New potential markers for the detection of boldenone misuse

Boldenone is one of the most frequently detected anabolic androgenic steroids in doping control analysis. Boldenone misuse is commonly detected by the identification of the active drug and its main metabolite, 5β-androst-1-en-17β-ol-3-one (BM1), by gas chromatography-mass spectrometry (GC-MS), after previous hydrolysis with β-glucuronidase enzymes, extraction and derivatization steps. However, some cases of endogenous boldenone and BM1 have been reported. Nowadays, when these compounds are detected in urine at low concentrations, isotope ratio mass spectrometry (IRMS) analysis is needed to confirm their exogenous origin. The aim of the present study was to identify boldenone metabolites conjugated with sulphate and to evaluate their potential to improve the detection of boldenone misuse in sports. Boldenone was administered to a healthy volunteer and urine samples were collected up to 56h after administration. After a liquid-liquid extraction with ethyl acetate, urine extracts were analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) using electrospray ionisation in negative mode by monitoring the transition of m/z 365-350, specific for boldenone sulphate. Boldenone sulphate was identified in the excretion study urine samples and, moreover, another peak with the same transition was observed. Based on the MS/MS behaviour the metabolite was identified as epiboldenone sulphate. The identity was confirmed by isolation of the LC peak, solvolysis and comparison of the retention time and MS/MS spectra with an epiboldenone standard. These sulphated metabolites have not been previously reported in humans and although they account for less than 1% of the administered dose, they were still present in urine when the concentrations of the major metabolites, boldenone and BM1, were at the level of endogenous origin. The sulphated metabolites were also detected in 10 urine samples tested positive to boldenone and BM1 by GC-MS. In order to verify the usefulness of these new metabolites to discriminate between endogenous and exogenous origin of boldenone, four samples containing endogenous boldenone and BM1, confirmed by IRMS, were analysed. In 3 of the 4 samples, neither boldenone sulphate nor epiboldenone sulphate were detected, confirming that these metabolites were mainly detected after exogenous administration of boldenone. In contrast, boldenone sulphate and, in some cases, epiboldenone sulphate were present in samples with low concentrations of exogenous boldenone and BM1. Thus, boldenone and epiboldenone sulphates are additional markers for the exogenous origin of boldenone and they can be used to reduce the number of samples to be analysed by IRMS. In samples with boldenone and BM1 at the concentrations suspicion for endogenous origin, only if boldenone and epiboldenone sulphates are present, further analysis by IRMS will be needed to confirm exogenous origin.     

Detection of altrenogest and its metabolites in post administration horse urine using liquid chromatography tandem mass spectrometry--increased sensitivity by chemical derivatisation of the glucuronic acid conjugate

Altrenogest (17alpha-allyl-17beta-hydroxyestra-4,9,11-trien-3-one) is a steroid used for the control of estrus in horses. This drug can potentially be abused in racehorses as the occurrence of estrus can alter their performance. This work describes an analytical method based on liquid chromatography-tandem mass spectrometry for the detection of altrenogest in horse urine down to a concentration of 13 pg/mL (0.042 nM). Furthermore, the qualitative aspect of metabolism of altrenogest in the horse has been studied. The main transformations that were found for this species were conjugation with glucuronic acid and sulfate. These phase II metabolites were identified by molecular mass and by comparison of their collision-induced dissociation product ion spectra with that of the synthetic aglycone at positive and negative potential, respectively. No phase I metabolites were discovered. In order to increase the ionisation in positive electrospray, a derivatisation procedure forming a basic oxime was tested. This process significantly increased the detection sensitivity for altrenogest glucuronide in horse urine.     

Criteria to distinguish between natural situations and illegal use of boldenone, boldenone esters and boldione in cattle 1. Metabolite profiles of boldenone, boldenone esters and boldione in cattle urine

