,17 alpha-diol isomers were predicted. Comparison of the experimentally determined MU value of the urinary metabolite with those of the theoretical values established the correct stereochemistry of the steroid. This method has been compared with the use of gas chromatography-mas spectrometry in the determination of the stereochemistry of unknown metabolites.

Some applications of chromatography to steroid analysis in the horse

Since 1974 an extensive research programme has been carried out in this laboratory in order to develop methods to detect and confirm the administration of both anabolic steroids and corticosteroids to the horse. Success has been achieved through the application of results obtained from detailed studies on metabolism and from the determination of enaogenous steroid profiles. Chromatography, in a number of different forms, has played a vital role in these studies and, where necessary, essential chromatographic techniques have been incorporated in routine screening and confirmatory analysis procedures.

The use of stable isotopes and gas chromatography/mass spectrometry in the identification of steroid metabolites in the equine

Stable isotope gas chromatography/mass spectrometry has been used successfully in the elucidation of structures of urinary steroid metabolites in the horse and in the identification of metabolites isolated from in vivo perfusion and in vitro incubation studies using equine tissue preparations. Deuterium-labeled steroids, testosterone, dehydroepiandrosterone, and 5-androstene-3 beta,17 beta-diol have been synthesized by base-catalyzed isotope exchange methods and the products characterized by gas chromatography/mass spectrometry. [16,16(-2)H2]Dehydroepiandrosterone (plus radiolabeled dehydroepiandrosterone) was perfused into a testicular artery of a pony stallion and was shown to be metabolized into 2H2-labeled testosterone, 4-androstenedione, isomers of 5-androstene-3,17-diol, 19-hydroxytestosterone, and 19-hydroxy-4-androstenedione. In further studies, equine testicular minces have been incubated with 2H2-labeled and radiolabeled dehydroepiandrosterone and 5-androstene-3 beta, 17 beta-diol. The metabolites, whose identity was confirmed by stable isotope gas chromatography/mass spectrometry, proved the interconversion of the two substrates, as well as formation of testosterone and 4-androstenedione. The aromatization of dehydroepiandrosterone was also confirmed, together with the formation of an isomer of 5(10)-estrene-3,17-diol from both substrates showing 19-demethylation without concomitant aromatization. In studies of the feto-placental unit, the allantochorion was shown to aromatize [2H5]testosterone to [2H4]estradiol, the loss of one 2H from the substrate being consistent with aromatization of the A ring. The formation of 6-hydroxyestradiol was also confirmed in this study. The same technique has been valuable in determining the structure of two metabolites of nandrolone isolated from horse urine.(ABSTRACT TRUNCATED AT 250 WORDS)

The biotransformation and urinary excretion of dexamethasone in equine male castrates

The pro-drugs of dexamethasone, a potent glucocorticoid, are frequently used as anti-inflammatory steroids in equine veterinary practice. In the present study the biotransformation and urinary excretion of tritium labelled dexamethasone were investigated in cross-bred castrated male horses after therapeutic doses. Between 40-50% of the administered radioactivity was excreted in the urine within 24 h; a further 10% being excreted over the next 3 days. The urinary radioactivity was largely excreted in the unconjugated steroid fraction. In the first 24 h urine sample, 26-36% of the total dose was recovered in the unconjugated fraction, 8-13% in the conjugated fraction and about 5% was unextractable from the urine. The metabolites identified by microchemical transformations and thin-layer chromatography were unchanged dexamethasone, 17-oxodexamethasone, 11-dehydrodexamethasone, 20-dihydrodexamethasone, 6-hydroxydexamethasone and 6-hydroxy-17-oxodexamethasone together accounting for approx 60% of the urinary activity. About 25% of the urinary radioactivity associated with polar metabolites still remains unidentified.

Studies into aromatase activity associated with fetal allantochorionic and maternal endometrial tissues of equine placenta. Identification of metabolites by gas chromatography mass spectrometry

Maternal endometrial and fetal allantochorionic tissues were separated manually from the placentae of seven healthy thoroughbred and three pony mares, ranging in gestational age from 100 to 318 days. The homogeneity of subcellular fractions prepared from these tissues was assessed initially using the marker enzymes, succinate dehydrogenase, NADPH cytochrome C reductase and lactate dehydrogenase for the mitochondrial, microsomal and cytosolic fractions, respectively. Light microscopy and histochemical analysis demonstrated that the separated fetal allantochorionic membrane, which is made up of allantoic and chorionic epithelia, contained no significant contamination of maternal tissues. The maternal endometrium, however, was found to contain appreciable amounts of fetal chorion torn off during the separation process. Tissue homogenates and subcellular fractions were incubated with testosterone together with [4-(14)C] and [(2)H5 or (2)H3] labelled analogues in either an NADPH (1 mM) or a NADPH-regenerating environment; control experiments (without additional cofactor) were also performed. After extraction of the tissue homogenates, neutral and phenolic (oestrogen) unconjugated steroids were separated by column chromatography. Radiolabelled studies revealed that in allantochorionic tissue incubations 67-77% of testosterone was converted to oestrogenic material, subcellular fractionation indicating that oestrogen production was largely confined to the microsomal fraction and time-course studies showing that the rate of formation appeared to be linear up to 90 min. In contrast, only 5-25% conversion occurred using maternal endometrial tissues, which could be accounted for by the contaminating presence of fetal chorion. No oestrogen production was detected in control incubations. These radiolabelled studies demonstrate that aromatase activity is located on the fetal allantochorionic surface and, together with the histochemical data, further delineate this activity to the chorion in mature equine placenta. Gas chromatographic-mass spectrometric (GC-MS) analysis of the phenolic extracts from allantochorionic tissue homogenate incubations indicated the presence of substrate-derived oestradiol-17beta (E2), 6-oxo-oestradiol-17beta (6-oxo-E2) and 6beta-hydroxyoestradiol-17beta (6beta-OH-E2). Whereas all three oestrogens were identified as metabolites from testosterone in incubations performed using allantochorionic tissue homogenates and post-mitochondrial suspensions (PMS), only E2 was identified from incubations performed using microsomal fractions prepared from this tissue. We conclude that both the microsomal and cytosol fractions are required for the conversion of E2 to the 6-oxygenated species in vitro. Using stable isotope-labelled substrates and GC-MS analysis the mechanism of formation of these metabolites from these in vitro incubation studies may be inferred. GC-MS analysis of the neutral extracts from allantochorionic tissue homogenate incubations confirmed the presence of small quantities of substrate-derived 5(10)-oestrenediols. No substrate-derived 5(10)-oestrene-3,17-diols were detected in extracts from microsomal preparations incubated in the absence of cytosol. These data suggest that demethylation of C19 steroids to produce C18 neutral steroids may require the synergistic action of enzymic activities that appear to reside both in the microsomal and cytosolic fractions of equine allantochorionic tissues.