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

A method for the screening and confirmatory analysis of beta-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.     

Detection and elimination profile of cathinone in equine after norephedrine (Propalin®) administration using a validated liquid chromatography–tandem mass spectrometry method

Cathinone is the principal psychostimulant present in the leaves of khat shrub, which are widely used in East Africa and the Arab peninsula as an amphetamine-like stimulant. Cathinone readily undergoes metabolism in vivo to form less potent cathine and norephedrine as the metabolites. However, the presence of cathine and norephedrine in biological fluids cannot be used as an indicator of cathinone administration. The metabolism of pseudoephedrine and ephedrine, commonly used in cold and allergy medications, also produces cathine and norephedrine, respectively, as the metabolites. Besides, cathine and norephedrine may also originate from the ingestion of nutritional supplemental products containing extracts of Ephedra species. In Canada, ephedrine and norephedrine are available for veterinary use, whereas cathinone is not approved for human or veterinary use. In this article, the detection of cathinone in equine after administration of norephedrine is reported. To the best of our knowledge, this is the first such report in any species where administration of norephedrine or ephedrine generates cathinone as the metabolite. This observation is quite significant, because in equine detection of cathinone in biological fluids could be due to administration of the potent stimulant cathinone or the nonpotent stimulant norephedrine. A single oral dose of 450 mg norephedrine was administered to four Standardbred mares. Plasma and urine samples were collected up to 120 h after administration. The amount of cathinone and norephedrine detected in post administration samples was quantified using a highly sensitive, specific, and validated liquid chromatography–tandem mass spectrometry method. Using these results, we constructed elimination profiles for cathinone and norephedrine in equine plasma and urine. A mechanism that generates a geminal diol as an intermediate is postulated for this in vivo conversion of norephedrine to cathinone. Cathinone was also detected in samples collected after a single intramuscular administration of 200 mg ephedrine and oral administration of 300 mg ephedrine in equine.

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Figure Electron density structure of cathinone

     

Doping control analysis of insulin and its analogues in equine urine by liquid chromatography-tandem mass spectrometry

Insulin and its analogues have been banned in both human and equine sports owing to their potential for misuse. Insulin administration can increase muscle glycogen by utilising hyperinsulinaemic clamps prior to sports events or during the recovery phases, and increase muscle size by its chalonic action to inhibit protein breakdown. In order to control insulin abuse in equine sports, a method to effectively detect the use of insulins in horses is required. Besides the readily available human insulin and its synthetic analogues, structurally similar insulins from other species can also be used as doping agents. The author's laboratory has previously reported a method for the detection of bovine, porcine and human insulins, as well as the synthetic analogues Humalog (Lispro) and Novolog (Aspart) in equine plasma. This study describes a complementary method for the simultaneous detection of five exogenous insulins and their possible metabolites in equine urine. Insulins and their possible metabolites were isolated from equine urine by immunoaffinity purification, and analysed by nano liquid chromatography-tandem mass spectrometry (LC/MS/MS). Insulin and its analogues were detected and confirmed by comparing their retention times and major product ions. All five insulins (human insulin, Humalog, Novolog, bovine insulin and porcine insulin), which are exogenous in horse, could be detected and confirmed at 0.05ng/mL. This method was successfully applied to confirm the presence of human insulin in urine collected from horses up to 4h after having been administered a single low dose of recombinant human insulin (Humulin R, Eli Lilly). To our knowledge, this is the first identification of exogenous insulin in post-administration horse urine samples.     

