Urinary Excretion of the ß-Adrenergic Feed Additives Ractopamine and Zilpaterol in Breast and Lung Cancer Patients.

ß2-Adrenergic agonists (ß-agonists) have been legally used in the U.S. for almost two decades to increase lean muscle mass in meat animals. Despite a cardiotoxic effect after high-dose exposure, there has been limited research on human ß-agonist exposures related to meat consumption. We quantified urinary concentrations of ractopamine and zilpaterol, two FDA-approved ß-agonist feed additives, and examined the extent to which the concentrations were associated with estimated usual meat intake levels. Overnight urine samples from 324 newly diagnosed breast cancer patients and spot urine samples from 46 lung cancer patients at the time of diagnosis, prior to treatment, were collected during 2006-2010 and 2014-2015, respectively. Urinary ractopamine and zilpaterol concentrations were measured by LC-MS/MS. Ractopamine and zilpaterol, respectively, were detected in 8.1% and 3.0% of the urine samples collected (n = 370). Only 1.1% (n = 4) of the urine samples had zilpaterol concentrations above the limit of quantification, with the mean value of 0.07 ng/mL in urine. The presence of detectable ractopamine and zilpaterol levels were not associated with meat consumption estimated from a food frequency questionnaire, including total meat (P = 0.13 and 0.74, respectively), total red meat (P = 0.72 and 0.74), unprocessed red meat (P = 0.74 and 0.73), processed red meat (P = 0.72 and 0.15), and poultry intake (P = 0.67 for ractopamine). Our data suggest that the amount of meat-related exposure of ß-agonists was low.

Detection of clenbuterol at trace levels in doping analysis using different gas chromatographic-mass spectrometric techniques.

This study demonstrates the development of a gas chromatography-triple quadrupole tandem mass spectrometry (GC-MS-MS) assay to detect clenbuterol in human urine and the comparison of this method with GC-MS techniques and gas chromatography-high resolution mass spectrometry (GC-HRMS) techniques. Urine samples were hydrolyzed with ß-glucuronidase, extracted with methyl tert-butyl ether and dried under nitrogen. The derivative reagent was N-methyl-N-(trimethylsilyl)-trifluoroacetamide with NH4I and was analyzed by GC-MS, GC-MS-MS and GC-HRMS. A validation study was conducted by GC-MS-MS. The analyses of clenbuterol using different mass spectrometric techniques were compared. The limit of detection (LOD) for clenbuterol in human urine was 2 ng/mL by GC-MS (selected ion monitoring mode: SIM mode), 0.06 ng/mL by GC-HRMS and 0.03 ng/mL by GC-MS-MS, respectively, while the LOD by GC-HRMS was 0.06. With GC-MS-MS, the intra-assay and inter-assay precisions were less than 15%, the recoveries were 86 to 112% and the linear range was 0.06 to 8.0 ng/mL. The GC-MS under SIM mode can be used as a screening tool to detect clenbuterol at trace levels in human urine. The GC-MS-MS and GC-HRMS methods can confirm clenbuterol when its concentration is below 2 ng/mL. The results demonstrate that the GC-MS-MS method is quite sensitive, specific and reliable for the detection of clenbuterol in doping analysis.

Myocardial toxicity in a group of greyhounds administered ractopamine.

Ractopamine, a synthetic ß(2)-adrenoceptor agonist, is widely used as a feed additive in the United States to promote a reduction in body fat and enhance muscle growth in cattle, pigs, and turkeys. It has the potential for illegal use in show and racing animals because it may affect performance via its ß-adrenergic agonist properties or anabolic activities. Nine greyhounds were orally administered 1 mg/kg of ractopamine to investigate the ability to detect the drug in urine. Postdosing, 7 of 9 dogs developed cardiac arrhythmias and had elevated troponin levels indicating myocardial damage. One dog necropsied 4 days postdosing had massive myocardial necrosis, mild to focally moderate skeletal muscle necrosis, and widespread segmental arterial mediolysis. A second dog necropsied 17 days postdosing had mild myocardial necrosis and fibrosis. Scattered arteries exhibited segmental medial and perimedial fibromuscular dysplasia. This is the first reported case of arterial, cardiac, and skeletal muscle damage associated with ractopamine.

Simultaneous determination of beta2-agonists in human urine by fast-gas chromatography/mass spectrometry: method validation and clinical application.