Boldenone is an androgenic steroid that improves the growth and food conversion in food producing animals. In most countries worldwide, this anabolic steroid is forbidden for meat production. Until recently, the control of its illegal use was based either on 17beta-boldenone or 17alpha-boldenone (its main metabolite in cattle) identification in edible tissues, hair, faeces or urine. Recent observations and data tend to demonstrate the natural occurrence (but not ubiquitous) in cattle of these steroids, making the analytical strategy of the control more complicated. We investigated the metabolism of boldenone in cattle after intramuscular and oral treatment of boldenone, boldenone esters and boldione. The central objective was to elucidate the structures of the main metabolites (phase I and phase II) in urine, with main objective to be further in position to compare boldenone urinary profiles of treated and non-treated animals. Nine metabolites have been identified, only four were present whatever the treatment and the administered boldenone source. Nevertheless, all of them have been detected at least once in non-treated animals which did not permit us to use them as biomarkers of an illegal treatment. At last, but not at least, all metabolites were found mainly glucuro-conjugated, and rarely sulfo-conjugated, with the only exception of 17beta-boldenone. Current investigations are showing the absence of 17beta-boldenone sulfoconjugate in non-treated animals; that would permit to distinguish non-treated from treated animals with boldione, boldenone and boldenone esters.     

Detection of endogenous boldenone in the entire male horses

Boldenone (1,2-dehydrotestosterone) is a common veterinary anabolic agent. Its structure is very similar to testosterone. Testosterone is endogenous in the horse, whereas there has been no report concerning the detection of endogenous boldenone. This paper reports the direct observation of sulphate conjugate of boldenone in equine urine from entires. The detection procedures involved solid-phase extraction, immunoaffinity column (IAC) purification, and then LC-MS-MS analysis on a Q-ToF instrument. The identification of boldenone sulphate has provided direct evidence for the endogenous nature of boldenone in entire male horses. Quantification data for the normal level of boldenone in Hong Kong racehorses will also be discussed.     

Criteria to distinguish between natural situations and illegal use of boldenone, boldenone esters and boldione in cattle: 2. Direct measurement of 17β-boldenone sulpho-conjugate in calf urine by liquid chromatography–high resolution and tandem mass spectrometry

Boldenone is banned in the European Union (Directive 96/22/EC) as growth promoter for meat producing animals. Boldione (ADD), boldenone and boldenone esters (mainly the undecylenate form) are commercially available as anabolic preparations, either to the destination of human, horse or cattle. Since the late 90s, the natural occurrence of boldenone metabolites has been reported in cattle. According to EU regulation, the unambiguous demonstration of boldenone administration in bovine urine should be provided on the basis of boldenone identification in the corresponding conjugate fraction. An analytical method has been developed and validated according to current standards with main concern to the measurement of intact 17β-boldenone-sulphate. The analytical procedure included direct extraction–purification of target analyte on octadecylsilyl cartridges and direct detection of phase II metabolite by liquid chromatography (negative electrospray), tandem mass spectrometry (QqQ) or high resolution mass spectrometry (Orbitrap™). Decision limit (CCα) and detection capability (CCβ) were respectively 0.2 μg L⁻¹ and 0.4 μg L⁻¹ on triple quadrupole and 0.1 μg L⁻¹ and 0.2 μg L⁻¹ on hybrid system. The method was successfully applied to the analysis of incurred samples collected in different experiments. 17β-Boldenone-sulphate was measurable up to 36 h after oral administration of boldione, and 30 days after 17β-boldenone undecylenate intra-muscular injection. This conjugate form was never detected in non-treated animals, confirming its status of definitive candidate marker for boldenone administration in calf.     

Criteria to distinguish between natural situations and illegal use of boldenone,boldenone esters and boldione in cattle: 2. Direct measurement of 17β-boldenone sulpho-conjugate in calf urine by liquid chromatography–high resolution and tandem mass spectrometry

Boldenone is banned in the European Union (Directive 96/22/EC) as growth promoter for meat producing animals. Boldione (ADD), boldenone and boldenone esters (mainly the undecylenate form) are commercially available as anabolic preparations, either to the destination of human, horse or cattle. Since the late 90s, the natural occurrence of boldenone metabolites has been reported in cattle. According to EU regulation, the unambiguous demonstration of boldenone administration in bovine urine should be provided on the basis of boldenone identification in the corresponding conjugate fraction. An analytical method has been developed and validated according to current standards with main concern to the measurement of intact 17β-boldenone-sulphate. The analytical procedure included direct extraction–purification of target analyte on octadecylsilyl cartridges and direct detection of phase II metabolite by liquid chromatography (negative electrospray), tandem mass spectrometry (QqQ) or high resolution mass spectrometry (Orbitrap?). Decision limit (CCα) and detection capability (CCβ) were respectively 0.2 μg L^?1 and 0.4 μg L^?1 on triple quadrupole and 0.1 μg L^?1 and 0.2 μg L^?1 on hybrid system. The method was successfully applied to the analysis of incurred samples collected in different experiments. 17β-Boldenone-sulphate was measurable up to 36 h after oral administration of boldione, and 30 days after 17β-boldenone undecylenate intra-muscular injection. This conjugate form was never detected in non-treated animals, confirming its status of definitive candidate marker for boldenone administration in calf.     

