Fast reliable assay for morphine and its metabolites using high-performance liquid chromatography and native fluorescence detection

A method for the fast analysis of morphine (M), normorphine (NM), morphine-3- and -6-glucuronides (M3G and M6G) and codeine (C) is described which has the advantages of sensitivity, speed and specificity. Dihydrocodeine and heroin can also be assayed. The method is based on extraction of the opiates from serum, plasma and cerebrospinal fluid using reversed-phase solid-phase extraction columns, followed by reversed-phase high-performance liquid chromatography with native fluorescence detection. The extraction step provides greater than 95% recovery, and the response of the detection system is linear from 0.5 to beyond 750 ng. The method allows analysis of M, NM, M3G, M6G and C. No other drugs have been found to interfere with the assay. The assay offers a quick, cheap and reliable method of specifically determining morphine and its metabolites, including the potent M6G, from a small sample volume; this will be of advantage to both clinician and basic scientist.     

Chromatographic determination of amines in biological fluids with special reference to the biological monitoring of isocyanates and amines. IV. Determination of 1,6-hexamethylenediamine in human urine using capillary gas chromatography and selective ion monitoring

A capillary gas chromatographic (GC) method was developed for the determination of 1,6-hexamethylenediamine (HDA) in hydrolysed human urine. The method was based on a derivatization procedure with heptafluorobutyric anhydride. The amides formed were determined using capillary GC with selected ion monitoring in the chemical ionization mode with ammonia as reagent gas. The overall recovery was 34% for a concentration of 100 micrograms/l of HDA in urine. The minimum detectable concentration in urine was below 0.5 microgram/l. The precision of the method was 5% (n = 9). Deuterium-labelled HDA [H2NC2H2(CH2)4C2H2NH2] was used as the internal standard. A male subject was exposed to hexamethylene diisocyanate (HDI) for 7.5 h in a test chamber. The average air concentration of HDI was ca. 30 micrograms/m3, which corresponds to ca. 85% of the threshold limit value in Sweden (35 micrograms/m3). The half time of urinary levels of HDA was ca. 1.4 h and more than 90% of the urinary elimination was completed within 4 h after the exposure. The amount of HDA excreted in urine was ca. 10 micrograms, corresponding to ca. 10% of the estimated inhaled dose of HDI.     

Detection of exogenous intake of natural corticosteroids by gas chromatography/combustion/isotope ratio mass spectrometry: application to misuse in sport

A detailed procedure for the analysis of exogenous hydrocortisone and cortisone in urine by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is proposed. As urinary levels of hydrocortisone are rather low for GC/C/IRMS analysis, the focus is on the main corticosteroid metabolites, tetrahydrocortisone (THE) and tetrahydrocortisol (THF). Following different solid phase extraction purifications, THE and THF are oxidized to 5beta-androstanetrione before analysis by GC/C/IRMS. Significant differences in delta(13)C per thousand values of synthetic natural corticosteroids and endogenous human corticosteroids have been observed. Therefore, a positive criterion, to detect exogenous administration of synthetic corticosteroids in anti-doping control, is proposed.     

Improved high-performance liquid chromatographic determination of ciprofloxacin and its metabolites in human specimens.

A simple approach to the quantitation of ciprofloxacin and its three metabolites, M1 (desethylene-ciprofloxacin), M2 (sulfo-ciprofloxacin) and M3 (oxo-ciprofloxacin), in human serum, urine, saliva and sputum is described. This assay allows the parent drug and its metabolites to elute and be resolved in a single chromatogram at 280 nm using a linear gradient. The procedure involved liquid-liquid extraction. Separation was achieved on a C18 reversed-phase column. The limit of detection of ciprofloxacin is 0.05 microgram/ml and that of its three metabolites is 0.25 microgram/ml. This method is sufficiently sensitive for pharmacokinetic studies.     