Stereospecific analysis of flurbiprofen and its major metabolites in plasma and urine by chiral-phase liquid chromatography

Summary Direct chiral-phase HPLC methods have been developed for the determination of flurbiprofen and its major metabolites, namely 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen, in biological fluids using a derivatized amylose chiral stationary phase (CSP; Chiral-pak AD). Quantification of all three analytes, both free and conjugated, in urine was carried out following liquid-liquid extraction using tandem ultraviolet (UV) and fluorescence detection. Determination of flurbiprofen and the 4′-hydroxy-metabolite in plasma utilized the same CSP but required modification in the mobile phase composition and sole use of fluorescence detection. The urine assay was linear (r>0.998) between 0.05–10 μg mL⁻¹, 0.1–20 μg mL⁻¹ and 0.01–2 μg mL⁻¹ for the enantiomers of flurbiprofen, 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen respectively. The plasma assay was linear (r>0.997) between 0.1–6 μg mL⁻¹ and 0.01–0.6 μg mL⁻¹ for the enantiomers of flurbiprofen and 4′-hydroxyflurbiprofen respectively. Both assays, typically yielded within- and between-day imprecision and accuracy values less than 10% for the enantiomers of the different analytes. Initial volunteer studies suggest that the disposition of flurbiprofen displays modest enantioselectivity in humans.     

In vitro metabolism studies of desoxy‐methyltestosterone (DMT) and its five analogues,and in vivo metabolism of desoxy‐vinyltestosterone (DVT) in horses

The positive findings of norbolethone in 2002 and tetrahydrogestrinone in 2003 in human athlete samples confirmed that designer steroids were indeed being abused in human sports. In 2005, an addition to the family of designer steroids called ‘Madol’ [also known as desoxy‐methyltestosterone ( DMT )] was seized by government officials at the US–Canadian border. Two years later, a positive finding of DMT was reported in a mixed martial arts athlete's sample. It is not uncommon that doping agents used in human sports would likewise be abused in equine sports. Designer steroids would, therefore, pose a similar threat to the horseracing and equestrian communities. This paper describes the in vitro metabolism studies of DMT and five of its structural analogues with different substituents at the 17α position (R ? H, ethyl, vinyl, ethynyl and ²H₃‐methyl). In addition, the in vivo metabolism of desoxy‐vinyltestosterone ( DVT ) in horses will be presented. The in vitro studies revealed that the metabolic pathways of DMT and its analogues occurred predominantly in the A‐ring by way of a combination of enone formation, hydroxylation and reduction. Additional biotransformation involving hydroxylation of the 17α‐alkyl group was also observed for DMT and some of its analogues. The oral administration experiment revealed that DVT was extensively metabolised and the parent drug was not detected in urine. Two in vivo metabolites, derived respectively from (1) hydroxylation of the A‐ring and (2) di‐hydroxylation together with A‐ring double‐bond reduction, could be detected in urine up to a maximum of 46 h after administration. Another in vivo metabolite, derived from hydroxylation of the A‐ring with additional double‐bond reduction and di‐hydroxylation of the 17α‐vinyl group, could be detected in urine up to a maximum of 70 h post‐administration. All in vivo metabolites were excreted mainly as glucuronides and were also detected in the in vitro studies. Copyright © 2015 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.     

Sensitive liquid chromatographic/tandem mass spectrometric method for the determination of beclomethasone dipropionate and its metabolites in equine plasma and urine

