Presence of ß2 -agonist growth promoters in human urine samples: GC-MS/MS evaluation of the excretion profiles of ractopamine administered in microdoses.

ß2 -adrenergic agonists having the potential to be misused to enhance performance for their thermogenic and anabolic properties are prohibited in sports. Clenbuterol, ractopamine and zilpaterol are utilised legally or illegally as growth promoters of animals raised for their meat. No withdrawal times are imposed for ractopamine prior to slaughter; residues are detected in meat of treated animals, which constitutes a risk of inadvertent consumption. Insufficient information is available on the fate of ractopamine in humans to implement efficient detection in athletes' urine samples. We have developed a confirmation procedure for total ractopamine in urine following the enzymatic hydrolysis of glucuronides and sulphates and the conversion to tri-TMS derivative (limit of identification at 0.15 ng/ml). The sulphates were found to form between 85% to 97% of ractopamine excreted in athletes' urine samples analysed routinely or in volunteers following the administration of a micro-dose of 2.5 µg. Peak levels were reached at 2 to 6 h and decreased rapidly below 1 ng/ml 10 h after dosing. With one exception, the highest level estimated in athletes' samples was 1.2 ng/ml. Zilpaterol was confirmed in a few urine samples collected in the USA and Mexico (highest level 2 ng/ml), while hundreds of athletes' samples were reported to contain clenbuterol by our laboratory over the past 7 years. Most of these cases originated from Mexico (n = 102) and Guatemala (n = 119), often clustered in events during which multiple samples were collected, and for the vast majority, in levels lower than 0.2 ng/ml.

An ultrasensitive colloidal gold immunosensor to simultaneously detect 12 beta (2)-adrenergic agonists.

In this study, we first prepared a selective monoclonal antibody against 12 beta (2)-adrenergic agonists (Salbutamol, Clenbuterol, Brombuterol, Clenpenterol, Mabuterol, Carbuterol, Cimbuterol, Mapenterol, Pirbuterol, Terbutaline, Cimaterol, and Clenproperol). Then three haptens were designed and derived, among which, haptenS3 used the amino group of the salbutamol analog to derive a carboxyl group containing a spacer, which is unique to this study. The half-maximal inhibitory concentration (IC50) values were 0.35 ng/mL (Salbutamol), 0.42 ng/mL (Clenbuterol), 0.78 ng/mL (Brombuterol), 0.88 ng/mL (Clenpenterol), 1.34 ng/mL (Mabuterol), 1.38 ng/mL (Carbuterol), 1.71 ng/mL (Cimbuterol), 2.24 ng/mL (Mapenterol), 2.25 ng/mL (Pirbuterol), 2.27 ng/mL (Terbutaline), 3.49 ng/mL (Cimaterol), and 4.89 ng/mL (Clenproperol). We further developed a monoclonal antibody-based colloidal gold immunochromatographic test strip for screening and detecting 12 beta (2)-adrenergic agonists in swine urine and lamb samples. The immunochromatographic method developed in this study is a suitable tool for the on-site rapid detection and screening of beta (2)-adrenergic agonists in swine urine and lamb samples.

Single-dose administration of clenbuterol is detectable in dried blood spots.

Clenbuterol is a ß2 -agonist prescribed for asthmatic patients in some countries. Based on its anabolic and lipolytic effects observed in studies on rodents and in livestock destined for food production, clenbuterol is abused by bodybuilders and athletes seeking leanness. Urinary clenbuterol analysis is part of routine doping analysis. However, the collection of urine samples is time-consuming and can be intimidating for athletes. Dried blood spot (DBS) appears attractive as an alternative matrix, but the detectability of clenbuterol in humans through DBS has not been investigated. This study evaluated if clenbuterol could be detected in DBS and urine collected from six healthy men after oral intake of 80 µg clenbuterol. The DBS and urine samples were collected at 0, 3, 8, 24, and 72 h post-ingestion, with additional urine collections on days 7 and 10. Using LC-MS/MS, it was shown that clenbuterol could be detected in all DBS samples for 24 h post-ingestion and with 50% sensitivity 3 days after ingestion. The DBS method was 100% specific. Evaluation of analyte stability showed that clenbuterol is stable in DBS for at least 365 days at room temperature when using desiccant and avoiding light exposure. In urine, clenbuterol was detectable for at least 7-10 days after ingestion. Urinary clenbuterol concentrations below 5 ng/mL were present in some subjects 24 h after administration. Collectively, these data indicate that DBS are suitable for routine doping control analysis of clenbuterol with a detection window of at least 3 days after oral administration of 80 µg.