Quantitation of pancuronium, 3-desacetylpancuronium, vecuronium, 3-desacetylvecuronium, pipecuronium and 3-desacetylpipecuronium in biological fluids by capillary gas chromatography using nitrogen-sensitive detection

A sensitive and specific capillary gas chromatographic (GC) assay was developed for the quantitation of the quaternary ammonium steroidal neuromuscular blocking drugs pancuronium (PANC), vecuronium (VEC) and pipecuronium (PIP), as well as the metabolites 3-desacetylpancuronium (3-desPANC) and 3-desacetylvecuronium (3-des VEC) in plasma, bile and urine; the putative metabolite 3-desacetylpipecuronium (3-des PIP) was extracted and quantitated only in urine. The procedure employed a single dichloromethane extraction of the iodide ion-pairs of the monoquaternary or bisquaternary ammonium compounds (including internal and external standards) from acidified, ether-washed biological fluid followed by the formation of stable O-tert.-butyldimethylsilyl derivatives at the 3-hydroxy steroidal position of the metabolites. An automated capillary GC system fitted with a nitrogen-sensitive detector and an integrator was then used to analyze and quantitate both parent compounds and their derivatized metabolites. Optimal extraction, derivatization and GC conditions, as well as short-term stability and recoveries of these drugs and metabolites in plasma, are reported. Electron ionization mass spectrometry combined with GC was used to confirm the identities of compounds eluted from the column. The assay demonstrated a 10(3)-fold linear range up to 5000 ng/ml for PANC, VEC, 3-des VEC and PIP, and lower limits of detection with adequate precision of 2 ng/ml for PANC, VEC and PIP, and 4 ng/ml for 3-des VEC; 3-des PANC was linear from 8 to 500 ng/ml while 3-des PIP was linear from 25 to 1000 ng/ml. The precision (coefficient of variation) of the calibration curves for underivatized drugs and their derivatized metabolites over the linear ranges was 2-20% and the reproducibility of the assay over a range of clinical concentrations of these drugs found in human plasma was 5-16% for PANC, 2-4% for VEC and 6-11% for PIP. No interferences were detected in the assay of plasma samples from 106 surgical patients.     

Simultaneous determination of perhexiline and its monohydroxy metabolites in biological fluids by gas chromatography-electron-capture detection

A rapid and sensitive method for the simultaneous determination of perhexiline and its cis-4-axial and trans-4-equatorial monohydroxy metabolites (M1 and M3, respectively) in human plasma, urine and bile is described. The assay utilises a single diethyl ether extraction, heptafluorobutyric acid anhydride derivatisation and separation and detection by gas chromatography-electron-capture detection. The limits of detection are 0.1 microgram/ml for perhexiline and 0.025 microgram/ml for the M1 and M3 metabolites. This method has been used in a five-day kinetic study of three healthy adult males who ingested a single 300-mg dose of perhexiline maleate. One of these volunteer subjects exhibited elevated plasma perhexiline and markedly reduced plasma and urinary M1 concentrations together with profoundly prolonged plasma and urinary M1 elimination times when compared with the other two subjects. These differences are thought to be of genetic origin. There were also obvious differences in urinary M3 concentrations which were discussed.     

High-performance liquid chromatographic method for the quantitation of quinidine and selected quinidine metabolites

A specific and sensitive assay for the separation and quantitation of quinidine, 3-hydroxyquinidine, quinidine-N-oxide, O-desmethylquinidine and dihydroquinidine is presented. The assay is shown to be sensitive to concentrations of 0.1 microgram/ml for all the above compounds when using a serum sample of 0.1 ml. The standard curve demonstrates linearity at concentrations from 0.1 to 5 micrograms/ml. The extraction procedure consists of adjusting the serum to an alkaline pH and extracting once with a mixture of methanol-dichloromethane (15:85). The organic extract is dried and the residue is solubilized in mobile phase. The chromatographic conditions are an isocratic delivery of the mobile phase 0.01 M K2HPO4-acetonitrile (96:4) through a C18 column at ambient temperature. Detection of the compounds of interest is by ultraviolet absorption at a wavelength of 210 nm. For each compound the inter-assay variation is less than 10% and the intra-assay variation is less than 15%. No interfering compounds were detected when a commercially prepared serum spiked with 28 commonly used therapeutic compounds was assayed by this method. The analytical method presented here for the isolation and quantitation of quinidine, several active metabolites, and dihydroquinidine has adequate sensitivity and specificity for monitoring the concentration of quinidine and quinidine metabolites in patient samples.     