A fast screening protocol was developed and validated for the simultaneous determination of 15 beta(2)-agonists in human urine (bambuterol, cimbuterol, clenbuterol, fenoterol, formoterol, isoproterenol, mapenterol, metaproterenol, procaterol, ractopamine, ritodrine, salbutamol, salmeterol, terbutaline, tulobuterol). The overall sample processing includes deconjugation with enzyme hydrolysis, liquid-liquid extraction, followed by derivatization of the extract and detection of beta(2)-agonists trimethylsilyl-derivatives by fast-gas chromatography/electron impact-mass spectrometry (fast-GC/EI-MS). Sample extraction and derivatization were optimized with the purpose of improving recoveries and reaction yields for a variety of analytes with different structures simultaneously, while keeping the procedure simple and reliable. Validation parameters were determined for each analyte under investigation, including selectivity, linearity, intra- and inter-assay precision, extraction recoveries and signal to noise ratio (S/N) at the lowest calibration level. Fast-GC/MS sequences, based on the use of short columns, high carrier-gas velocity and fast temperature ramping, allow considerable reduction of the analysis time (7 min), while maintaining adequate chromatographic resolution. The overall GC cycle time was less than 9 min, allowing a processing rate of 6 samples/h. High MS-sampling rate, using a benchtop quadrupole mass analyzer, resulted in accurate peak shape definition under both scan and selected ion monitoring modes, and high sensitivity in the latter mode. The method was successfully tested on real samples arising from clinical treatments. Copyright (c) 2009 John Wiley & Sons, Ltd.

Analysis of beta-agonists and beta-blockers in urine using hollow fibre-protected liquid-phase microextraction with in situ derivatization followed by gas chromatography/mass spectrometry.

A method using hollow fibre-protected liquid-phase microextraction (HF-LPME) with in situ derivatization followed by gas chromatography/mass spectrometry (GC/MS) was established for the analysis of beta-agonists and beta-blockers in urine. Because it can simultaneously extract and derivatize compounds of interest by methylbenzol and N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) in HF-LPME, the approach overcomes the drawbacks of considerable time-consuming and tedious operation, meanwhile improves enrichment multiple. The optimized conditions were extraction for 20 min at 35 degrees C with 5.0 microL of mixed extraction solvent (methylbenzol/MSTFA=1:1, v/v) with stirring speed of 925 rpm in 5.0 mL sample under pH 12.0 and 14% (w/v) NaCl. The method provided very wide linear ranges (0.25-400 ng mL(-1)) and low detection limits in the range of 0.08-0.10 ng mL(-1) for clenbuterol, metoprolol and propranolol while enrichment factors reached up to 256. The analytes could be determined in spiked urine by the method with high extraction efficacy (93.79-109.04% recoveries) and precision (<9.70% RSD). It has a satisfactory result for metoprolol in practical human urine samples for a single-dose administration of 50 mg after 36 h. The proposed method only needs few microliters of organic solvent and derivatizing agent; the operation is simple, convenient and rapid for the trace analysis of beta-agonists and beta-blockers in biological fluids; it can be readily generalized for high sample throughput. So, it is hopeful that the study will facilitate the monitoring of beta-agonists and beta-blockers in the competition sports.

A screening method for the simultaneous detection of glucocorticoids, diuretics, stimulants, anti-oestrogens, beta-adrenergic drugs and anabolic steroids in human urine by LC-ESI-MS/MS.

This paper presents a general screening method, based on liquid chromatography/mass spectrometry (LC/MS), for the simultaneous detection in human urine of 72 xenobiotics (21 diuretics, 16 synthetic glucocorticoids, 17 beta-adrenergic drugs, 10 stimulants, 5 anti-oestrogens and 3 anabolic steroids), excreted free or as glucuro-conjugates in urine. Although the method has been specifically designed and evaluated in view of its potential application to anti-doping analyses, it can also be effective in other areas of analytical toxicology. Sample preparation was based on two liquid/liquid separation steps (performed at alkaline and at acid pH, respectively) of hydrolyzed human urine, and then an assay by LC/MS-MS in positive and negative ionization mode using an electrospray ionization source (ESI) and multiple reaction monitoring (MRM) as the acquisition mode. The overall time needed for an LC run was less than 15 minutes. All compounds showed good reproducibility in terms of both the retention times (CV%<1) and the relative abundances of the diagnostic transitions (CV%<10). The limits of detection (LOD) were in the range of 1-50 ng/mL for glucocorticoids, anti-oestrogens and steroids, and 50-500 ng/mL for diuretics, beta-adrenergic drugs and stimulants, thus satisfying the minimum required performance limits (MRPL) set by the World Anti-Doping Agency (WADA) for the accredited anti-doping laboratories.

