Determination of two mercapturic acids related to crotonaldehyde in human urine: influence of smoking.

Crotonaldehyde, an alpha,beta-unsaturated aldehyde, and a potent alkylating agent, is present in many foods and beverages, ambient air and tobacco smoke. A previous study indicated that two metabolites, 3-hydroxy-1-methylpropylmercapturic acid (HMPMA) and 2-carboxyl-l-methylethylmercapturic acid (CMEMA), were excreted in rat urine after subcutaneous injection of crotonaldehyde. Herein, we report the development of a method based on liquid chromatography with tandem mass spectrometry (LC-MS/MS) and deuterated analytes as internal standards, for the determination of HMPMA and CMEMA in human urine. The limits of quantification of the method were 92 and 104 ng/mL for HMPMA and CMEMA, respectively. The calibration curves for both compounds were linear up to 7500 ng/mL with R2 >0.99. It was found that cigarette smokers excreted about three to five-fold more HMPMA, and only slightly elevated amounts of CMEMA, in their urine compared to nonsmokers. In smokers, we also found significant correlations between the urinary excretion levels of HMPMA (but not CMEMA) and several markers of exposure for smoking, including the daily cigarette consumption, carbon monoxide in exhaled breath, salivary cotinine, and nicotine plus five of its major metabolites in urine. Smoking cessation or switching from smoking conventional cigarettes to experimental cigarettes with lower crotonaldehyde delivery led to significant reductions of urinary HMPMA excretion, but not CMEMA excretion. Alcohol consumption did not influence either urinary HMPMA or CMEMA excretion. We conclude that HMPMA is a potentially useful biomarker for smoking-related exposure to crotonaldehyde.

Atypical time course of clinical signs in a dog poisoned by strychnine.

The general health of a German shepherd dog had deteriorated slightly when it was found after being loose for one hour. After 10 hours of observation, the dog showed signs of pain for the first time and signs of poisoning, such as tenseness of muscles, slight opisthotonus, regurgitation, salivation, mydriasis, dyspnoea and cyanosis, were observed; it died 15 minutes after showing the first clinical signs but it had no seizures or tetanic spasms at any time. A postmortem examination did not reveal any pathological changes. A screening test for alkaloids was positive for strychnine (strychnidin-10-one). The presence of strychnine was confirmed and its concentration was determined by gas chromatography/mass spectrometry in urine (728.5 ng/ml) and in the stomach contents (44.6m microg/g). No strychnine was detected in the dog's serum, but traces of brucine (2,3-dimethoxystrychnidin-10-one), the dimethoxy derivative of strychnine, were detected. This case was compared with other strychnine poisonings recorded in the authors' laboratory over the previous six years, taking into account the species, type of samples, the clinical signs and their duration, the postmortem findings, and the concentrations of strychnine. This was the only case to show such an atypical time course of clinical signs.

Elimination of doxepin isomers from the horse following intravenous application.

The tricyclic antidepressant doxepin, representing a 5:1 mixture of trans- and cis-isomers, owns tranquilizing properties. This compound has been associated with illicit medication of racing horses, and therefore should be considered in doping control. Because analysis of doxepin in equine body fluids has not been documented in the literature, a highly sensitive analytical method was developed to individually monitor the doxepin isomers in blood and urine of horses by the use of gas chromatography/mass spectrometry. Following a dose of 1 mg doxepin-HCl/kg intravenously (i.v.), both the isomers were quantified for up to 24 h in serum of horses (n=4). The beta-half-lives of the trans- and cis-isomers were 3.5 and 3.1 h, respectively. The ratio of the trans/cis-isomers was found to be constant (4.7:1) during drug elimination and thus corresponded to the original composition of the antidepressant. Up to 12 h following administration low trans-isomer concentrations in an average range of 2-6 ng/mL were detected in urine of each of the horses, while the cis-isomer was only present in two of four horses for up to 8 and 12 h, respectively. In serum, mean trans-isomer concentrations exceeded urine levels maximally 120-fold after 3 h and at least sixfold after 12 h. As serum exhibits considerably higher concentrations of the doxepin isomers as compared with urine, blood of horses is the recommended body fluid when screening for the antidepressant.

[Detection of levamisole and ivermectin in organ samples from a dead collie]. / Nachweis von Levamisol und Ivermectin in Organproben eines verendeten Collies.