Clenbuterol: a new toxic substance in paediatrics.

A 13-year-old girl presented to the emergency department with acute onset of chest pain, nausea and tremor. The patient denied drug ingestion, and urine toxicology was negative. ECG demonstrated sinus tachycardia, prolonged QTc (541 ms) and ST depression. Laboratory testing demonstrated metabolic acidosis, hypokalaemia, hypophosphataemia and hyperglycaemia. She was commenced on continuous cardiac monitoring and treated with intravenous fluids and electrolyte replacement. Presenting features and laboratory abnormalities resolved within 48 hours. The National Poisons Information Service and Clinical Biochemistry were integral to her management, advising the clinical team on the likeliest aetiology. Five weeks after discharge, urine toxicology, using mass spectrometry, identified clenbuterol. Clenbuterol is an oral ß2-agonist with anabolic and lipolytic effects that is misused as a performance and image enhancing drug. Clinicians must be aware of the increasing availability of these drugs and their potential for causing harm in children and adolescents.

Selective extraction and detection of ß-agonists in swine urine for monitoring illegal use in livestock breeding.

The illegal use of ß-agonists often endangers animal-derived food safety. In this study, a selective detection method for ß-agonists in swine urine was established via the combination of polymeric ionic liquid-molecularly imprinted graphene oxide-miniaturized pipette tip solid-phase extraction and high-performance liquid chromatography. It is worth noting that this method relied mainly on the designed adsorbent, which presented a rich adsorption mechanism, fast mass transfer rate, and high selectivity, and was successfully utilized in the selective extraction of ß-agonists from swine urine samples. The proposed method has low LOD (0.20-0.56 ng/mL), high recovery (94.9-107.9%), and high reusability (4 times, 91.9-108.8%), which indicates its high potential as a selective, sensitive, accurate, and nonfatal method for monitoring the illegal use of ß-agonists in the livestock breeding stage.

Simultaneous Detection of Multiple ß-Adrenergic Agonists with 2-Directional Lateral Flow Strip Platform.

Clenbuterol (CL), salbutamol (SAL) and ractopamine (RAC) are the three common ß-adrenergic agonists, which are the main hazards in food safety and affect human health through the food chain. A convenient and efficient method is urgently required to perform on-site detection of multiple ß-adrenergic agonists to avoid frequent poisoning incidents. In this paper, a 2-directional lateral flow strip technique (2-directional LFS) is developed for rapid and simultaneous detection of CL, SAL and RAC with single sampling. Compared to the conventional lateral flow strip, this 2-directional LFS technique can realize simultaneous detection of three or more target analytes without any change of intrinsic simplicity of LFS. Furthermore, this 2-directional LFS can effectively avoid the potential intrinsic cross-reactivity among the reagents to analogues. Under the optimized conditions, CL, SAL and RAC were all successfully determined with satisfactory results in both buffer and urine samples with the detection limit as low as 0.5 ng/mL. This 2-directional LFS technique can revolutionize the commercial single-analyte LFS products and can effectively widen the applications of the classic LFS in various fields.

The risk of higenamine adverse analytical findings following oral administration of plumula nelumbinis capsules.

Higenamine is a ß2-agonist that has been included in the Prohibited List of the World Anti-Doping Agency (WADA) since 2017. Meanwhile, it exists in plumula nelumbinis, a part of the lotus seed, and is commonly used as an ingredient in cuisines, herbal medicines, and nutritional supplements in China and other countries in East Asia. Therefore, an evaluation of the risk of an adverse analytical finding (AAF) of higenamine caused by plumula nelumbinis products is necessary in doping control. In this study, 14 volunteers took plumula nelumbinis capsules orally (0.34 g/caplet, 6 caplets/day, 7 days), and another 11 volunteers ingested higenamine tablets (three 5 mg tablets/day for 7 days). Urine samples were collected over a period of 14 days. All urine samples were subjected to quantitative dilute-and-shoot analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analytical results showed that urinary higenamine concentrations exceeded the WADA reporting limit (10 ng/mL) during the drug period in most sample groups. The maximum higenamine concentration observed in the plumula nelumbinis capsule group was 500 ng/mL. Based on confidence interval theory, appropriate data were used to establish mathematical models. The models reflected that the higenamine concentration in urine can exceed the WADA reporting limit with a high probability after taking plumula nelumbinis capsules. In conclusion, oral administration of plumula nelumbinis capsules showed a high risk of an AAF due to higenamine.