Beclomethasone dipropionate (BDP) is a potent pro-drug to beclomethasone (BOH) and is used in the treatment of chronic and acute respiratory disorders in the horse. The therapeutic dose of BDP (325 microg per horse) by inhalation results in very low plasma and urinary concentrations of BDP and its metabolites that pose a challenge to detection and confirmation by equine forensic laboratories. To solve this problem, a method involving the use of a liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) was developed for the detection, confirmation and quantification of the analytes in equine samples. Ammonium formate or acetate buffer added to LC mobile phase favored the formation of [M + H](+) ions from BDP and its metabolites, whereas formic acid led to the formation of sodium and potassium adduct ions ([M + Na](+), [M + K](+)) together with [M + H](+) ions. Acetonitrile, on the other hand, favored the formation of abundant solvent adduct ions [M + H + CH(3)CN](+) with the analytes under electrospray ionization (ESI) and atmospheric pressure chemical ionization conditions. In contrast, methanol formed much less solvent adduct ions than acetonitrile. The solvent adduct ions were thermally stable and could not be completely desolvated under the experimental conditions, but they were very fragile to collision-induced dissociation (CID). Interestingly, these solvent adduct ions were observed on a triple-quadrupole mass spectrometry but not on an ion trap instrument where helium used as a damping gas in the ion trap might cause the solvent adduct ions desolvated by collision. By CID studies on the [M + H](+) ions of BDP and its metabolites, their fragmentation paths were proposed. In equine plasma at ambient temperature over 2 h, BDP and B21P were hydrolyzed in part to B17P and BOH, respectively, but B17P was not hydrolyzed. Sodium fluoride added to equine plasma inhibited the hydrolysis of BDP and B21P. The matrix effect in ESI was evaluated in equine plasma and urine samples. The method involved the extraction of BDP and its metabolites from equine plasma and urine samples by methyl tert-butyl ether, resolution on a C(8) column with a mobile phase gradient consisting of methanol and ammonium formate (2 mmol l(-1), pH 3.4) and multiple reaction monitoring for the analytes on a triple-quadrupole mass spectrometer. The detection limit was 13 pg ml(-1) for BDP and B17P, 25 pg ml(-1) for BOH and 50 pg ml(-1) for B21P in plasma and 25 pg ml(-1) for BOH in urine. The method was successfully applied to the analysis of equine plasma and urine samples for the analytes following administration of BDP to horses by inhalation. B17P, the major and active metabolite of BDP, was detected and quantified in equine plasma up to 4 h post-administration by inhalation of a very low therapeutic dose (325 microg per horse) of BDP.     

Identification of a tolfenamic acid metabolite in the horse by gas chromatography-mass spectrometry.

A tolfenamic acid metabolite, a hydroxylated product, has been identified in equine plasma and urine samples using gas chromatography-mass spectrometry in the electron-impact and chemical-ionization modes. The method also allows the qualitative monitoring of the elimination of the drug and its metabolites from plasma. The two compounds are detected up to 48 and 24 h, respectively, after a single oral administration of a 30 mg/kg dose. The simultaneous detection of the two products increases the reliability of anti-doping control analysis.     

Quantitation of anabolic hormones and their metabolites in bovine serum and urine by liquid chromatography-tandem mass spectrometry

A specific and sensitive method based on tandem mass spectrometry with on-line high-performance liquid chromatography using atmospheric pressure chemical ionisation (LC–APCI-MS–MS) for the quantitation of anabolic hormone residues (17β-19-nortestosterone, 17β-testosterone and progesterone) and their major metabolites (17-19-nortestosterone and 17-testosterone) in bovine serum and urine is reported. [²H₂]17β-Testosterone was used as internal standard. The analytes were extracted from urine (following enzymatic hydrolysis) and serum samples by liquid–liquid extraction and purified by C₁₈ solid-phase extraction. Ionisation was performed in a heated nebulizer interface operating in the positive ion mode, where only the protonated molecule, [M+H]⁺, was generated for each analyte. This served as precursor ion for collision-induced dissociation and two diagnostic product ions for each analyte were identified for the unambiguous hormone confirmation by selected reaction monitoring LC–MS–MS. The overall inter-day precision (relative standard deviation) ranged from 6.37 to 2.10% and from 6.25 to 2.01%, for the bovine serum and urine samples, respectively, while the inter-day accuracy (relative error) ranged from −5.90 to −3.18% and from −6.40 to −2.97%, for the bovine serum and urine samples, respectively. The limit of quantitation of the method was 0.1 ng/ml for all the hormones in bovine serum and urine. On account of its high sensitivity and specificity the method has been successfully used to confirm illegal hormone administration for regulatory purposes.     