LC-MS/MS characterization of the urinary excretion profile of the mycotoxin deoxynivalenol in human and rat

The understanding of mycotoxins transfer to biological fluids is challenged by the difficulties in performing and replicating in vivo experiments as well as the lack of suitable methods of analysis to detect simultaneously a range of chemically different metabolites at trace levels. LC-MS/MS has been used herein to study the urinary excretion profile of the mycotoxin deoxynivalenol in human and Wistar rat. Deoxynivalenol and deoxynivalenol glucuronide were found in both human and rat urines, whereas de-epoxydeoxynivalenol and its glucuronide conjugate were only detected in rat urine. The presence of two deoxynivalenol glucuronide isomers in Wistar rat urine has been shown for the first time. Structure confirmation of the detected metabolites was provided by the analysis of fragmentation patterns. A solid phase extraction clean up procedure allowing recoveries in the range 72-102% for deoxynivalenol, de-epoxydeoxynivalenol, and their glucuronide conjugates was optimized. A multiple reaction monitoring method for the simultaneous determination of all investigated metabolites was elaborated allowing the direct detection of deoxynivalenol metabolites without the hydrolysis step. Deoxynivalenol urinary levels in the range 0.003-0.008 μg/ml were detected in healthy human subjects, whereas deoxynivalenol and de-epoxynivalenol levels between 1.9-4.9 μg/ml and 1.6-5.9 μg/ml, respectively were found in administered rat urine. These findings emphasize the relevance of the highly selective and sensitive LC-MS/MS technique for the direct detection and characterization of deoxynivalenol metabolites in complex biological matrices.     

Liquid chromatography-tandem mass spectrometry analysis of anisodamine and its phase I and II metabolites in rat urine

A sensitive and specific method for the analysis of anisodamine and its metabolites in rat urine by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) was developed. Various extraction techniques (free fraction, acid hydrolyses and enzyme hydrolyses) and their comparison were carried out for investigation of the metabolism of anisodamine. After extraction procedure the pretreated samples were injected on a reversed-phase C18 column with mobile phase (0.2 ml/min) of methanol/0.01% triethylamine solution (adjusted to pH 3.5 with formic acid) (60:40, v/v) and detected by MS/MS. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular masses (DeltaM), retention-times and full scan MS(n) spectra with those of the parent drug. At least 11 metabolites (N-demethyl-6beta-hydroxytropine, 6beta-hydroxytropine, tropic acid, N-demethylanisodamine, hydroxyanisodamine, anisodamine N-oxide, hydroxyanisodamine N-oxide, glucuronide conjugated N-demethylanisodamine, sulfate conjugated and glucuronide conjugated anisodamine, sulfate conjugated hydroxyanisodamine) and the parent drug were found in rat urine after the administration of a single oral dose 25mg/kg of anisodamine. Hydroxyanisodamine, anisodamine N-oxide and the parent drug were detected in rat urine for up 95 h after ingestion of anisodamine.     

Study of natural and artificial corticosteroid phase II metabolites in bovine urine using HPLC-MS/MS