Analysis of tretoquinol and its metabolites in human urine by liquid chromatography-tandem mass spectrometry.

Tretoquinol (trimetoquinol), a ß2-agonist, has been explicitly listed on the World Anti-Doping Agency Prohibited List 2019 since January 2019; however, it has been distributed as an antiasthmatic on the medical market. This study aimed to develop a liquid chromatography-tandem mass spectrometric method for the quantification of tretoquinol (free form plus glucuronide) in human urine for doping control purposes. An excretion study (n = 6) of tretoquinol hydrochloride hydrate (6 mg) was performed, and urine samples were collected prior to oral administration and during the first 48 h, along with spot urine samples at 7 and 14 days after administration. All the urine samples were analysed using the developed method. The limit of detection for the developed method was 0.03 ng/mL. The inter-day precision for the target analyte was excellent (2.7% to 9.2%), and the inter-day accuracy of target analyte was -0.6% to -3.6%. In all subjects, tretoquinol (free form plus glucuronide conjugate) was identified up to 48 h after administration. The maximum concentrations were in the range of 12.4-78.8 ng/mL and the mean concentration was 55.3 ng/mL. The metabolites O-methylated tretoquinol, tretoquinol sulphate and O-methylated tretoquinol sulphate could be also identified in human urine after administration. The longest-lasting urinary metabolite of tretoquinol currently known, O-methylated tretoquinol, is also likely to be a useful marker in doping controls.

Ultra-fast retroactive processing of liquid chromatography high-resolution full-scan Orbitrap mass spectrometry data in anti-doping screening of human urine.

RATIONALE: Retroactive analysis of previously tested urine samples has become an important sports anti-doping tool. Retroactive reprocessing of old data files acquired from a generic screening procedure can reveal detection of initially unknown substances, like illegal drugs and newly identified metabolites. METHODS: To be able to efficiently search through hundreds to thousands of liquid chromatography high-resolution full-scan Orbitrap mass spectrometry data files of anti-doping samples, a combination of MetAlign and HR_MS_Search software has been developed. MetAlign reduced the data size ca 100-fold making possible local storage of a massive volume of data. RESULTS: The newly developed HR_MS_Search module can search through the reduced data files for new compounds (mass or isotope pattern) defined by mass windows and retention time windows. A search for 33 analytes in 940 reduced data files lasted 10 s. The output of the automatic search was compared to the standard manual routine evaluation. The results of searching were evaluated in terms of false negatives and false positives. The newly banned b2-agonist higenamine and its metabolite coclaurine were successfully searched in reduced data files originating from a testing period for which these substances were not banned, as an example of retroactive analysis. CONCLUSIONS: The freeware MetAlign software and its automatic searching module HR_MS_Search facilitated the retroactive reprocessing of reduced full-scan high-resolution liquid chromatography/mass spectrometry screening data files and created a new tool in anti-doping laboratories' network.

Sensitive and Matrix-Tolerant Lateral Flow Immunoassay Based on Fluorescent Magnetic Nanobeads for the Detection of Clenbuterol in Swine Urine.

The lack of sensitivity and poor matrix tolerance are the main bottlenecks of the lateral flow immunoassay (LFIA). Here, a sensitive and matrix-tolerant method that integrated immunomagnetic separation and fluorescent lateral flow immunoassay (IMS-FLFIA) based on fluorescent magnetic nanobeads was developed to detect the clenbuterol (CLE) residue in swine urine. The limit of detection (LOD) of IMS-FLFIA is 4 times lower than that of traditional colloidal gold LFIA. This method, which exhibits similar LOD and linearity range in both phosphate-buffered saline and urine swine, is highly correlated with liquid chromatography-tandem mass spectrometry for the detection of real swine urine samples. The result indicated that IMS-FLFIA has a universal resistance to the swine urine matrix. The merits of this assay, high sensitivity, matrix tolerance, accuracy, and specificity, ensure a promising future in detection of veterinary drug residues.