Improved gas chromatographic assay for the simultaneous determination of nitroglycerin and its mono- and dinitrate metabolites.

A sensitive, specific capillary gas chromatographic-electron-capture detection method for the simultaneous determination of nitroglycerin (GTN), 1,2- and 1,3-glyceryl dinitrate (1,2-GDN and 1,3-GDN, respectively) and 1- and 2-glyceryl mononitrate (1-GMN and 2-GMN, respectively) is reported. The minimum quantifiable concentration for GTN, GDNs and GMNs is 0.4 ng/ml in plasma, with extraction recoveries for GMNs greater than 76% and for GTN and the GDNs greater than 95%. Over the full range of quantifiable concentrations the inter-run assay precision and accuracy were less than 13 and 11%, respectively, for all five nitrates. Similar intra-run assay precision and accuracy values were found. The method was employed in the preliminary in vitro examination of GTN, GDN and GMN kinetics in human blood. Following addition of GTN to human blood, the ratio of 1,2-GDN to 1.3-GDN maximum concentrations (Cmax) was ca. 7:1, reflecting preferential denitration of the GTN molecule at the primary positions, while the Cmax ratio for 2-GMN to 1-GMN in this system was ca. 6:1, representing a highly selective if not specific primary denitration of the 1,2-GDN molecule. Following the intravenous administration of 1,2-GDN to five healthy male volunteers, 2-GMN/1-GMN Cmax ratios averaged 8.8:1, representing a highly selective but not specific formation of 2-GMN from the 1,2-GDN molecule. The assay will find utility in in vitro studies attempting to address the molecular pharmacology of GTN and its metabolites, and in in vivo clinical pharmacology studies attempting to address the relationship between pharmacokinetics and pharmacodynamics of GTN and its metabolites.     

Determination of diclofensine, an antidepressant agent, and its major metabolites in human plasma by high-performance liquid chromatography with fluorometric detection

A sensitive and selective high-performance liquid chromatographic assay was developed for the determination of diclofensine (I) and its key metabolites in human plasma. The assay involves deproteinization of plasma, overnight Glusulase incubation to hydrolyze the major metabolite (I-B-glucuronide), extraction of the parent compound and its deconjugated metabolites (I-A, I-B and I-C) from the alkalinized aqueous phase into diethyl ether-ethanol (95:5), the residue of which (containing compounds I, I-A, I-B and I-C) is alkylated with 2-iodopropane dissolved in acetone, using solid potassium hydroxide as a catalyst. The compounds are extracted from the reaction mixture into diethyl ether, after adding ethanol-water-acetic acid (55:40:5), the residue of which is dissolved in 0.05 M sulfuric acid, and reacted with mercuric acetate at 100 degrees C, which oxidizes tertiary tetrahydroisoquinolines to their 3,4-dihydroisoquinoline derivatives, followed by a photochemical reaction in the same solution to form intensely fluorescent isoquinolinium derivatives. An aliquot of this reaction mixture is injected onto a reversed-phase high-performance liquid chromatography column (5-microns Nova-Pac C13 phase in a radial compression cartridge, 10 cm X 8 mm), using the mobile phase 0.25 M triethylammonium phosphate (pH 2.5)-0.25 M acetic acid-methanol-acetonitrile-tetrahydrofuran (150:350:125:375:25). The void volume (Vo) is approximately 1.4 min and the retention times (tR) of the respective isoquinolium derivatives of diclofensine (I) are ca. 3.5 min, internal standard (II) ca. 4.2 min, nordiclofensine (I-A) ca. 5 min, while the phenolic metabolites I-B and I-C give peaks at 6.4 min and 10.4 min, respectively. The derivatives are detected by fluorescence. The method was used to determine plasma concentrations of the parent drug (I) and its major phenolic metabolite I-B (aglycone) in plasma in two normal volunteers following a single oral 45-mg dose and following seven consecutive days of oral dosing of 45 mg three times a day as part of a multiple ascending dose tolerance study.     