Corticosteroid compounds are widely used therapeutically for their anti-inflammatory properties and sometimes as growth promoters in food producing animals. In the field of drug residue analysis, knowledge of the main metabolic pathways of target analytes improves the efficiency of the corresponding control. Thus, phase II metabolism of corticosteroids, for which very little literature is available, was investigated in cattle. An LC-MS/MS detection method was developed for five commercially available conjugated corticosteroids, permitting direct monitoring during the development of their separation on anion exchange SPE. This separation method is further applicable to other potential urinary conjugated corticosteroids. Because our purpose was not to identify all the existing corticosteroid phase II metabolites, but to obtain their total relative proportions, enzymatic hydrolysis was optimized and performed on each separated fraction (glucuronides and sulfates). Finally, the phase II metabolic profiles of natural and artificial corticosteroids in bovine urine were studied and compared. LC-MS/MS detection with negative electrospray ionization appeared efficient for both glucuronide and sulfate conjugated corticosteroids, and quaternary ammonium stationary phase permitted their effective separation. The experimental design used for optimization of the enzymatic hydrolysis with a purified Helix pomatia preparation demonstrated optimal values for pH 5.2, temperature of 50 degrees C and incubation duration of 4h. Results on bovine urine samples collected on two animals before and after dexamethasone administration showed important differences regarding the proportion of total conjugated forms between endogenous cortisol, endogenous tetrahydrocortisol, and exogenous dexamethasone. This proportion appeared significantly higher for tetrahydrocortisol (40-65%) than cortisol (2-8%) or dexamethasone (4-27%). This innovative methodology demonstrates the suitability of anion exchange SPE and LC-MS/MS for the study of steroid hormones phase II metabolism, and appears promising to investigate metabolic profile differences linked to the hormone administration mode or origin, with direct application in the field of doping controls.     

Determination of a prostaglandin D2 antagonist and its acyl glucuronide metabolite in human plasma by high performance liquid chromatography with tandem mass spectrometric detection--a lack of MS/MS selectivity between a glucuronide conjugate and a phase I metabolite

A method for the determination of a prostaglandin D(2) receptor antagonist (I, a compound being evaluated for the prevention of niacin induced flushing) and its acyl glucuronide metabolite (II) in human plasma is presented. The method utilized high performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection using an atmospheric pressure chemical ionization (APCI) interface operated in the positive ionization mode. The product ion was a radical cation generated via a homolytic bond cleavage. A chemical analog of the drug was used as internal standard (III). The acyl glucuronide metabolite (II) was detected using the same precursor-to-product ion transition used for the parent compound after chromatographic separation of I and II. Drug and metabolite were extracted using semi-automated, 96-well format solid phase extraction (SPE), and chromatography was performed using a reverse phase analytical column with an isocratic mobile phase. The chromatographic retention factor (k') of II was found to be highly sensitive to mobile phase formic acid concentration. An adjustment in mobile phase formic acid concentration improved the chromatographic separation between II and a mono-hydroxylated metabolite after an unexpected lack of MS/MS selectivity between the two molecules was observed. The dependence of retention factor on formic acid concentration (k' increased as formic acid concentration decreased) was thought to indicate polar interactions between II and the stationary phase. The stability of II in spiked human plasma was determined. The rate of hydrolysis back to parent compound was relatively low (approximately 0.1 and 0.5% per hour at room temperature and 4 degrees C, respectively) indicating that significant changes in analyte concentrations did not occur during sample processing. The concentration range of the assay was 10-2500 ng/mL for both drug and glucuronide metabolite.     

Determination of Serotonin and Dopamine Metabolites in Human Brain Microdialysis and Cerebrospinal Fluid Samples by UPLC-MS/MS: Discovery of Intact Glucuronide and Sulfate Conjugates

An UPLC-MS/MS method was developed for the determination of serotonin (5-HT), dopamine (DA), their phase I metabolites 5-HIAA, DOPAC and HVA, and their sulfate and glucuronide conjugates in human brain microdialysis samples obtained from two patients with acute brain injuries, ventricular cerebrospinal fluid (CSF) samples obtained from four patients with obstructive hydrocephalus, and a lumbar CSF sample pooled mainly from patients undergoing spinal anesthesia in preparation for orthopedic surgery. The method was validated by determining the limits of detection and quantification, linearity, repeatability and specificity. The direct method enabled the analysis of the intact phase II metabolites of 5-HT and DA, without hydrolysis of the conjugates. The method also enabled the analysis of the regioisomers of the conjugates, and several intact glucuronide and sulfate conjugates were identified and quantified for the first time in the human brain microdialysis and CSF samples. We were able to show the presence of 5-HIAA sulfate, and that dopamine-3-O-sulfate predominates over dopamine-4-O-sulfate in the human brain. The quantitative results suggest that sulfonation is a more important phase II metabolism pathway than glucuronidation in the human brain.     

Plasma and urinary markers of oral testosterone undecanoate misuse.

