Edward Houghton

Immunoaffinity chromatography combined on-line with high-performance liquid chromatography–mass spectrometry for the determination of corticosteroids

On-line coupled immunoaffinity chromatography-reversed-phase high-performance liquid chromatography (IAC-HPLC) with detection by quadrupole ion trap mass spectrometry using a particle beam interface has been developed for the determination of the steroids, dexamethasone and flumethasone. HEMA (polyhydroxyethylmethacrylate) was evaluated as a support material for the anti-dexamethasone antibodies used in IAC. Antibody cross-reactivity and non-specific binding have been investigated for the HEMA bound anti-dexamethasone IAC column. The on-line IAC-HPLC-MS determination of dexamethasone and flumethasone in post-administration equine urine samples showed precisions (R.S.D.) of 8.0 and 7.1%, respectively, with limits of detection in the range 3-4 ng/ml.

Preliminary study of the metabolism of 17α-methyltestosterone in horses utilizing gas chromatography–mass spectrometric techniques

Little is known about the metabolism of 17 alpha-alkyl anabolic steroids in horses. In this study, the metabolism of 17 alpha-methyltestosterone is investigated by oral administration of a (1 + 1) mixture of the steroid and its deuteriated analogue. Both compounds were synthesized from dehydroisoandrosterone (DHA), using a Grignard reaction followed by an Oppenauer oxidation. Post-administration urine extracts were analysed by gas chromatography--mass spectrometry (GC-MS) using both electron impact (IE) and chemical ionization (CI). Interpretation of the data was facilitated by observation of the fragment ions present in the mass spectra. Notably, the D-ring fragment ions were indicative of 15- or 16-hydroxylation, where 16-hydroxy metabolites showed ion pairs at m/z 218/221 and at m/z 231/234 while 15-hydroxy compounds gave the 231/234 ion pair alone. Unaltered D-rings showed fragment ions at m/z 143/146. The data showed that the main phase 1 metabolic processes were partial and complete reduction of the 3-oxo-4-ene group, 15-hydroxylation, 16-hydroxylation, 17-epimerization and hydroxylation at at least two other undetermined sites, postulated as the 6 and 11 positions. Phase 2 metabolism, in the form of glucuronide and sulfate formation, was also common. The information provided by this investigation will result in improved effectiveness of confirmatory analytical procedures for 17 alpha-alkyl anabolic steroids.

The use of stable carbon isotope analysis to detect the abuse of testosterone in cattle

The use of stable carbon isotope analysis to detect the administration of anabolic steroids to cattle was investigated. Samples were extracted by solid-phase extraction on C18 cartridges. Stable isotope ratios (13C:12C) were measured by gas chromatography-isotope ratio mass spectrometry (GC-IRMS) of the underivatised extracts. A programmed temperature vaporiser (PTV) injector was installed in the GC-IRMS system, which conferred a number of advantages. First, it allowed large volumes of sample to be injected whilst the injector liner was cool. The solvent was subsequently vented to the atmosphere prior to transfer of the sample to the GC column. Thus a significantly greater amount of sample could be presented for analysis, thereby increasing the sensitivity. Second, by this means virtually all the solvent could be removed prior to analysis. This eliminates solvent peak tailing, which can be a major problem in GC-IRMS. Finally, the PTV allowed the use of higher initial GC oven temperatures, which in turn facilitated the analysis of underivatised steroids by reducing the GC run time and improving the chromatographic peak shape. The carbon isotope composition of 5 beta-androstane-3 alpha,17 alpha-diol, the major metabolite of testosterone found in bovine bile, was measured in bile samples from untreated cattle and from cattle injected intramuscularly with testosterone or a mixture of testosterone esters. There was considerable inter-animal variation in the values obtained and there was no significant difference between samples from treated and untreated animals. However, when the isotopic composition of the metabolite was normalised with respect to that of an endogenous reference compound (cholesterol) in the same sample, the difference between treated and untreated animals become statistically significant.

Study of the metabolism of testosterone, nandrolone and estradiol in cattle†

The current metabolism study was undertaken to identify key analytes in urine, plasma and bile following testosterone, nandrolone and estradiol administrations to cull cows, heifers and steers. This information will be used to develop confirmatory analysis procedures. In the present study, mixtures (1:1) of testosterone, nandrolone or estradiol and their deuterium labelled analogues were administered to cull cows, heifers and steers. Two analogues of deuterium labelled testosterone were synthesised and administered, to facilitate identification of metabolites. Following administration, urine, plasma and bile samples were collected and subjected to solid phase extraction. The extracts were derivatised and analysed by GC-MS. The major analytes derived from the administered steroids were identified on the basis of the twin ion peaks produced for their non-labelled and deuterium labelled analogues and their stereochemistries determined by comparison of retention times with appropriate reference standards. Using suitable internal markers, excretion profiles for the major analytes in urine and plasma have been determined and levels in isolated bile samples estimated. This work is on-going, and this paper is a summary of some of the studies completed to date.

