Detection of residues in urine and tissues of sheep treated with trace levels of dietary ractopamine HCl1,2

Abstract

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.

Assessment of the withdrawal period for ractopamine hydrochloride in the goat and sheep

“…To produce an accurate, simple assay with high recoveries, sample components were separated on two adsorption columns: an HPLC column and SPE column (17,18). The recovery of ractopamine from plasma was 88-99% (19,20), consistent with reported recoveries of the half-life in these two species (21,22). and supporting suggestions that it is more extensively distributed in goat tissues than in sheep tissues (23,24).…”

Detection and quantification of residues in sheep exposed to trace levels of dietary zilpaterol HCl

“…On adaptation days 6 and 7 head gates were closed at approximately 0900 h and sheep were held until micturition occurred (typically less than 1.5 h). Urine was collected into 400 mL polypropylene beakers as described by Smith et al (2016).…”

“…Wethers were provided ad libitum access to grass/ alfalfa hay, and water and zilpaterol treatments were provided in 1.75 kg allotments of test feed at approximately 0730 h daily (Study days 0 to11) for 12 consecutive days. Feeding and sample collection was essentially identical to that described by Smith et al (2016) with the exception that pre-dose samples were collected from each sheep prior to the initiation of zilpaterol HCl exposures. The first post-dosing urine sample was collected on Study Day 0 within an hour or two of zilpaterol exposure.…”

“…Thus, the sensitivity of a given assay, not the nature of an exposure event (purposeful or accidental), may determine whether a given sample contains violative residues. To illustrate, Smith et al (2016) demonstrated that sheep exposed to ultra-low levels of dietary ractopamine (10 ng per kg body weight (bw) per day; 0.5 µg per sheep per day) excreted sporadically detectable urinary…”

“…To date, few studies have correlated actual animal exposure levels with the practical detection of residues using routinely tested matrices and commonly used screening or determinative analytical methods. For β-agonist compounds, minimum doses (see discussions by Smith 1998;Smith et al 2016) are required to elicit clinical or growth promoting effects, so there is no performance or economic incentive to expose animals below such doses. Analytical methods used to enforce policies against therapeutic or growth effects are typically sensitive enough to detect animal exposures well below doses labelled for production purposes.…”