Orally administered testosterone undecanoate (TU), an anabolic, androgenic steroid, can potentially be abused by athletes. Indirect evidence for detecting oral TU intake could be deduced from the changes in steroid profile post-administration. Direct evidence could be obtained by detection of unchanged TU in plasma. To this end, both urinary and plasma steroid profiles of six healthy male subjects given a single oral dose of 120 mg of TU were studied by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/tandem mass spectrometry (GC/MS/MS). The increased concentration of glucuronidated testosterone in plasma appears to be the most characteristic sign of oral TU intake. The testosterone glucuronide (TG)/nonconjugated testosterone (T) ratio, TG/17-hydroxyprogesterone (17OHP) ratio, and TG/luteinizing hormone (LH) ratio were observed to be significantly elevated above their basal levels for 10 h, 10 h, and 6 h, respectively. Urinary ratios of TG/epitestosterone glucuronide (EG) were found to be higher than the cut-off value of 6 for the period 4 approximately 8 h post-administration, but only in three subjects. One subject failed to respond with respect to all of the above-mentioned indirect markers, as TG was not significantly increased in either plasma or urine. Unchanged TU was directly detected in plasma of all six subjects from 1 approximately 1.5 h to 4 approximately 6 h after oral TU intake by GC/MS/MS, providing unequivocal proof of exogenous testosterone intake. Distinct and complementary markers for detecting oral TU intake could be obtained from plasma and urine, respectively.     

A new sulphate metabolite as a long-term marker of metandienone misuse

Metandienone is one of the most frequently detected anabolic androgenic steroids in sports drug testing. Metandienone misuse is commonly detected by monitoring different metabolites excreted free or conjugated with glucuronic acid using gas chromatography mass spectrometry (GC–MS) and liquid chromatography tandem mass spectrometry (LC–MS/MS) after hydrolysis with β-glucuronidase and liquid–liquid extraction. It is known that several metabolites are the result of the formation of sulphate conjugates in C17, which are converted to their 17-epimers in urine. Therefore, sulphation is an important phase II metabolic pathway of metandienone that has not been comprehensively studied. The aim of this work was to evaluate the sulphate fraction of metandienone metabolism by LC–MS/MS. Seven sulphate metabolites were detected after the analysis of excretion study samples by applying different neutral loss scan, precursor ion scan and SRM methods. One of the metabolites (M1) was identified and characterised by GC–MS/MS and LC–MS/MS as 18-nor-17β-hydroxymethyl-17α-methylandrost-1,4,13-triene-3-one sulphate. M1 could be detected up to 26 days after the administration of a single dose of metandienone (5 mg), thus improving the period in which the misuse can be reported with respect to the last long-term metandienone metabolite described (18-nor-17β-hydroxymethyl-17α-methylandrost-1,4,13-triene-3-one excreted in the glucuronide fraction).     

Studies on anabolic steroids--12. Epimerization and degradation of anabolic 17 beta-sulfate-17 alpha-methyl steroids in human: qualitative and quantitative GC/MS analysis.

The epimerization and dehydration reactions of the 17 beta-hydroxy group of anabolic 17 beta-hydroxy-17 alpha-methyl steroids have been investigated using the pyridinium salts of 17 beta-sulfate derivatives of methandienone 1, methyltestosterone 4, oxandrolone 7, mestanolone 10 and stanozolol 11 as model compounds. Rearrangement of the sulfate conjugates in buffered urine (pH 5.2) afforded the corresponding 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes in a ratio of 0.8:1. These data indicated that both epimerization and dehydration of the 17 beta-sulfate derivatives were not dependent upon the respective chemical features of the steroids studied, but were instead inherent to the chemistry of the tertiary 17 beta-hydroxy group of these steroids. Interestingly, in vivo studies carried out with human male volunteers showed that only methandienone 1, methyltestosterone 4 and oxandrolone 7 yielded the corresponding 17-epimers 2, 5 and 8 and the 18-nor-17,17-dimethyl-13(14)-enes 3, 6 and 9 in ratios of 0.5:1, 2:1 and 2.7:1, respectively. No trace of the corresponding 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes derivatives of mestanolone 10 and stanozolol 11 was detected in urine samples collected after administration of these steroids. These data suggested that the in vivo formation of the 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes derivatives of 17 beta-hydroxy-17 alpha-methyl steroids is also dependent upon phase I and phase II metabolic reactions other than sulfation of the tertiary 17 beta-hydroxyl group, which are probably modulated by the respective chemical features of the steroidal substrates. The data reported in this study demonstrate that the 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes are not artifacts resulting from the acidic or microbial degradation of the parent steroids in the gut as previously suggested by other authors, but arise from the rearrangement of their 17 beta-sulfate derivatives. Unchanged oxandrolone 7 was solely detected in the unconjugated steroid fraction whereas unchanged steroids 1, 4 and 11 were recovered from the glucuronide fraction. These data are indirect evidences suggesting that the glucuronide conjugates of compounds 1 and 4 are probably enol glucuronides and that of compound 11 is excreted in urine as a N-glucuronide involving its pyrazole moiety. The urinary excretion profiles of the epimeric and 18-nor-17,17-dimethyl-13(14)-ene steroids are presented and discussed on the basis of their structural features.     

