Horse hair analysis has proved to be a useful tool in testing for doping agents alternatively to urine due to its long window of detection. The aim of this study was to develop and validate an LC-MS/MS method for the detection and quantification of 13 frequently prescribed veterinary drugs. The method should be applied to 12 mane and tail hair samples from seven horses. Based on a hair growth rate of about 2cm per month time of drug administration should be compared with the drug concentration found in the corresponding hair segment.

Mane and/or tail hair were obtained from horses having received medical treatment. Time of drug administration was reported by the horse holders. Hair samples were washed with water and acetone, dried and cut in 2 or 4-cm-segments depending on total hair length (16cm to 70cm). Segments were cut into snippets and extracted for four hours with methanol under sonication. The extracts were analyzed by LC-ESI-MS/MS using a Dionex Ulti- Mate 3000 HPLC system and an Applied Biosystems 5500 QTrap linear ion trap triple quadrupole mass spectrometer operated in multiple reaction monitoring mode. Analytes were separated on a Kinetex C18-column (2,6μm, 50mm x 2,1mm). The method was validated for the detection and quantification of tramadol, butorphanol, morphin, buprenorphine, acepromazine, chlorpromazine, fluphenazine, detomidine, flunixin, phenylbutazone, firocoxib, meloxicam and ketoprofen according to international guidelines.

All analytes except fluphenazine and acepromazine fulfilled the main validation criteria. Two and 4-cm segments allowed a monitoring of one to two months period of drug administration. In fair mane and tail hair samples of horses no drugs could be detected. Concentrations in dark hair samples ranged up to 39pg/mg for flunixin, 1.8pg/mg for detomidine, 4pg/mg for butorphanol, 1.1pg/mg for tramadol, 1.4pg/mg for morphine, 1.2pg/mg for acepromazine, 1050pg/mg for phenylbutazone and below the limit of quantification for meloxicam. In some cases, drugs were detected in the hair segment corresponding to the time of medical treatment. Drugs could be also detected in the vicinal segments. This drug distribution patterns suggest transport of drugs along the hair shaft.

The study presents a sensitive method for the validated quantification of frequently prescribed veterinary drugs in horse hair. Hair analysis for these drugs could be used to test for medical treatment of horses to obtain indication of the horse's fitness, e.g. before purchase.