Determination of gamma-hydroxybutyric acid in human urine by capillary electrophoresis with indirect UV detection and confirmation with electrospray ionization ion-trap mass spectrometry

γ-Hydroxybutyric acid (GHB), a minor metabolite or precursor of γ-aminobutyric acid (GABA), acts as a neurotransmitter/neuromodulator via binding to GABA receptors and to specific presynaptic GHB receptors. Based upon the stimulatory effects, GHB is widely abused. Thus, there is great interest in monitoring GHB in body fluids and tissues. We have developed an assay for urinary GHB that is based upon liquid–liquid extraction and capillary zone electrophoresis (CZE) with indirect UV absorption detection. The background electrolyte is composed of 4 mM nicotinic acid (compound for indirect detection), 3 mM spermine (reversal of electroosmosis) and histidine (added to reach a pH of 6.2). Having a 50 μm I.D. capillary of 40 cm effective length, 1-octanesulfonic acid as internal standard, solute detection at 214 nm and a diluted urine with a conductivity of 2.4 mS/cm, GHB concentrations ≥2 μg/ml can be detected. Limit of detection (LOD) and limit of quantitation (LOQ) were determined to be dependent on urine concentration and varied between 2–24 and 5–60 μg/ml, respectively. Data obtained suggest that LOD and LOQ (both in μg/ml) can be estimated with the relationships 0.83 κ and 2.1 κ, respectively, where κ is the conductivity of the urine in mS/cm. The assay was successfully applied to urines collected after administration of 25 mg sodium GHB/kg body mass. Negative electrospray ionization ion-trap tandem mass spectrometry was used to confirm the presence of GHB in the urinary extract via selected reaction monitoring of the m/z 103.1→m/z 85.1 precursor–product ion transition. Independent of urine concentration, this approach meets the urinary cut-off level of 10 μg/ml that is required for recognition of the presence of exogenous GHB. Furthermore, data obtained with injection of plain or diluted urine indicate that CZE could be used to rapidly recognize GHB amounts (in μg/ml) that are ≥ 4 κ.     

Application of liquid separation techniques to the determination of the main urinary nicotine metabolites

A rapid procedure for the analysis of the main nicotine metabolites (cotinine, trans-3′-hydroxycotinine) in urine has been worked out. The procedure includes isolation of nicotine and its metabolites from urine by means solid–liquid extraction technique using resin Amberlite XAD-2 and then quantitation by the use of thin-layer chromatography with densitometry (in reflection mode). GC–MS was applied to confirm the results obtained by TLC. The procedure was applied to the analysis of cotinine concentrations in urine samples taken from children living in Upper Silesia region (Poland). Among 444 investigated children we did not find cotinine almost in 60% but in 15% of this population, there were children who could have been exposed to cigarette smoke.     

Chromatographic separation and enantiomeric resolution of flurbiprofen and its major metabolites

Summary The chromatographic separation and resolution of the enantiomers of flurbiprofen and its two major metabolites, 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen was investigated using four different approaches: reversed-phase HPLC after pre-column derivatization with (R)-1-(naphthen-1-yl)ethylamine; reversed-phase HPLC using hydroxypropyl-β-cyclodextrin as a chiral mobile phase additive; chiral-phase HPLC using either an α₁-acid glycoprotein CSP (Chiral-AGP) or an amylose tris(3,5-dimethylphenylcarbamate) CSP (Chiralpak AD). Of all the approaches, only the direct method using the Chiralpak AD CSP demonstrated separation and enantiomeric resolution of all three analytes within an acceptable run time of 45 minutes. Enantiomeric resolution values of 1.67,3.67 and 3.44 were obtained for flurbiprofen, 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen respectively. Semi-preparative isolation of the individual enantiomers of both metabolites, followed by CD analysis, revealed that the elution order on the AD CSP wasR-beforeS-enantiomer for both metabolites and the same as that observed for flurbiprofen. The metabolite elution order was subsequently confirmed on the analysis of urine samples obtained from a healthy volunteer following oral administration of the individual drug enantiomers.     