Detection of clenbuterol in heroin users in twelve postmortem cases at the Philadelphia medical examiner's office.

The presence of clenbuterol, a beta2-adrenergic agonist banned for human use in the United States because of its serious side effects, is reported in a series of 12 postmortem cases in which the cause of death was attributed to illicit drug use. During the first three months of 2007, postmortem specimens from cases previously screening positive for opiates or fentanyl were screened specifically for clenbuterol using enzyme-linked immunosorbent assay. Confirmation of clenbuterol was performed using solid-phase extraction, derivatization with trimethylboroxine, and analysis utilizing a gas chromatograph-mass spectrometer (GC-MS) operated in the full-scan mode. The limits of detection and quantitation in blood were 2.5 and 5 ng/mL, respectively. Linearity was from 5 to 100 ng/mL. Clenbuterol was positive in 12/106 (11%) drug-related cases and in 12/575 (2.1%) of the total cases tested. In each of the 12 cases positive for clenbuterol, heroin use was either confirmed by the presence of 6-acetylmorphine or strongly suspected by the presence of morphine with a history of heroin abuse. Because the use of clenbuterol in the United States is restricted to veterinary medicine, its detection is an unexpected finding. Its presence in these cases serves as a caution to emergency room physicians and toxicologists to consider and test for clenbuterol when treating a suspected heroin user who presents atypically. This is the first known series of clenbuterol-positive cases of illicit drug users to be reported from a medical examiner's toxicology laboratory.

Introduction of HPLC/orbitrap mass spectrometry as screening method for doping control.

A new doping control screening method has been developed, for the analysis of doping agents in human urine, using HPLC/orbitrap with in-source collision-induced dissociation and atmospheric pressure chemical ionization. The developed method allows the detection of 29 compounds, including agents with antiestrogenic activity, beta(2) agonists, exogenous anabolic steroids, and other anabolic agents. The mass accuracy of this method is better at 2 ppm using an external reference. The detection limit for all compounds tested was better than 100 pg/ml. The recoveries of most analytes were above 70%. The measured median repeatability values for doping agents included in the method at concentrations of 1 and 10 ng/ml were 21 and 17%, respectively. The relative standard deviation (RSD) of the intraday precision (n = 6) ranged from RSD = 16-22%, whereas the interday precision (n = 18), ranged from RSD = 17-26%, depending on the solute concentration investigated.

A descriptive study of an outbreak of clenbuterol-containing heroin.

STUDY OBJECTIVE: Illicit drugs may be adulterated with substances other than the sought-after substance of abuse. Although the true incidence and clinical effects of this practice are unknown, geographically disparate outbreaks of clinically significant adulteration continue to occur. We report on a recent outbreak of clenbuterol-adulterated heroin occurring along the East Coast of the United States. METHODS: After identification of index cases, 5 US poison centers collaborated with state and territorial health departments to alert the public of clenbuterol-tainted heroin. A case definition of clenbuterol-tainted heroin toxicity was promulgated, and emergency departments (EDs) were asked to contact poison centers when cases were identified. RESULTS: We identified 34 probable or confirmed ED presentations in 5 states during a 6-month period. Thirteen of the 34 patients met the criteria for "confirmed" exposures. Clenbuterol was identified in the blood and or urine of 12 of these 13 patients. Clenbuterol concentrations ranged from 2.4 to 26 ng/mL in the blood and 9.4 to 12,526 ng/mL in the urine. Symptoms included nausea, chest pain, palpitations, dyspnea, and tremor. Physical findings included significant tachycardia, hypotension, and laboratory evidence of hyperglycemia, hypokalemia, and increased lactate levels. Six patients demonstrated biochemical evidence of myocardial injury. Ten patients received beta-adrenergic antagonists without adverse effect. CONCLUSION: The adulteration of heroin by clenbuterol was associated with sympathomimetic effects, metabolic acidosis, and myocardial injury. The report also highlights how collaborative efforts among poison centers using the Centers for Disease Control and Prevention's Epi-X system rapidly identified a disease outbreak.