The detection of androstenedione abuse in sport: a mass spectrometry strategy to identify the 4‐hydroxyandrostenedione metabolite

Studies have shown that the administration of androstenedione (ADIONE) significantly increases the urinary ratio of testosterone glucuronide to epitestosterone glucuronide (T/E) – measured by gas chromatography/mass spectrometry (GC/MS) – in subjects with a normal (≈1) or naturally high (>1) initial values. However, the urinary T/E ratio has been shown not to increase in subjects with naturally low (<1) initial values. Such cases then rely on the detection of C₆‐hydroxylated metabolites shown to be indicative of ADIONE administration. While these markers may be measured in the routine GC/MS steroid profile, their relatively low urinary excretion limits the use of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to specifically confirm ADIONE administration based on depleted ¹³C content. A mass spectrometry strategy was used in this study to identify metabolites of ADIONE with the potential to provide compound‐specific detection. C₄‐hydroxylation was subsequently shown to be a major metabolic pathway following ADIONE administration, thereby resulting in urinary excretion of 4‐hydroxyandrostenedione (4OH‐ADIONE). Complementary analysis of 4OH‐ADIONE by GC/MS and GC/C/IRMS was used to confirm ADIONE administration. Copyright © 2008 Commonwealth of Australia. Published by John Wiley & Sons, Ltd.     

In vivo biotransformation of 17 alpha-methyltestosterone in the horse revisited: identification of 17-hydroxymethyl metabolites in equine urine by capillary gas chromatography/mass spectrometry

The in vivo phase I biotransformation of 17 alpha-methyltestosterone in the horse leads to the formation of a complex mixture of regio- and stereoisomeric C(20)O(2), C(20)O(3) and C(20)O(4) metabolites, excreted in urine as glucuronide and sulphate phase II conjugates. The major pathways of in vivo metabolism are the reduction of the A-ring (di- and tetrahydro), epimerisation at C-17 and oxidations mainly at C-6 and C-16. Some phase I metabolites have been identified previously by positive ion electron ionisation capillary gas chromatography/mass spectrometry (GC/EI + MS) mainly from the characteristic fragmentation patterns of their methyloxime-trimethylsilyl ether (MO-TMS), enol-TMS or TMS ether derivatives. Following oral administration of 17 alpha-methyltestosterone to two castrated thoroughbred male horses, the glucuronic acid conjugates excreted in post-administration urine samples were selectively hydrolysed by E. coli beta-glucuronidase enzymes. Unconjugated metabolites and the steroid aglycones obtained after enzymatic deconjugation were isolated from urine by solid-phase extraction, derivatised as MO-TMS ethers and analysed by GC/EI + MS. In addition to some of the known metabolites previously identified from the characteristic mass spectral fragmentation patterns of 17 alpha-methyl steroids, some isobaric compounds exhibiting a diagnostic loss of 103 mass units from the molecular ions with subsequent losses of trimethylsilanol or methoxy groups and an absence of the classical D-ring fragment ion were detected. From an interpretation of their mass spectra, these compounds were identified as 17-hydroxymethyl metabolites, formed in vivo in the horse by oxidation of the 17-methyl moiety of 17 alpha-methyltestosterone. This study reports on the GC/EI + MS identification of these novel 17-hydroxymethyl C(20)O(3) and C(20)O(4) metabolites of 17 alpha-methyltestosterone excreted in thoroughbred horse urine.     

Metabolic studies with phenobarbitone, primidone and their N-alkyl derivatives: quantification of substrates and metabolites using chemical ionization gas chromatography-mass spectrometry

Metabolic studies with phenobarbitone, primidone and some of their N-alkyl derivatives required the concurrent assay of any mixture of these substrates (twelve compounds) and their major metabolites (an additional twenty-two compounds) in urine. The method described in the present report met this requirement by incorporating two complementary derivatization techniques into a gas chromatographic-mass spectrometric (GC-MS) assay procedure. Following hydrolysis of conjugates with beta-glucuronidase, urine samples were extracted with ethyl acetate (3 X 5 ml). The combined extracts were dried over sodium sulphate, divided into two equal portions, and the solvent was removed. One residue was derivatized by propylation using 1-iodopropane with base catalysis. The other residue was silylated using methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide. The derivatives in each case were analysed by GC-MS, using temperature-programmed packed-column GC and chemical ionization MS. Mass spectra were acquired over an appropriate mass range, and peak areas for the compounds of interest were determined from specific mass chromatograms. Satisfactory precision, accuracy, specificity and sensitivity were obtained for all analytes. All compounds produced satisfactory derivatives by at least one procedure; twelve compounds could be analysed by both techniques. The method illustrates the utility of chemical ionization GC-MS for the simultaneous quantitative analysis of multiple related analytes in complex biological samples.     