Development and validation of a hydrophilic interaction liquid chromatography with tandem mass spectrometry method for the simultaneous detection and quantification of etilefrine and oxilofrine in equine blood plasma and urine

A sensitive hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method was developed and validated for the simultaneous detection and quantification of etilefrine and oxilofrine in equine blood plasma and urine. The method is highly sensitive and specific with good precision and accuracy. In plasma the limit of detection and limit of quantification are 0.03 and 0.1 ng/mL, respectively, for both analytes. In urine the limit of detection and limit of quantification are 0.3 and 1 ng/mL, respectively, for both analytes. The suitability of the method for doping control analysis in equine species is demonstrated by analyzing postadministration samples collected after a single intravenous administration of 50 mg etilefrine to a standardbred mare. Etilefrine was detected up to 120 h in urine and up to 48 h in plasma. Etilefrine is highly conjugated in equine urine whereas it exists in the free form in equine plasma. Therefore, enzyme hydrolysis prior to sample preparation is recommended for the detection and quantification of etilefrine and oxilofrine in equine urine.     

Determination of pinazepam and its metabolites in serum, urine and brain by gas—liquid chromatography and mass spectrometry

A sensitive and specific assay, involving electron capture gas—liquid chromatography, has been developed for the identification of pinazepam and its metabolites in serum, urine and brain samples from dogs and rats after single or repeated oral administration of the drug. Serum and urine samples from healthy humans after a single oral administration have also been analysed. The identity of gas—liquid chromatographic peaks has been established by mass spectrometry. In blood serum and brain, only pinazepam and its N-depropargylated product (demethyldiazepam) were found; from urine, 3-hydroxypinazepam and oxazepam were also recovered. The sensitivity of the gas—liquid chromatographic method is of the order of 5–10 ng of pinazepam and 15–20 ng of the other three benzodiazepines per ml of serum.     

Encapsulation and characterization of flurbiprofen loaded poly(є-caprolactone)–poly(vinylpyrrolidone) blend micropheres by solvent evaporation method

Flurbiprofen loaded PCL/PVP blend microspheres were prepared by o/w solvent evaporation method using various concentrations of gelatin as emulsifying agent. Microsphere recovery decreased with a decrease in the concentration of the emulsifier in the dispersion. Encapsulation efficiency and drug loading of microspheres increased with decrease in concentration of emulsifying agent. Hydration rate, encapsulation efficiency and drug loading of microspheres increased with increase in concentration of PVP. Rheological properties showed free flowing nature of microspheres. SEM (Scanning electron microscope) revealed microspheres were discrete, spherical and became porous with decrease in concentration of emulsifying agent but smooth with higher concentration of emulsifying agent. FTIR (Fourier transform infrared spectroscopy) spectra of pure and encapsulated flurbiprofen in all formulation showed no significant difference in characteristic peaks, suggesting stability of flurbiprofen during encapsulation process. X-RD (X-ray powder diffractometry) of pure flurbiprofen shows sharp peaks, which decreases on encapsulation, indicating dispersion at molecular level and hence decrease in the crystallinity of drug in microspheres. Microspheres showed an enteric nature at pH 1.2 and a sustained release pattern at pH 6.8. Rapid drug release was observed in microspheres with higher concentration of PVP (polyvinylpyrrolidone), PVP acts as channeling agent. Formulation with low concentration of emulsifying agent also showed a fast release due to porous structure. Drug release kinetics followed zero order at pH 1.2 while at pH 6.8 Higuchi model was best fitted and was found non fickian.     

Characterization of an avidin-bonded column for direct injection in reversed-phase high-performance liquid chromatography

An automatic method for the determination of metabolites of Ropivacaine in urine was set up. It utilizes supported liquid membrane extraction for sample clean-up and enrichment, followed by ion-pair chromatography determination using UV detection. The extraction was very selective with no observed interfering compounds from the urine matrix, permitting simple isocratic chromatographic analysis. The detection limits for spiked urine samples were 2–18 nM for the different compounds. The repeatability was 1–3% (RSD) with an internal standard that was also extracted, and about twice without this standard. A throughput of 3.3 samples per hour was achieved and the liquid membrane was stable for more than a week.     