A collie, known for its breed-dependent adverse reaction to ivermectin, was without any clinical signs. The dog was prophylactically treated with 3 mg/kg KG (s.c.) of levamisole. Within 15 minutes, the dog showed convulsions, vomitus, and dyspnea, and perished 2.5 hours after injection of the drugs. The pathological findings were not informative as to the cause of death, and with regard to the adverse reactions, additional application of ivermectin was not excluded. Therefore, organ samples were submitted for toxicological analysis of both levamisole and ivermectin. For detection of levamisole and ivermectin, modified GC/MS and HPLC procedures were developed. Concentrations up to 535 micrograms levamisole and up to 26 ng ivermectin were found per g tissue. Both analytical methods are sensitive enough to detect these drugs after application of low doses. This study elucidates that combination of low-dosed ivermectin and levamisole is no recommendable means against adverse effects of ivermectin, with respect to collies. Moreover, the synergistic effects of ivermectin and levamisole suggests the same drug incompatibility in other dog breeds and animal species.

Development of a method for determination of cyanide concentrations in serum and rumen fluid of cattle.

OBJECTIVES: To develop a reliable method for measurement of cyanide concentrations in cattle, using gas chromatography-mass spectrometry (GC-MS), and establish reference ranges of cyanide concentrations in cattle. ANIMALS: 52 Fleckvieh cattle. PROCEDURE: Cattle were allocated to 3 groups; 12 were fed leguminous grass and hay, 36 were fed whole-maize and corn-cob silages, and 4 were fed other feedstuffs. Samples of blood, rumen fluid, and liver were collected at time of slaughter. Serum, rumen fluid, and liver homogenate were assayed for cyanide content, using a derivatization procedure. A technique for analysis by GC-MS that used selected ion monitoring was developed. RESULTS: Compared with a spectrophotometric method, detection of cyanide in serum and rumen fluid by use of GC-MS was selective and sensitive, with a limit of detection of 0.7 microM. Spectrophotometric analysis yielded false-negative and false-positive results. Thus, the GC-MS method was used for subsequent analysis. In all cattle except 1, cyanide concentration ranged from < 0.7 to 35 microM in serum and from < 0.7 to 28 microM in rumen fluid; cyanide concentration in that 1 animal was 206 microM. Cattle fed clover, grass, grass hay, and clover hay had 8.3- to 8.6-fold higher mean cyanide concentrations in rumen fluid and serum than cattle fed whole-maize and corn-cob silages. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this study suggest a reference range that should be useful for aiding in the diagnosis of cyanide poisoning. Also, cattle can apparently accommodate a serum cyanide concentration of 206 microM without adverse effects.

[Pyrogallol concentrations in rumen content, liver and kidney of cows at pasture]. / Pyrogallolkonzentrationen in Panseninhalt, Leber und Niere von Weidekühen.

Pyrogallol (1, 2, 3-trihydroxybenzene), the decomposition product of hydrolysable tannins in oak bark, leaves and acorns, is suspected to be poisonous to animals. The aim of our investigations was to correlate clinical signs and pathological findings with pyrogallol concentrations in organs of poisoned and healthy animals. In a field study, pyrogallol concentrations were determined in liver, kidney, and rumen from seven cattle. In a herd of twelve cows, five animals suffered from hemorrhagic diarrhea, anorexia, weakness, rumen stasis, dyspnoea, and colic symptoms. Death was observed in five cows within five weeks after repeated intake of green acorns and oak leaves. Toxicological analyses of rumen content, liver, and kidney specimens of one cattle confirmed the suspicion of pyrogallol contamination. In this animal, values ranged from 6 to 13 ng pyrogallol per gram specimen. In control cattle, concentrations were clearly lower than in perished cattle. Under antioxidative work-up conditions, detection limit was 0.6 ng/g in rumen content and 1.0 ng/g in liver and kidney, respectively.

Pyrogallol poisoning of pigeons caused by acorns.

Green acorns are known to contain high concentrations of pyrogallol. Here, we describe an extended case report of two pigeons found dead with a filled muscular stomach of acorns. The following pathologic findings were observed: irritation of mucosal membranes in the gastrointestinal tract, blackish discolored chyme, hyperemic organs, and general edemas. The muscular stomach (ventriculus) was filled with pieces of acorns, and the abdominal cavity contained bloody aqueous fluid. In order to uncover the cause of death, we determined pyrogallol in liver and kidney of one dead pigeon and in ventriculus contents of both pigeons by gas chromatography/mass spectrometry. A further aim of our study was to compare pathologic findings and pyrogallol concentrations in kidney, liver, and ventriculus of poisoned pigeons with those of healthy pigeons. The pyrogallol concentrations in samples of dead pigeons were 16-1200-fold higher than in control animals fed grass and maize-corn. Altogether, the acorn-filled ventriculus, the pathologic findings, the well nourished state, and the high pyrogallol concentrations in the dead pigeons suggest an acute pyrogallol poisoning by acorn. With respect to controls, we conclude that pyrogallol concentrations of 6 ng/g of kidney, 8 ng/g of liver, and 2 ng/g of gastric content do not affect the health of pigeons.