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.

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.

On-line immunoaffinity chromatography—high-performance liquid chromatography—mass spectrometry for the determination of dexamethasone

The determination of the synthetic corticosteroid dexamethasone in equine urine is described using coupled on-line immunoaffinity chromatography–high-performance liquid chromatography (IAC–HPLC) with detection by UV or mass spectrometry. The limit of detection for dexamethasone with UV detection was 30 µg l–1 for a 10 ml urine sample. Atmospheric pressure chemical ionization mass spectrometry, with single ion monitoring, gave a limit of detection of 0.1 µg l–1 for dexamethasone. The determination of dexamethasone by on-line IAC–HPLC–MS has been demonstrated in post-administration equine urine samples.

Metabolism of methyltestosterone in the greyhound

Gas chromatography/mass spectrometry and selective derivatisation techniques have been used to identify urinary metabolites of methyltestosterone following oral administration to the greyhound. Several metabolites were identified including reduced, mono-, di- and trihydroxylated steroids. The major metabolites observed were 17alpha-methyl-5beta-androstane-3alpha-17beta-diol, 17alpha-methyl-5beta-androstane-3alpha,16alpha,17beta-triol, and a further compound tentatively identified as 17alpha-methyl-5z-androstane-6z,17beta-triol. The most abundant of these was the 17alpha-methyl-5beta-androstane-3alpha,16alpha,17beta-triol. This metabolite was identified by comparison with a reference standard synthesised using a Grignard procedure and characterised using trimethylsilyl (TMS) and acetonide-TMS derivatisation techniques. There did not appear to be any evidence for 16beta-hydroxylation as a phase I metabolic transformation in the greyhound. However, significant quantities of 16alpha-hydroxy metabolites were detected. Selective enzymatic hydrolysis procedures indicated that the major metabolites identified were excreted as glucuronic acid conjugates. Metabolic transformations observed in the greyhound have been compared with those of other mammalian species and are discussed here.

Analysis of endogenous human endothelin peptides by high-performance liquid chromatography and mass spectrometry

Summary: The three isoforms of both endothelin (ET) and big ET are not readily distinguishable by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) alone. We have previously used high-performance liquid chromatography (HPLC) linked with RIA to determine ET isoforms in various tissue extracts, and cell supernatants. Our aim was to confirm that the isoforms of ET secreted by human umbilical vein endothelial cells (HUVECs) are ET-1 and big ET-1, using HPLC linked to electrospray mass spectrometry (ESI-MS). Authentic synthetic peptide standards were used to obtain the specific ions for ET isoforms and to calculate their retention times on the HPLC system. ET-1, [Met7]-sulfoxy ET-1, ET-2, ET-3, and big ET-1 could all be separated by HPLC and by specific ions on ESI-MS. Pooled supernatant (500 ml) from 50 cultures of HUVECs was applied first to an anti-C-terminal ET-1 immunoaffinity column and then to an anti-C-terminal big ET-1 immunoaffinity column. The eluates were solid phase-extracted and then injected onto an HPLC system linked to ESI-MS. Peaks of mass intensity at the specific ions and retention times corresponding to ET-1, [Met7]-sulfoxy ET-1, and big ET-1 were found in these eluates. However, no signal was present for ET-2 or ET-3. These data confirm that HUVECs in culture produce ET-1, [Met7]-sulfoxy ET-1, and big ET-1.

DISCRIMINATION OF MAMMALIAN GROWTH HORMONES BY PEPTIDE-MASS MAPPING

Recognition by the legal authorities that growth hormones (GHs) may be abused to improve sporting performance and/or physique has led to the implementation of controls that make it an offence to produce, supply, possess or import and export GHs, with intent to supply, without the authority to do so. A method is described for the discriminatory analysis of human, equine, porcine and bovine GHs for forensic purposes. Peptide-mass mapping by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry following tryptic digestion gave sequence coverages of 97.4%, 93.7%, 94.2% and 90.6% for human, equine, porcine and bovine GHs respectively. The tryptic-mass maps generated were sufficient to discriminate between the four hormones analysed and thus provide unambiguous identification of each individual GH. Identification of the N-terminal peptides of recombinant equine and porcine GHs, which possess additional methionine residues, within the tryptic-mass maps may provide the basis of a test to indicate exogeneous administration rather than endogenous secretion of GH in performance dogs and horses.