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.     

Chromatographic Detection of Trimetoquinol (Inolin®) and its Major Urinary Metabolites in the Horse: A Preliminary Report

Trimetoquinol (TMQ) (1-(3,4,5-trimethoxybenzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, m.w. 345) is the prototype tetrahydroisoquinoline pharmaceutical. TMQ is marketed as a bronchodilator in human medicine; in horse racing, TMQ is listed as an Association of Racing Commissioners International (ARCI) class 3 foreign substance. As such, TMQ is considered to have the potential to affect racing performance in horses, and a validated qualitative confirmatory method is required to regulate its use in racing. We selected 8 g kg^–1 of TMQ IV as a safe and effective dose for studies on its metabolism and analytical detection in horses. We developed a solid phase extraction method for recovery of TMQ and its metabolites from equine urine, identified suitable high performance liquid chromatographic conditions for these substances and our internal standard, papaverine, and developed a highly sensitive ESI(+)-LC-MS-MS method (estimated LOD, 100 pg mL^–1) for TMQ and its major metabolites in equine urine. Multiple Reaction Monitoring (MRM) analysis of unhydrolyzed post-administration urine showed small amounts of unchanged TMQ, along with glucuronide, methylated, and sulfated metabolites, with glucuronide metabolites predominating. Following glucuronidase hydrolysis, recovered parent TMQ peaked at relatively high concentrations (>300 ng mL^–1) within 1 h of administration and thereafter declined. The methylated metabolites of TMQ peaked later and at comparable total concentrations, and thereafter declined more slowly. These data suggest that glucuronide hydrolysis of post-administration urine samples will allow recovery of readily identifiable quantities of parent TMQ. These findings, combined with the highly sensitive LC-MS-MS detection of parent TMQ described herein suggest that glucuronide hydrolysis of post-administration urine, followed by LC-MS-MS or other analysis, will allow effective regulatory control of this agent in racing horses.Published as # 351 from the Equine Pharmacology, Therapeutics and Toxicology Program at the Maxwell H. Gluck Equine Research Center and Department of Veterinary Science, University of Kentucky. Published as Kentucky Agricultural Experiment Station Article # 04-14-048 with the approval of the Dean and Director, College of Agriculture and the Kentucky Agricultural Experimental Station.Revised: 8 June and 12 July 2004     

超高效液相色谱-四极杆-飞行时间质谱检测和鉴定猪尿中氯丙那林的主要代谢产物

采用超高效液相色谱-四极杆-飞行时间质谱(UPLC/Q-TOF MS)检测和鉴定了猪尿中氯丙那林的主要代谢产物,并讨论了氯丙那林在猪体内的主要代谢途径。按10 mg/kg(b. w.)的剂量口服灌食氯丙那林,分别采集给药前及给药后的猪尿液样品。采用UPLC/Q-TOF MS对样品进行分析,并应用质量亏损过滤和离子色谱峰提取等数据处理技术,在给药后24 h内的猪尿中检测和鉴定了9种氯丙那林的代谢产物,其中,Ⅰ相代谢产物2种,Ⅱ相代谢产物7种。然后,根据氯丙那林原形和代谢产物的碎片离子特征,对代谢产物的结构进行鉴定。最后,根据所鉴定的代谢产物,推测氯丙那林在猪体内的代谢途径包括苯环羟基化、β -羟基和仲氨基的葡萄糖醛酸轭合、羟基化后的葡萄糖醛酸和硫酸轭合等。研究结果表明,羟基化氯丙那林及其轭合产物的相对含量大于60%,明显高于氯丙那林原形及其轭合产物,是尿液中的主要代谢产物。本研究将为确定氯丙那林在动物体内的残留标示物及加强对氯丙那林非法使用的监控提供科学依据。