Methadone screening of racehorses.

The misuse of opiates in racehorses relates to their effect of increasing locomotor activity. Because methadone, a narcotic analgesic, has been suspected of use as a doping compound in the past, it was added to the list of banned drugs and should be considered in doping control. Because the literature fails to provide information on detection of methadone in blood or urine of horses, an enzyme-linked immunosorbent assay was developed to monitor this narcotic in equine body fluids. Combined with high-performance liquid chromatography, the immunoassay also served to confirm positives indicated by screening. Following intravenous administration of methadone (0.1 mg/kg), the drug was found for up to 24 h in blood but was never identified in urine (10-pg/mL detection limit). Thus, urine is dismissed as a source of methadone control, and the use of blood to screen racehorses for this narcotic analgesic is suggested.

Excretion of the anabolic steroid boldenone by racing pigeons.

OBJECTIVES: To detect the anabolic steroid bolden-one and to monitor its elimination in the droppings (hereafter referred to as feces) of pigeons treated with the drug. ANIMALS: 8 female pigeons ("Texas" race, 500 +/- 20 g). PROCEDURE: 4 pigeons were given boldenone-17-undecylenate (10 mg/kg of body weight, IM). Feces were collected over defined periods, freeze-dried, extracted with buffer (pH 7.2), and centrifuged. Total immunoreactivity in the supernatant was determined directly by use of an ELISA, and individual boldenone concentration was measured by use of a high-performance liquid chromatography (HPLC)/ELISA after solvent extraction of the aqueous phase. An additional 4 pigeons received a 1-mg/kg dose of the drug. RESULTS: Screening of feces from boldenone-treated pigeons revealed detection of the drug up to 49 days after its administration. The free parent compound was detected during the same period at a constant value of 12 ng/g of lyophilized feces. Positive results predicted by screening were reliably confirmed by the HPLC/ ELISA. Treatment of pigeons with a lower dosage (1 mg/kg) yielded positive results for 31 days. CONCLUSIONS: Illegal medication of pigeons with the anabolic steroid boldenone can be uncovered by screening of feces, using a specific ELISA. Confirmation analysis by use of 2 HPLC systems combined with ELISA reliably yields evidence of drug misuse. Owing to the multiple immunoreactive material, the apparent boldenone concentrations registered by screening markedly exceeded the immunoreactivity attributed to boldenone recovered by HPLC/ELISA. Because the test yields positive results in pigeons for at least 31 days after a single treatment, even at a low dosage of the 17-undecylenate preparation (1 mg/kg), the proposed boldenone test procedure is recommended for doping control in racing pigeons.

[Cortisol levels in blood and urine of trotting horses]. / Cortisolkonzentrationen im Blut und Harn von Trabrennpferden.

Statistical analysis of normally occurring cortisol levels in serum and urine of horses served to recommend thresholds for this corticosteroid in these body fluids, as application of exogenous cortisol as well as ACTH may elevate the cortisol concentrations above the proposed threshold. The present study contributes to the general issue of how to establish thresholds for trotting horses upon sportive examination. 100 randomly selected post competition serum and urine samples, respectively, were submitted to cortisol analysis by means of HPLC. Concentrations of the endogenous corticosteroid in serum and urine followed a log-normal distribution with mean values of 61 and 49 ng/ml, respectively. The probability was 1: 100,000 to exceed concentration limits of 230 (serum) and 394 ng/ml (urine). Designation of thresholds for cortisol has proven problematic and is discussed here.

[Detection of doping compounds in the racing pigeon]. / Nachweis von Dopingsubstanzen in der Brieftaube.