Combined thin-layer chromatography-photography-densitometry for the quantitation of cyclophosphamide and its four principal urinary metabolites

A novel method for the quantitative determination of the anti-cancer drug cyclophosphamide and its principal urinary metabolites 4-oxocyclophosphamide, carboxyphosphamide, phosphoramide mustard and bis(2-chloroethyl)amine has been devised. The assay combines adsorption of drug-related material onto Amberlite XAD-2 and thin-layer chromatography with spot visualization using 4-(4-nitrobenzyl)pyridine, rapid photography and densitometry. The intra-assay coefficient of variation for each compound was less than 6%. The limit of detection of the assay was 1 microgram ml-1 for cyclophosphamide, phosphoramide mustard and bis(2-chloroethyl)amine and 0.5 microgram ml-1 for 4-oxocyclophosphamide and carboxyphosphamide. The method was validated for cyclophosphamide and 4-oxocyclophosphamide using gas chromatography. It is concluded that the method provides the first means of determining the full metabolic spectrum for cyclophosphamide in patients without recourse to the administration of radioisotopically labelled drug.     

Development and validation of a gas chromatography/mass spectrometry metabonomic platform for the global profiling of urinary metabolites

This paper presents a simple and reliable gas chromatography/mass spectrometry (GC/MS) method for the metabonomic analysis of human urine samples. The sample preparation involved the depletion of excess urea via treatment with urease and subsequent protein precipitation using ice-cold ethanol. An aliquot of the mixture was separated, dried, trimethylsilyl (TMS)-derivatized and 1.0 microL of the derivatized extract was injected into the GC/MS system via split injection (1:10). Approximately 150 putative metabolites belonging to different chemical classes were identified from the pooled human urine samples. All the identified metabolites were selected to evaluate precision and stability of the GC/MS assay. More than 95% of the metabolites demonstrated good reproducibility, with intra-day and inter-day precision values below 15%. Metabolic profiling of 53 healthy male and female urine samples in combination with pattern recognition techniques was performed to further validate the GC/MS metabolite profiling assay. Principal component analysis (PCA) followed by orthogonal partial least squares analysis (OPLS) revealed differences between urinary metabolite profiles of healthy male and female subjects. This validated GC/MS metabolic profiling method may be further applied to the metabonomic screening of urinary biomarkers in clinical studies. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Liquid chromatographic analysis of prednisolone, prednisone and their 20-reduced metabolites in perfusion media

A reversed-phase liquid chromatographic assay was developed to quantitate prednisolone, prednisone and the 20 alpha-dihydro and 20 beta-dihydro reduced metabolites of both parent compounds in tissue culture media from in vitro perfusions of the human placental lobule. Steroids were extracted from perfusate, using reversed-phase cartridges, with average recoveries of 95.2% or greater. The internal standard for the analyses was 6 alpha-methylprednisolone. In this assay cortisol coelutes with prednisolone, however, no other significant interferences were found. Assay of each steroid was linear in the range 0-1 microgram/ml. Intra-assay coefficients of variation were measured at 10 and 750 ng/ml with ranges of 3.4% (20 alpha-dihydroprednisone) to 8.8% (20 beta-dihydroprednisolone) and 4.1% (20 beta-dihydroprednisone) to 8.8% (prednisone). The corresponding inter-assay coefficients of variation were 3.3% (20 alpha-dihydroprednisone) to 9.1% (20 beta-dihydroprednisolone) and 1.9% (prednisolone) to 3.5% (prednisone). The analyses utilized two C18 columns which were linked together and maintained at 40 degrees C.     

Plasma level monitoring of D,L-verapamil and three of its metabolites by reversed-phase high-performance liquid chromatography.