Simultaneous determination of ß-agonists on hexagonal boron nitride nanosheets/multi-walled carbon nanotubes nanocomposite modified glassy carbon electrode.

ß-Agonists are illegally consumed in various products such as food and animal and effect the nutrition distribution owing to change of body fat. In addition, they result in acute poisoning and several symptoms such as muscular tremors and nervousness. A new electrochemical approach based on two-dimensional hexagonal boron nitride (2D-hBN) nanosheets decorated functionalized multi-walled carbon nanotubes (f-MWCNTs) was presented for simultaneous determination of ß-agonists such as phenylethanolamine A (PEA), clenbuterol (CLE), ractopamine (RAC) and salbutamol (SAL) in urine samples. X-ray diffraction (XRD) method, Raman spectroscopy, scanning electron microscope (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used characterizations of nanomaterials. After that, 2D-hBN/f-MWCNTs nanocomposite modified glassy carbon electrode (GCE) was prepared for simultaneous determination of ß-agonists. 1.0 × 10-12-1.0 × 10-8 M and 1.0 × 10-13 M were founded as the linearity range and the detection limit (LOD) for PEA, CLE, RAC and SAL. Finally, the prepared electrochemical sensor was used for urine sample analysis in presence of ascorbic acid (AA) and uric acid (UA).

The use of hair as a long-term indicator of low-dose ß2 agonist treatments in cattle: Implications for growth-promoting purposes monitoring.

The objective of this study was to assess the feasibility of using hair as a long-term indicator of cocktail (low-dose ß2 agonists) treatments in cattle. Six male Simmental cattle were treated with a mixture of low-dose clenbuterol, ractopamine, and salbutamol at dosages of 5.3, 223.3, and 50.0 µg/kg, respectively. The trial lasted for 112 days and included 28 days of treatment and 84 days of withdrawal. Plasma and urine samples taken during the treatment period contained the highest residues, with maximum concentrations of clenbuterol, ractopamine, and salbutamol in plasma of 1.49 ng/mL (Day 21), 43.78 (Day 14) ng/mL, and 8.07 ng/mL (Day 7), respectively, and in urine of 62.40 ng/mL (Day 28), 3995.77 ng/mL (Day 28), and 503.72 ng/mL (Day 1), respectively. On day 42 of withdrawal, the residues of all three ß2 agonists in plasma were below the limit of quantification (LOQ; 0.3 ng/mL for clenbuterol, and 0.5 ng/mL for ractopamine and salbutamol), and in urine samples were below or near the LOQ (the highest being ractopamine at 1.10 ng/mL). The highest concentrations of clenbuterol, ractopamine, and salbutamol in hair were 88.36, 1351.92, and 100.58 ng/g, respectively, on day 14 of withdrawal; and the residues were long-lasting, with 7.64, 28.55, and 8.77 ng/g, respectively, on day 84 of withdrawal. The results of this study demonstrate that hair could be utilized as a long-term indicator of the use of a combination of low-dose ß2 agonists in cattle, which could have implications for growth-promoting purposes monitoring.

Colorimetric detection of the ß-agonist ractopamine in animal feed, tissue and urine samples using gold-silver alloy nanoparticles modified with sulfanilic acid.

A highly sensitive, selective and simple method was proposed for colorimetric detection of ractopamine on the basis of the interaction between ractopamine and sulfanilic acid-modified gold-silver alloy nanoparticles (AuAgNPs). The AuAgNPs were prepared by the reduction of HAuCl4 and AgNO3 with sodium citrate in aqueous medium and further modified by sulfanilic acid. The interaction of ractopamine with sulfanilic acid induced rapid aggregation of sulfanilic acid-modified AuAgNPs along with an optical colour change, leading to precise quantification which could be detected by absorptiometry. Under the optimum conditions, the absorbance ratio (A600/A435) of sulfanilic acid-modified AuAgNPs exhibited a linear relationship with the concentration of ractopamine in the range of 4.5-31.6 ng/mL. The detection limit of ractopamine was 1.5 ng/mL. The established novel colorimetric detection method showed high selectivity towards ractopamine. The method was successfully applied to detect ractopamine in spiked pork, swine feed and swine urine samples with excellent recoveries from 94.4% to 112.5%. These results demonstrated that the proposed new method has a good potential for practical applications.