Development of a lateral-flow assay for rapid screening of the performance-enhancing sympathomimetic drug clenbuterol used in animal production; food safety assessments.

A lateral-flow assay that could provide visual evidence of the presence of clenbuterol in swine urine was developed. Colloidal gold was prepared and conjugated with anti-clenbuterol monoclonal antibody. Immunochromatographic test strips were produced, and then, 210 samples were tested on these strips. Analysis was completed in 10 min. Detection limit was 3 ppb of clenbuterol. Parallel GC-MS data indicated that clenbuterol rapid detection strip had no false negative. The false positive rate was 4.4%. Immunochromatographic strip has great applied value in the food safety field because it possesses benefits of sensitivity, stability, reproducibility, ease of use and inexpensive.

Validation and application of a screening method for beta2-agonists, anti-estrogenic substances and mesocarb in human urine using liquid chromatography/tandem mass spectrometry.

Electrospray ionization (ESI) mass spectra of 15 anti-estrogenic substances, beta2-agonists and mesocarb were investigated in terms of fragmentation patterns. On the basis of this product ion information, a simultaneous screening method for anti-estrogenic substances, beta2-agonists and mesocarb was developed for doping control purposes. After hydrolysis, liquid-liquid extraction was adopted for the sample preparation. The recoveries for all compounds were 30 and 96%. A single liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis could be performed in 13 min for the analysis of 15 anti-estrogenic substances, beta2-agonists and mesocarb. A quantitative analysis was also validated. Inaccuracies were below +/-12% and precisions varied from 0 to 15.8%. The limit of detection was below 10 ng/mL except formestane (300 ng/mL) and aminoglutethimide (100 ng/mL). The validated method was applied for the analysis of excretion samples.

Liquid chromatography/electrospray ionization tandem mass spectrometric screening and confirmation methods for beta2-agonists in human or equine urine.

Electrospray ionization (ESI) mass spectra of 19 common beta(2)-agonists were investigated in terms of fragmentation pattern and dissociation behavior of the analytes, proving the origin of fragment ions and indicating mechanisms of charge-driven and charge-remote fragmentation. Based on these data, liquid chromatographic/ESI tandem mass spectrometric (LC/ESI-MS/MS) screening and confirmation methods were developed for doping control purposes. These procedures employ established sample preparation steps including either acidic or enzymatic hydrolysis, alkaline extraction and, in the case of equine urine specimens, acidic re-extraction of the analytes. In addition, a degradation product of formoterol caused by acidic hydrolysis during sample preparation could be identified and utilized as target compound in screening and also confirmation methods. The screening procedures cover 18 or 19beta(2)-agonists, the estimated limits of detection of which for equine and human urine samples vary between 2 and 100 ng ml(-1) and between 2 and 50 ng ml(-1), respectively. A single LC/MS/MS analysis can be performed in 9 min.

Determination of salbutamol using on-line solid-phase extraction and sequential injection analysis. Comparison of chemiluminescence and fluorescence detection.

Determination of salbutamol using sequential injection analysis (SIA) with chemiluminescence and fluorescence detection has been devised. The chemiluminescence signal was emitted during the oxidation of salbutamol by potassium permanganate in sulfuric acid medium. Sodium polyphosphate was used as chemiluminescence enhancer. The fluorescence signal (excitation wavelength 230 nm) was also measured in sulfuric acid medium. Both detection techniques were compared with respect to the application of the methods to the determination of salbutamol in biological materials. The sample pre-treatment takes place directly in the SIA system, when salbutamol is adsorbed on the solid-phase (Baker-carboxylic acid) microcolumn integrated into the system. Sulfuric acid serves both as the reagent and the eluent. The lab-made SIA system consisted of a 2.5-mL Cavro syringe pump, ten-port Vici Valco selection valve and Spectra-Physics FS 970 fluorescence detector, which was lab-modified for chemiluminescence detection. The system was controlled by a PC using originally compiled LabVIEW-supported software. Concentrations, volumes of reagents and flow rates were optimised by a simplex method. Salbutamol was determined in the linear range 0.05-10 microg mL(-1) (RSD 1.53%), with the detection limit (3 sigma) 0.03 microg mL(-1) and sample throughput of 42 samples per hour with chemiluminescence detection in standard solutions. The fluorescence detection enabled the determination of salbutamol in standard solutions in the linear range 0.5-100 microg mL(-1) (RSD 2.69%), with the detection limit 0.2 microg mL(-1) and sample throughput of 24 h(-1). The proposed methods were applied to the determination of salbutamol in human serum and urine. However, serum is a very complicated matrix and the SIA-SPE analysis did not provide satisfactory results. It was possible to determine salbutamol in human urine using this technique. Better recovery was achieved with fluorescence detection.