Integrated analytical approach in veal calves administered the anabolic androgenic steroids boldenone and boldione: urine and plasma kinetic profile and changes in plasma protein expression

Surveillance of illegal use of steroids hormones in cattle breeding is a key issue to preserve human health. To this purpose, an integrated approach has been developed for the analysis of plasma and urine from calves treated orally with a single dose of a combination of the androgenic steroids boldenone and boldione. A quantitative estimation of steroid hormones was obtained by LC-APCI-Q-MS/MS analysis of plasma and urine samples obtained at various times up to 36 and 24 h after treatment, respectively. These experiments demonstrated that boldione was never found, while boldenone alpha- and beta-epimers were detected in plasma and urine only within 2 and 24 h after drug administration, respectively. Parallel proteomic analysis of plasma samples was obtained by combined 2-DE, MALDI-TOF-MS and muLC-ESI-IT-MS/MS procedures. A specific protein, poorly represented in normal plasma samples collected before treatment, was found upregulated even 36 h after hormone treatment. Extensive mass mapping experiments proved this component as an N-terminal truncated form of apolipoprotein A1 (ApoA1), a protein involved in cholesterol transport. The expression profile of ApoA1 analysed by Western blot analysis confirmed a significant and time dependent increase of this ApoA1 fragment. Then, provided that further experiments performed with a growth-promoting schedule will confirm these preliminary findings, truncated ApoA1 may be proposed as a candidate biomarker for steroid boldenone and possibly other anabolic androgens misuse in cattle veal calves, when no traces of hormones are detectable in plasma or urine.     

Analytical strategies for the direct mass spectrometric analysis of steroid and corticosteroid phase II metabolites

The use of steroid hormones as growth promoters remains illegal in Europe. A classical approach used to control their utilization consists to measure the parent drug in target biological matrices. However, this strategy may fail when the parent drug is submitted to extensive metabolism reactions. For urine and tissue samples, chemical or enzymatic hydrolysis is usually applied in order to deconjugate glucuronide and sulfate phase II metabolites. But this treatment lead to the loss of information such as nature and relative proportions of the different conjugated forms, which can be useful, for example, to discriminate an endogenous production from an exogenous administration for natural hormones, or for other clinical or biochemical specific applications. For these purposes, direct measurement of conjugated metabolites using liquid chromatography-tandem mass spectrometry may represent a solution of choice. In this context, the mass spectrometric behavior of 14 steroid and corticosteroid phase II metabolites after electrospray ionization was investigated. Their fragmentation pathways in tandem mass spectrometry revealed some specificities within the different group of conjugates. A specific acquisition program (MRM mode) was developed for the unambiguous identification of the studied reference compounds. A more generic method (Parent Scan mode) was also developed for fishing approaches consisting to monitor several fragment ions typical of each conjugate class. A reverse phase HPLC procedure was also proposed for efficient retention and separation of the studied compounds. Finally, a protocol based on quaternary amine SPE was developed, permitting the separation of free, glucuronide, and sulfate fractions. Preliminary results on biological samples demonstrated the suitability of this analytical strategy for direct measurement of dexamethasone glucuronide and sulfate residues in bovine urine.     

Development of an HPLC-MS/MS method for the selective determination of paracetamol metabolites in mouse urine

An HPLC-MS/MS method has been developed for the selective quantitative analysis of paracetamol and its two major metabolites. The use of tandem MS enabled the detection and quantitation of metabolites in small sample sizes with high sensitivity and selectivity. Isocratic elution using acetonitrile and water containing formic acid combined with electrospray-tandem MS enabled the separation and accurate quantitation of each analyte and the internal standard 3-acetamidophenol. The on-column limits of detection for paracetamol, paracetamol sulfate, and paracetamol glucuronide were 2.4, 1.2, and 1.2 pmol, respectively. The method was applied to quantitate paracetamol and its metabolites in mouse urine. It is highly specific, sensitive, and easily adaptable to measure these analytes in biological fluids of other animals.     