Multiclass screening in urine by comprehensive two-dimensional liquid chromatography time of flight mass spectrometry for residues of sulphonamides, beta-agonists and steroids.

Nowadays routine residue monitoring involves the analysis of many compounds from different classes, mainly in urine. In the past two decades, developments heavily focused on the use of mass spectrometers (MS) and faster and more sensitive MS detectors have reached the market. However, chromatographic separation (CS) was rather ignored and the cognate developments in CS were not in line. As a result, residue analysis did not improve to the extent anticipated. CS by LC x LC is a promising technique and will enable a further increase in the range of compounds and compound classes that can be detected in a single run. In the present study, a self-built LC x LC system, using a 10 port valve, was connected to a single quadrupole MS with electrospray interface. Standards containing a mixture of sulphonamides, ß-agonists and (steroid) hormones, 53 compounds, in total, were analysed. Results demonstrated that these compounds were well separated and could be detected at low levels in urine, i.e. limit of detection (LOD) from 1 µg L-1 for most ß-agonists to 10 µg L-1 for some sulphonamides and most hormones. To enhance the sensitivity, optimisation was performed on an advanced commercial LC x LC system connected to a full scan accurate MS. This ultimately resulted in a fast high throughput untargeted method, including a simple sample clean-up in a 96-well format, for the analysis of urine samples.

Analysis for higenamine in urine by means of ultra-high-performance liquid chromatography-tandem mass spectrometry: Interpretation of results.

Higenamine (Norcoclaurine) is a very popular substance in Chinese medicine and is present in many plants. The substance may be also found in supplements or nutrients, consumption of which may result in violation of anti-doping rules. Higenamine is prohibited in sport at all times and included in Class S3 (ß-2-agonists) of the World Anti-Doping Agency (WADA) 2017 Prohibited List. The presence of higenamine in urine samples at concentrations greater than or equal to 10 ng/mL constitutes an adverse analytical finding (AAF). This work presents a new metabolite of higenamine in urine sample which was identified by means of ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Samples were prepared according to 2 protocols - a Dilute and Shoot (DaS) approach and a method involving acid hydrolysis and double liquid-liquid extraction (LLE). To meet the requirements typical for a confirmatory analysis, the screening procedure was further developed. In samples prepared by the DaS method, 2 peaks were observed; the earlier one was specific for higenamine and the later one unknown. MS scan analysis showed mass about 80 Da higher than that of higenamine. In turn, in samples prepared in accordance with the protocol involving hydrolysis, an increase in the area under peak for higenamine was observed, while the second peak was absent. It seems that the described strategy of detection of higenamine in urine avoids false negative results.

Magnetic graphene dispersive solid phase extraction-ultra performance liquid chromatography tandem mass spectrometry for determination of ß-agonists in urine.

In this study, a magnetic graphene-based dispersive solid phase extraction method was first developed for extraction of ß-agonists in urine. During the experiments, the absorbent amount, sample pH, extraction time, elution solution and elution time were optimized respectively. The optimized extraction method was finished within 10min, and showed high enrichment factors for 9 ß-agonists (20-26 folds). Furthermore, this absorbent could be reused for at least 60 times. Then this extraction method was combined with ultra performance liquid chromatography triple quadrupole tandem mass spectrometry to determine the 9 drugs in urine. The limits of detection for the 9 drugs were in a range of 0.015-0.023ngmL-1, and the recoveries from the standards fortified blank urine were in a range of 60.2%-109.4%. Therefore, this method could be used as a simple, rapid, sensitive and accurate tool to determine trace level of ß-agonists in urine.

Pseudo template molecularly imprinted polymer for determination of 14 kind of ß-agonists in animal urine by ultra-high-performance liquid chromatography-tandem mass spectrometry.