A high-performance liquid chromatographic assay was developed for determination of verapamil, norverapamil (M1) and its N-dealkylated metabolites (M2 and M3) in plasma. Plasma samples were vortex-mixed, deproteinized and centrifuged. The analysis was performed on a C18 reversed-phase column with fluorimetric detection. Since the polarity of verapamil and norverapamil differs considerably from that of M2 and M3, two different eluents were used for rapid high-performance liquid chromatographic separation. The eluent for the separation of verapamil and norverapamil was acetonitrile-0.07% orthophosphoric acid (33:67, v/v), and for M2 and M3 acetonitrile-0.07% orthophosphoric acid (25:75, v/v). The high-performance liquid chromatographic assay allowed rapid, sensitive and reliable quantitation of verapamil and three of its metabolites in plasma without an extraction procedure. The limit of detection was less than 5 ng/ml (plasma) for all compounds. No interferences with other commonly co-administered drugs was observed. Plasma concentrations of verapamil and its metabolites were determined in 21 patients receiving a continuous infusion of verapamil for tachyarrhythmia of acute onset. The steady-state plasma concentration data of verapamil and its three main metabolites in these patients gave evidence that the plasma concentration of verapamil and its active metabolite norverapamil was primarily determined by the extent of the formation of M2.     

Detection of sibutramine administration: a gas chromatography/mass spectrometry study of the main urinary metabolites

A gas chromatographic/mass spectrometric (GC/MS) study aimed at identifying the metabolites of sibutramine (1-(4-chlorophenyl)-N,N-dimethyl-alpha-(2-methylpropyl)cyclobutanemethanamine) in urine is described. Urinary excretion of sibutramine metabolites following the oral administration of a single dose of sibutramine was followed by GC/MS analysis. After identification of the chromatographic signals corresponding to the six main urinary metabolites, the fragmentation pattern was studied in electron ionization (EI) mode after derivatization to the corresponding methyl and trimethylsilyl derivatives. Urine samples were pretreated according to a reference procedure (liquid/liquid separation, enzymatic hydrolysis, pre-concentration under a stream of nitrogen and derivatization, either under thermal incubation and by microwave irradiation). All sibutramine metabolites were excreted as glucuroconjugates, and retain the chiral carbon present in the sibutramine skeleton. The metabolites identified included mono-desmethylsibutramine (nor-sibutramine), bi-desmethylsibutramine (nor-nor-sibutramine), and the corresponding hydroxylated compounds, the hydroxylation taking place either on the cyclobutane or on the isopropyl chain. The excretion profiles of the different metabolites were also evaluated. From an analytical point of view, the method can be applied to different fields of forensic analytical toxicology, including anti-doping analysis. Although the lack of certified reference materials does not allow a precise determination of the limits of detection (LODs) of all the sibutramine metabolites, an estimation taking into account the response factor of similar compounds ensures that all metabolites are still clearly detectable in a range of concentrations between 10 and 50 ng/mL, thus satisfying the minimum required performance limits (MRPLs) of the World Anti-Doping Agency (WADA).     

Cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography for analysis of morphine and its four metabolites in human urine

A cation-selective exhaustive injection and sweeping micellar EKC (CSEI-Sweep-MEKC) was established to analyze morphine and its four metabolites, including codeine, normorphine (NM), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G). After SPE, the urine samples were analyzed by this CE method. The phosphate buffer (75 mM, pH 2.5) containing 30% methanol was first filled into an uncoated fused-silica capillary (40 cm, 50 microm id), then a high-conductivity buffer (120 mM phosphate, 10.3 kPa for 99.9 s) followed. The pretreated urine sample was loaded by electrokinetic injection (10 kV, 600 s). The stacking and separation were performed by using phosphate buffer (25 mM, pH 2.5) containing 22% methanol and 100 mM SDS at -20 kV, and detected at 200 nm. During method validation, calibration plots were linear (r > or = 0.998) over a range of 30-3000 ng/mL for morphine, NM, and codeine, 100-2000 ng/mL for M6G, and 80-3200 ng/mL for M3G. The LODs (S/N = 5, sampling 600 s at 10 kV) were 10 ng/mL for morphine, NM, and codeine, 35 ng/mL for M6G, and 25 ng/mL for M3G. This stacking CE method could increase 2500-fold sensitivity of codeine, when comparing with CZE. Five addicts' urine specimens were analyzed. Their results were compared with those of LC-MS-MS, and showed good coincidence. This method could be feasible for monitoring morphine and its metabolites in forensic interest and pharmacokinetic investigations.     