Direct determination of anabolic steroid conjugates in human urine by combined high-performance liquid chromatography and tandem mass spectrometry

A novel screening procedure for the sulfate and glucuronide conjugates of testosterone (T) and epitestosterone (E) in human urine was developed based on liquid-solid extraction and microbore high-performance liquid chromatography combined on-line with ion-spray tandem mass spectrometry. Confirmation of the sulfate and glucuronide conjugates of testosterone and epitestosterone isolated frrm normal human urine was acheived by selected reaction monitoring of characteristic product ions of the parent compounds. Endogenous levels of the steroid conjugates are detected in normal male urine and an increase is observed when the sample is fortified with authentic analytical standards of the conjugates. Calibration curves of all steroid conjugates in urine are linear over a range of twenty. Deuterated internal standards of testosterone glucuronide and epitestosterone sulfate were used for quantitation of the endogenous conjugates. T/E ratios were determined based on the glucuronide fractions of six replicates from a normal male and were shown to be statistically reproducible and below the accepted T/E threshold of 6:1. Sulfate conjugates were shown to be present at significantly lower levels in the urine. The method has potential as an alternative for monitoring anabolic steroid conjugates in human urin.     

Multi residue screening of intact testosterone esters and boldenone undecylenate in bovine hair using liquid chromatography electrospray tandem mass spectrometry

The abuse of esters of natural androgenic steroids in cattle fattening and sports is hard to control via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. In veterinary control strange findings of 17beta-testosterone and 17alpha-testosterone in urine are often ignored because of the lack of statistically sound reference data of naturally occurring levels. An interesting alternative for inconclusive urine analyses in veterinary control can be provided by the analysis of the administered steroids themselves, i.e. the analysis of intact steroid esters in hair. Unfortunately, the analysis of intact steroid esters is complicated not only by the vulnerability of the esters which precludes alkaline hydrolysis of the hair, but also by the wide polarity range of short and long-chain esters yielding very poor recoveries for either the one or the other. In this study, a multi-steroid esters LC/MS/MS screening method is presented for trace analysis of the synthetic intact esters of 17beta-testosterone and the undecylenate ester of 17beta-boldenone in bovine hair. The method, requiring only 200 mg of pulverised hair, features a mild digestion procedure using tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and the use of four deuterium-labelled steroid esters as internal standards covering the wide polarity range of the analytes. In spiked hair samples for most of the analytes the limit of detection and the accuracy using isotope dilution were 2-5 ng/g and 97-105%, respectively. The applicability was demonstrated using hair samples from a controlled experiment in which six bovines were injected intramuscularly with two different doses of two commercial mixtures of testosterone esters, and with two different doses of boldenone undecylenate. Depending on the dose all administered testosterone- and boldenone esters were found to be incorporated in bovine hair following a single intramuscular injection, except testosterone propionate which dose might have been too low.     

Liquid chromatography-tandem mass spectrometry analysis of nitazoxanide and its major metabolites in goat

A rapid, sensitive and specific liquid chromatography-electrospray ionization (ESI) tandem mass spectrometry (LC-MS-MS) method has been developed for the identification of nitazoxanide metabolites in goat plasma and urine. The purified samples was separated using an XTerra MS C8 column with the mobile phase consisted of acetonitrile and 10-mM ammonium acetate buffer (pH 2.5) followed a linear gradient elution, and detected by MS-MS. Identification and structural elucidation of the metabolites were performed by comparing their retention-times, full scan, product ion scan, precursor ion scan and neutral loss scan MS-MS spectra with those of the parent drug or other available standard. Four metabolites (tizoxanide, tizoxanide glucuronide, tizoxanide sulfate and hydroxylated tizoxanide sulfate) were found and identified in goat after single oral administration of 200mg/kg dose of nitazoxanide. In addition, the possible metabolic pathway was proposed for the first time. The results proved that the established method was simple, reliable and sensitive, revealing that it could be used to rapid screen and identify the structures of active metabolites responsible for pharmacological effects of nitazoxanide and to better understand its in vivo metabolism.