To overcome the doping problems in racing pigeons it requires reliable methods to detect illegally medicated drugs. Difficulties applying specifically to pigeons caused the present investigation, since urine and blood commonly analyzed cannot be gathered from pigeons. Drug detection is complicated by those compounds exhibiting an extremely high potency such as the anabolic boldenone, the glucocorticoid prednisolone, and the bronchodilator clenbuterol. The material for analysis selected was faeces of pigeons treated with the doping substances under investigation. The freeze-dried material was subjected to liquid/liquid extraction (Extrelut), and the extracts were submitted to high performance liquid chromatography. Identification of drugs present in the fractions was carried out by specific antibodies (ELISA), and their relative chromatographic retentions were calculated by means of internal standards. The results obtained provide an overall information about elimination kinetics of the three drugs examined. Thus, boldenone, prednisolone, and clenbuterol were detected in the faeces up to 7 weeks, 2 and 1 day, respectively (limits of detection amounted to 0.1-4 ng per g of the freeze-dried faeces.) The detection periods correspond to the periods of pharmacological action of the individual compounds. The results reported here exemplify the proof of doping by examination of faeces from pigeons treated with very potent drugs. High performance liquid chromatography combined with ELISA turned out to be a suitable technique to detect illegal medication in racing pigeons.

Prothipendyl: detection and elimination in the horse--a case report.

The azaphenothiazine neuroleptic prothipendyl (Dominal) is suspected to be administered illegally at low doses to race-horses to improve their performance. Since for this species pharmacokinetic data of the drug are missing we studied its elimination from blood and urine in a standard-bred mare. At a low (subtherapeutic) dose (i.v., 0.24 mg/kg) the horse is described to be less excited while locomotor activity and attention remain unaffected. In contrast, sedation and ataxia are brought about at 1 mg/kg (therapeutic dose). Identification of prothipendyl given i.v. at subtherapeutic doses was achieved in blood only by means of gas chromatography-mass spectrometry (GC-MS), while the neuroleptic was found both in blood and urine upon 1 mg/kg. Quantification of the neuroleptic was carried out by virtue of triflupromazine as internal standard with the MS operating in the selected ion monitoring (SIM) mode. Under these conditions, the detection limit was 10 ng/ml body fluid. Disappearance of prothipendyl from blood was determined in the horse studied from terminal elimination process, yielding a t1/2 of 2.4 h. The results suggest that for detection of prothipendyl in the horse--in contrast to phenothiazine neuroleptics--screening of blood is preferred over urine as the drug was not recovered in urine after administration of subtherapeutic doses.

Detection of clenbuterol (Ventipulmin) in the horse.

An enzyme linked immunosorbent assay (ELISA) was developed to detect the beta 2-agonist clenbuterol in equine blood and urine. The antiserum was raised in rabbits, employing clenbuterol-diazo-BSA as antigen. Clenbuterol-diazo-horseradish peroxidase served as enzyme conjugate. The concentration of clenbuterol to decrease tracer binding by 50% (IC50 value) was found to be 27.50 +/- 4.20 pg/well (1.37 ng/ml). The antibody cross-reacted with salbutamol (30%), terbutaline (14%) and cimaterol (1%). Horse serum was used directly to screen for clenbuterol, while urine was employed diluted. Positive screening results were confirmed by means of two independent HPLC systems combined with off-line detection by the clenbuterol-ELISA. Salbutamol served as internal standard to ascertain relative retention of the drug. The detection limit of clenbuterol in serum and urine amounted to 0.04 ng/ml. In addition, GC/MS technique was applied to detect clenbuterol in urine samples by a newly developed derivatization method. Confirmation of intravenously given clenbuterol in serum of horses treated with Ventipulmin (0.8 microgram clenbuterol.HCl/kg) was achieved by HPLC/ELISA up to 24 h, in urine up to 96 h. After oral administration, the beta 2-agonist was detected in serum for 48 h and in urine for 75 h.

Identification and verification of the anabolic steroid boldenone in equine blood and urine by HPLC/ELISA.

An enzyme linked immunosorbent assay (ELISA) was developed to detect the anabolic steroid boldenone in equine blood and urine. The polyclonal antiserum was raised in rabbits, employing boldenone-17-hemisuccinate-bovine serum albumin as antigen. Boldenone-17-hemisuccinate-horseradish peroxidase served as enzyme conjugate. Sensitivity of the assay was 26.0 +/- 3.0 pg/well. Among the endogenous steroids tested only progesterone and testosterone exhibited moderate cross-reactivities, 3.4 and 2.5%, respectively. These cross-reactivities are of no importance for the boldenone assay. For the reduction of background levels, screening for boldenone of equine serum was performed after extraction. Urine samples were determined directly after dilution, omitting hydrolysis of boldenone conjugates. Positive screening results were confirmed by means of two independent HPLC systems combined with off-line detection, employing the boldenone ELISA. Methandienone served as internal standard to ascertain retention factors. In horses treated with boldenone-17-undecylenate the presence of boldenone in serum was confirmed up to 28 days and in unhydrolyzed urine up to 56 days post applicationem.