In vitro and in vivo studies of androst‐4‐ene‐3,6,17‐trione in horses by gas chromatography–mass spectrometry

This paper describes the application of gas chromatography–mass spectrometry (GC‐MS) for in vitro and in vivo studies of 6‐OXO in horses, with a special aim to identify the most appropriate target metabolite to be monitored for controlling the administration of 6‐OXO in racehorses. In vitro studies of 6‐OXO were performed using horse liver microsomes. The major biotransformation observed was reduction of one keto group at the C3 or C6 positions. Three in vitro metabolites, namely 6α‐hydroxyandrost‐4‐ene‐3,17‐dione (M1), 3α‐hydroxyandrost‐4‐ene‐6,17‐dione (M2a) and 3β‐hydroxyandrost‐4‐ene‐6,17‐dione (M2b) were identified. For the in vivo studies, two thoroughbred geldings were each administered orally with 500 mg of androst‐4‐ene‐3,6,17‐trione (5 capsules of 6‐OXO_®) by stomach tubing. The results revealed that 6‐OXO was extensively metabolized. The three in vitro metabolites (M1, M2a and M2b) identified earlier were all detected in post‐administration urine samples. In addition, seven other urinary metabolites, derived from a further reduction of either one of the remaining keto groups or one of the remaining keto groups and the olefin group, were identified. These metabolites included 6α,17β‐dihydroxyandrost‐4‐en‐3‐one (M3a), 6,17‐dihydroxyandrost‐4‐en‐3‐one (M3b and M3c), 3β,6β‐dihydroxyandrost‐4‐en‐17‐one (M4a), 3,6‐dihydroxyandrost‐4‐en‐17‐one (M4b), 3,6‐dihydroxyandrostan‐17‐one (M5) and 3,17‐dihydroxyandrostan‐6‐one (M6). The longest detection time observed in urine was up to 46 h for the M6 metabolite. For blood samples, the peak 6‐OXO plasma concentration was observed 1 h post administration. Plasma 6‐OXO decreased rapidly and was not detectable 12 h post administration. Copyright © 2009 John Wiley & Sons, Ltd.     

Metabolic studies of turinabol in horses

Turinabol (4-chloro-17alpha-methyl-17beta-hydroxy-1,4-androstadien-3-one) is a synthetic oral anabolic androgenic steroid. As in the case of other anabolic steroids, it is a prohibited substance in equine sports. The metabolism of turinabol in human has been reported previously; however, little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of turinabol in racehorses with an objective to identify the most appropriate target metabolites for detecting turinabol administration. For the in vitro studies, turinabol was incubated with fresh horse liver microsomes. Metabolites in the incubation mixture were isolated by liquid-liquid extraction and analysed by gas chromatography-mass spectrometry (GC-MS) after trimethylsilylation. The results showed that the major biotransformation of turinabol was hydroxylation at the C6, C16 and C20 sites to give metabolites 6beta-hydroxyturinabol (M1), 20-hydroxyturinabol (M2), two stereoisomers of 6beta,16-dihydroxyturinabol (M3a, M3b) and 6beta,20-dihydroxyturinabol (M4). The metabolite 6beta-hydroxyturinabol was confirmed using an authentic reference standard. The structures of all other turinabol metabolites were tentatively identified by mass spectral interpretation. For the in vivo studies, two horses were administered orally with turinabol. Pre- and post-administration urine samples were collected for analysis. Free and conjugated metabolites were isolated using solid-phase extraction and analysed by GC-MS as described for the in vitro studies. The results revealed that turinabol was extensively metabolised and the parent drug was not detected in urine. Two metabolites detected in the in vitro studies, namely 20-hydroxyturinabol and 6beta,20-dihydroxyturinabol, these were also detected in post-administration urine samples. In addition, 17-epi-turinabol (M5) and six other metabolites (M6a-M6c and M7a-M7c), derived from D-ring hydroxylation and A-ring reduction, were also detected. Except for 17-epi-turinabol, none of these metabolites has ever been reported in any species. All in vivo metabolites were detected within 48 h after administration.