Diagnostic evidence for the presence of beta-agonists using two consecutive derivatization procedures and gas chromatography-mass spectrometric analysis.

A GC-MS procedure for the detection of different beta-agonists in urine samples based on two consecutive derivatization steps is described. The derivatization procedure is based on the consecutive formation of cyclic methylboronate derivatives followed by a second derivatization step with MSTFA on the same extract, forming TMS derivatives. Injections in the GC-MS system may be carried out after each one of the derivatization steps, obtaining enough information for unambiguous identification. Limits of detection for the two derivatization steps ranged from 0.5 to 5 ng/ml. This procedure was tested with the beta-agonists bambuterol, clenbuterol, fenoterol, formoterol, salbutamol, salmeterol, alpha-hydroxy-salmeterol and terbutaline.

3-O-methyldobutamine, a major metabolite of dobutamine in humans.

Dobutamine is a synthetic ionotropic catecholamine commonly used to treat heart failure and shock. The catabolic fate of dobutamine in humans has yet to be reported, although formation of 3-O-methyldobutamine represents the principal pathway of dobutamine disposition in the dog. Herein, we describe the isolation and identification of 3-O-methyldobutamine in the urine of children receiving infusions of racemic dobutamine. In a 9-year-old child with heart failure approximately 80% of dobutamine administered intravenously at steady state was detected in the urine. Forty-seven percent of infused dobutamine was identified as 3-O-methyldobutamine and its acid-hydrolyzed derivatives, the latter mostly conjugated with sulfate (33%). Thirty-two percent consisted of acid-hydrolyzed dobutamine metabolites, primarily conjugated with sulfate (16%). Sonicates of human blood mononuclear cells catalyzed the formation of 3-O-methyldobutamine from dobutamine and S-adenosylmethionine in vitro. These findings indicate that formation of 3-O-methyldobutamine constitutes a major pathway of dobutamine metabolism in humans.

Screening of beta-2 agonists and confirmation of fenoterol, orciprenaline, reproterol and terbutaline with gas chromatography-mass spectrometry as tetrahydroisoquinoline derivatives.

A new method for a comprehensive screening and confirmation of beta-2 agonists in human urine is presented based on gas chromatography-low-resolution mass spectrometry (GC-MS) using electron impact ionisation (EI). After hydrolysis of the conjugates with beta-glucuronidase/arylsulfatase a derivatisation step with formaldehyde converts fenoterol, orciprenaline, reproterol and terbutaline to one derivative, a tetrahydroisoquinoline, while the other beta-2 agonists remain unchanged. Liquid-liquid extraction and trimethylsilylation follow. The tetrahydroisoquinoline derivatives show good gas chromatographic and mass spectrometric behaviour. The detection limit of these four beta-2 agonists in the screening using low-resolution mass spectrometry is 10 ng/ml of urine. The other beta-2 agonists are detected as parent compounds with the same recovery after sample preparation with and without formaldehyde. The EI mass spectra of the tetrahydroisoquinoline derivatives are presented.

Derivatization procedures for the detection of beta(2)-agonists by gas chromatographic/mass spectrometric analysis.