A pseudo template molecularly imprinted polymer (MIP) for selective extraction of ß-agonists was prepared using 4-hydroxyphenethyl alcohol as pseudo template molecule, methyl acrylic acid as functional monomer, and ethylene glycol dimethacrylate as cross-linker. Selectivity and adsorption capacity of prepared MIP were studied in detail. MIP selectively adsorbed 14 types of ß-agonists. Advantages of the prepared polymer include non-template leakage in purification of target analytes and cheap cost. MIP was used to purify ß-agonists in urine samples. The developed method was combined with ultra-performance liquid chromatography-tandem mass spectrometry. In optimum conditions, recoveries of the developed method ranged from 84.2% to 109.8% with low coefficients of variation at below 15%. Limits of detection and quantification for 14 target ß-agonists in urine samples reached below 0.05ng/mL and 0.1ng/mL, respectively. The developed method based on MIP enrichment and purification were successfully applied to analysis of target analytes in 50 actual urine samples.

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.

Preparation of selective magnetic dispersive solid-phase sorbent and its application for recognition clenbuterol from bovine urine.

A new kind of selective magnetic dispersive solid-phase sorbent based on multiple Fe3O4 nanospheres as the core structure and molecular imprinted material as the shell structure was synthesized with tert-butylamine and 2-chloroaniline as the templates. The obtained multicore-shell-structured sorbent was spherical (diameter distribution 25-90µm) with porous morphologies, thus incorporating strong magnetic properties and specific molecular recognition coupled with rapid adsorption and dynamic equilibrium. The sorbent was applied for rapid and selective screening of clenbuterol (CLB) in bovine urine samples. Good linearity was obtained in the range 1.25-200ngmL(-1) with the average recovery at three spiked levels ranging from 91.4% to 105.3%. The proposed method significantly improved the purification and extraction efficiency of CLB in urine samples and eliminated the effect of template leakage during quantitative analysis.

Detection of residues in urine and tissues of sheep treated with trace levels of dietary ractopamine HCl.

Qualitative assays are sometimes used as the sole basis for detecting drug residues in live animals or in animal products. Such assays have become increasingly sensitive as detection technologies have improved, yet the limitations of such assays to discriminate purposeful and accidental drug exposures remain poorly defined. A study was conducted to determine the ability of a ractopamine lateral flow assay to accurately detect incurred ractopamine residues in contaminated feeds and in sheep fed trace quantities of ractopamine HCl. False positive and negative samples were determined using a quantitative liquid chromatography-tandem mass spectrometric (LC-MS/MS) method. Ractopamine HCl was fed to sheep at 0 (Zero), 1 (Low), 10 (Med), or 100 (High) µg/kg of diet ( = 4 per level, 0.5 kg of feed/d) for 7 consecutive d and urine was collected daily about ∼16 h post exposure. On-site lateral flow assays were able to reliably (0% false negatives) detect 20 µg of ractopamine HCl per kg of feed. Urine from treated sheep tested positive for ractopamine residues by lateral flow assay in 7.4 (Zero), 0 (Low), 82 (Med), and 86% (High) of the urine samples from each group. Parent ractopamine was below the assay limit of quantification (LOQ, 0.7 ng/mL) in all urine samples using LC-MS/MS. After hydrolysis of ractopamine conjugates, total ractopamine (parent + hydrolyzed metabolites) in urine of Low animals was always less than the LOQ, but in 7 of 28 samples were above the limit of detection (LOD, 0.22 ng/mL). In contrast, urine in Med animals contained 1.08 to 9.13 ng/mL of total ractopamine, while urine of High animals contained 4.85-32.82 ng/mL of total ractopamine. Ractopamine is rapidly eliminated; nevertheless, > 80% of urine samples from sheep exposed to 5 µg/d (M) of ractopamine HCl had detectable residues by the screening assay and a 100% of samples had measurable ractopamine using LC-MS/MS methods. Tissue residues of ractopamine were not detected in any of the sheep. The sensitivity with which the rapid, qualitative assay detected ractopamine was sufficient to reveal trace ractopamine exposures; these data suggest that the use of qualitative tests to indicate purposeful treatment of animals (i.e., for doping or growth enhancement), in the absence of collaborating quantitative data, is inappropriate.