Acetylsalicylic acid and blood coagulation in the horse.

Equine blood may contain salicylic acid (SA) taken up as free acid or represents the metabolite of acetylsalicylic acid (ASA). To obtain information of SA in race horses we screened blood samples of trotting-horses routinely drawn to be analyzed for doping substances. The individual values determined followed a Gaussian distribution displaying a geometric mean of 19 ng SA per ml serum. A probit analysis revealed linear relationship (r = 0.995). Additional studies examined the antithrombotic efficacy of ASA in the horse. An oral dose of 300 mg ASA considerably elevated the bleeding time for more than 2 hours with concomitant SA serum levels between 800 and 1000 ng/ml. It is concluded that salicylate levels even below 1 microgram/ml serum bring about considerable pharmacologic effects such as prolongation of bleeding time, decrease in blood viscosity and possibly dilatation of blood vessels. These effects may improve the tissue supply with blood including oxygen.

Detection of methandienone (methandrostenolone) and metabolites in horse urine by gas chromatography-mass spectrometry.

The metabolic transformation of methandienone (I) in the horse was investigated. After administration of a commercial drug preparation to a female horse (0.5 mg/kg), urine samples were collected up to 96 h and processed without enzymic hydrolysis. Extraction was performed by a series of solid-liquid and liquid-liquid extractions, thus avoiding laborious purification techniques. For analysis by gas chromatography-mass spectrometry, the extracts were trimethylsilylated. Besides the parent compound I and its C-17 epimer II, three monohydroxylated metabolites were identified: 6 beta-hydroxymethandienone (III), its C-17 epimer (IV) and 16 beta-hydroxymethandienone (V). In addition, three isomers of 6 beta,16-dihydroxymethandienone (VIa-c) were discovered. Apparently, reduction of the delta 4 double bond of 16 beta-hydroxymethandienone (V) in the horse yields 16 beta,17 beta-dihydroxy-17 alpha-methyl-5 beta-androst-1-en-3-one (VII). Reduction of the isomers VIa-c results in the corresponding 6 beta,16,17-trihydroxy-17-methyl-5 beta-androst-1-en-3-ones (VIIIa-c). The data presented here suggest that screening for the isomers of VI and VIII, applying the selected-ion monitoring technique, will be the most successful way of proving methandienone administration to a horse.

Detection of diuretics in horse urine by GC/MS.

The use of diuretics in horses subject to doping control is prohibited. Thus, a sensitive screening procedure is required to identify the chemically different diuretics. We communicate here a method to detect three commonly employed acidic diuretics: bumetanide, ethacrynic acid, and furosemide. A liquid-liquid extraction on Extrelut 3 was performed at weak acidic and basic conditions using ethyl acetate as organic solvent. For analysis by GC, the diuretics were methylated on-column in the presence of MSTFA/TMAH, avoiding the commonly employed highly toxic derivatizing agent methyl iodide. For identification of diuretics, we used a mass selective detector operating in the SIM (selected ion monitoring) mode. Confirmation analysis may be obtained with a full scan run. Recoveries for the individual drugs ranged from 31 to 48% at the 100-ng/mL level for 3 mL urine, using calibration curves of drug standards with linearity from 2.5 to 20 ng injected. The limit of detection amounts to 40 ng/mL for the three diuretics. The method permits rapid and sensitive detection of diuretics in horse urine and is recommended for doping control.

[Detection of dexamethasone in horses]. / Nachweis von Dexamethason im Pferd.

Due to their marked antiinflammatory effect, synthetic corticosteroids are used to mask illness, especially lameness in horses. The detection of these drugs in equine body fluids requires accurate methods, particularly where misuse of corticosteroids is suspected. Gas chromatography/mass spectrometry (GC/MS) is well established as a reliable technique for the identification of drugs in biological fluids. Using GC/MS, we determined dexamethasone levels in horse urine and serum after intravenous application of a therapeutic dose. Dexamethasone was detectable, in serum for up to six hours, and in urine for up to 32 hours, after its administration. These findings indicate that serum measurements are unreliable for the detection of corticosteroid abuse, and demonstrate urine to be a more suitable body fluid for investigation. Nevertheless, it should be emphasized that, regardless of the technique employed, the clinical effects of dexamethasone last longer than 32 hours; thus, failure to detect dexamethasone does not disprove corticosteroid abuse.