An evaluation of derivatization procedures for the detection of beta(2)-agonists is presented. The study was performed with the beta(2)-agonists bambuterol, clenbuterol, fenoterol, formoterol, salbutamol, salmeterol and terbutaline. Different derivatizating agents were employed, aiming to obtain derivatives with high selectivity to be used in the gas chromatographic/mass spectrometric analysis of beta(2)-agonists in biological samples. Trimethylsilylation was compared with different agents and the role of some catalysts was evaluated. Acylation, combined trimethylsilylation and acylation, and the formation of cyclic methylboronates were also studied. Sterical hindrance caused by different substituents at the nitrogen atom of the beta-ethanolamine lateral chain of beta(2)-agonist molecules is mainly responsible for differences in the abundances of the derivatives obtained. The use of catalysts produces an increase in the derivatization yield, especially for compounds with low steric hindrance (substituents with primary and secondary carbon atoms). The formation of trimethylsilyl (TMS) ethers is not influenced by structural molecular differences when only hydroxy groups are involved in derivatization. Combined trimethylsilylation and acylation showed that compounds with a secondary carbon atom linked to the nitrogen atom form mainly N-TFA-O-TMS derivatives, with a small amount of N-TMS-O-TMS derivatives. Compounds with tert-butyl substituents at the amino group (bambuterol, salbutamol and terbutaline) formed O-TMS derivatives as the main products, although a limited amount of trifluoroacylation at the nitrogen atom also occurred. Cyclic methylboronates were formed with bambuterol, clenbuterol, formoterol, salbutamol and salmeterol. Owing to hydroxy substituents in unsuitable positions for ring formation, this procedure was not effective for fenoterol and terbutaline. Mass spectra of different derivatives and tentative fragmentation profiles are also shown. For screening purpose (e.g. sports drug testing), derivatization with MSTFA or BSTFA alone is recommended as a comprehensive derivatization technique for beta(2)-agonists owing to minimal by-product formation; formation of cyclic methylboronates can be useful for confirmation purposes. Detection limits were obtained for the TMS and cyclic methylboronate derivatives using the derivatizing reagents MSTFA and trimethylboroxine, respectively. For most of the compounds, lower detection limits were found for the TMS derivatives.

Determination of beta2-agonists in bovine urine: comparison of two extraction/clean-up procedures for high-resolution gas chromatography-mass spectrometry analysis.

Two extraction/clean-up analytical procedures were investigated and compared regarding their recovery and matrix-purification efficiency for screening beta2-agonist residues in fortified bovine urine by high-resolution gas chromatography-mass spectrometry (GC-MS). The first procedure, based on an analytical method originally developed for detecting anabolic steroids, consists of the employment of the nonionic resin, Amberlite XAD-2, a styrene-divinylbenzene copolymer for solid-phase extraction (SPE), followed by liquid-liquid extraction with diethyl ether. The second focuses on the use of a mixed SPE cartridge (reversed-phase and ion-exchange sorbent, Bond Elut Certify). In both cases, the trimethylsilylated derivatives were analyzed by GC-MS with an ion-trap detector. Clenbuterol, salbutamol, and terbutaline were used to spike urine samples during the comparison experimental phase. Afterwards, tulobuterol, mabuterol, mapenterol, cimbuterol, and brombuterol were included in the evaluation of the second procedure (the Bond Elut Certify procedure). At this stage, the detection was accomplished by GC-MS (quadrupole mass analyzer) with selective ion monitoring acquisition. The isotopic dilution method with the hexadeuterated analogues of clenbuterol and salbutamol was applied to prepare calibration curves and calculate recovery percentages. With XAD-2 resin, terbutaline and salbutamol (resorcinol and phenol-type beta2-agonists, respectively) could not be detected at 20 ng/mL or at 40 ng/mL. In spite of clenbuterol having been detected at 20 ng/mL, the results obtained were not reproducible. The use of the reversed-phase and ion-exchange sorbent Bond Elut Certify allowed multiresidue detection and showed several advantages for the screening of clenbuterol such as higher recoveries, cleaner final extracts, reduced sample preparation time, less labor intensive, and easier solvent consumption and disposal. Recoveries over 88% (concentrations ranging from 0.5 to 10 ppb) and limits of detection equal to 0.5 ppb were met for all the beta2-agonists studied with the last method.

Positive chemical ionization and tandem mass spectrometric fragmentation for the gas chromatographic analysis of beta-agonists using the ion trap technique.

A procedure is described for the determination of three characteristic beta-agonists (clenbuterol, terbutalin and salbutamol) based on the formation of the corresponding protonated molecules and related collisional experiments. Quantification was carried out on selected collisional fragments and the reproducibility of the relative abundances of these fragments was estimated. The performance of the method was tested on bovine urine samples spiked at the lowest level of 0.2 ng ml(-1) in each of the chosen compounds.