No abstract

The use of nicotine stimulants in horses is generally banned in horse racing and equestrian sports-accidental consumption of tobacco products is one of the possible causes of nicotine exposure in horses. The authors recently reported a comprehensive metabolic study of nicotine in equines, differentiating between nicotine exposure and sample contamination by means of a nicotine biomarker trans-3 0hydroxycotinine. To identify potential biomarkers for the differentiation of genuine nicotine administration and consumption of tobacco products, tobacco leaves (equivalent to 250 mg of nicotine) were nasoesophageally administered to three thoroughbred mares. Quantification methods of anatabine in plasma and urine were newly developed and validated and successfully applied to postadministration samples. Previously reported simultaneous quantification methods of eight target analytes including nicotine and its metabolites in plasma and urine were also applied to the samples.The results demonstrate that both trans-3 0 -hydroxycotinine and anatabine could be used as potential biomarkers in equine urine and plasma to indicate recent exposure to tobacco products in horses. As well, trans-3 0 -hydroxycotinine had the longest halflife as a detectable metabolite in urine and plasma. To our knowledge, this is the first report of a comprehensive study of tobacco product detection in horses.

Rationale For the purpose of doping control, this is the first report of accurate quantification of four critical structural isomers of nicotine metabolites (trans‐3′‐hydroxycotinine, cis‐3′‐hydroxycotinine, 5′‐hydroxycotinine, and N′‐hydroxymethylnorcotinine) in equine plasma and urine for the establishment of their elimination profiles. Besides, the pharmacokinetic studies of trans‐3′‐hydroxycotinine and N′‐hydroxymethylnorcotinine in equine plasma and urine are also presented for the first time. Methods The accurate quantification methods of the aforementioned four structural isomers in horse plasma and urine were successfully developed and validated using the solid‐phase extractions followed by liquid chromatography/tandem mass spectrometry analysis. Baseline chromatographic separation was achieved to completely differentiate these isomers, which shared the same selected reaction monitoring transition. Such methods were applied to post‐administration samples obtained from the nicotine and tobacco leaf administration studies for the establishment of pharmacokinetic profiles. Results N′‐Hydroxymethylnorcotinine could be quantified for the longest period, ranging from 48 to 72 h in plasma and 96 h in urine after a single administration of 250 mg of nicotine and an equivalent amount of nicotine in tobacco leaves. In terms of detection, both N′‐hydroxymethylnorcotinine and trans‐3′‐hydroxycotinine could be detected up to the last sample collection time point (96 h), indicating that they are the most appropriate biomarkers for nicotine exposure. Conclusions N′‐Hydroxymethylnorcotinine and trans‐3′‐hydroxycotinine were detected longest in plasma and urine samples after both nicotine and tobacco leaf administrations, and N′‐hydroxymethylnorcotinine was deemed most appropriate as a monitoring target due to its relatively higher abundance and slower elimination rate. These two biomarkers could also be used to differentiate sample contamination by tobacco products and genuine nicotine exposure to horse regardless of intentionality.

Procaterol (PCR) is a beta-2-adrenergic bronchodilator widely used in Japanese racehorses for treating lower respiratory disease. The pharmacokinetics of PCR following single intravenous (0.5 μg/kg) and oral (2.0 μg/kg) administrations were investigated in six thoroughbred horses. Plasma and urine concentrations of PCR were measured using liquid chromatography-mass spectrometry. Plasma PCR concentration following intravenous administration showed a biphasic elimination pattern. The systemic clearance was 0.47 ± 0.16 L/h/kg, the steady-state volume of the distribution was 1.21 ± 0.23 L/kg, and the elimination half-life was 2.85 ± 1.35 h. Heart rate rapidly increased after intravenous administration and gradually decreased thereafter. A strong correlation between heart rate and plasma concentration of PCR was observed. Plasma concentrations of PCR after oral administration were not quantifiable in all horses. Urine concentrations of PCR following intravenous and oral administrations were quantified in all horses until 32 h after administration. Urine PCR concentrations were not significantly different on and after 24 h between intravenous and oral administrations. These results suggest that the bioavailability of orally administrated PCR in horses is very poor, and the drug was eliminated from the body slowly based on urinary concentrations. This report is the first study to demonstrate the pharmacokinetic character of PCR in thoroughbred horses.