Article pubs.acs.org/JAFC
Ultratrace LC-MS/MS Analysis of Segmented Calf Hair for Retrospective Assessment of Time of Clenbuterol Administration in Agriforensics Wilco F. Duvivier,† Teris A. van Beek,† Thijs Meijer,‡ Ruth J. P. Peeters,† Maria J. Groot,‡ Saskia S. Sterk,‡ and Michel W. F. Nielen*,†,‡ †
Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands RIKILT Wageningen UR, P.O. Box 230, 6700 AE Wageningen, The Netherlands
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ABSTRACT: In agriforensics, time of administration is often debated when illegal drug residues, such as clenbuterol, are found in frequently traded cattle. In this proof-of-concept work, the feasibility of obtaining retrospective timeline information from segmented calf tail hair analyses has been studied. First, an ultraperformance liquid chromatography−tandem mass spectrometry (UPLC-MS/MS) hair analysis method was adapted to accommodate smaller sample sizes and in-house validated. Then, longitudinal 1 cm segments of calf tail hair were analyzed to obtain clenbuterol concentration profiles. The profiles found were in good agreement with calculated, theoretical positions of the clenbuterol residues along the hair. Following assessment of the average growth rate of calf tail hair, time of clenbuterol administration could be retrospectively determined from segmented hair analysis data. The data from the initial animal treatment study (n = 2) suggest that time of treatment can be retrospectively estimated with an error of 3−17 days. KEYWORDS: agriforensics, hair, β-agonist, mass spectrometry, liquid chromatography, veterinary control
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pigmentation.25 The prolonged detectability of drugs in hair versus blood or urine makes it a powerful option for forensic investigations.26,27 Because the average hair growth rate of human head hair is known, analysis of longitudinal segments can be applied to achieve a retrospective timeline of drug intake.28,29 Longitudinal hair analysis enables correlation of, for example, drug-facilitated crime with the time of drug administration.28 Typically, 1−3 cm longitudinal hair segments are used for drug analysis, corresponding to multiple months of hair growth.30,31 Over the past 10 years, several attempts have been made to apply segmented hair analysis to animals, but primarily for horses.32−40 Dunnett et al. published several papers describing segmented analysis of different drugs in horse hair, mainly focused on antibiotic residues. The distributions among tail hairs were consistent with the time of drug administration, although the used segment sizes (2.5−5 cm) were somewhat large compared to human forensic applications.32−35 Similar approaches on horse hair have been reported by Anielski et al.36 and Gray et al.39 for the detection of anabolic steroids and related compounds. Furthermore, segmented hair analysis has also been used to retrospectively determine stress factors in the hair of bears38 and orangutans.40 The smallest segment length used in previous animal studies is 2 cm,32−36,38−40 which does not allow a very accurate estimation of time of drug administration. Until now, segmented hair analysis has not been applied to bovine (calf) hair or to the illegal use of β-agonists such as clenbuterol.
INTRODUCTION Clenbuterol is a β-agonist approved for therapeutic use in some countries. However, it can also be used illegally, as a growth promoter for cattle. It increases the muscle-to-fat ratio and stimulates overall animal growth.1−3 Clenbuterol-contaminated meat can cause food intoxication with severe consequences such as heart disorders,4,5 and it has therefore been banned for use as a growth promoter in cattle in the European Union since 1988.6 Many different methods for the analysis of clenbuterol and other β-agonist residues in different matrices have been published over the years.1,2,7−24 Among these methods are approaches based on immunoassays,7 receptor assays,24 or biosensors,1,8,9,21 but these methods lack either sensitivity or selectivity. To overcome these problems, gas chromatography− mass spectrometry (MS) and liquid chromatography (LC)− tandem MS (MS/MS) have been used to analyze a large range of β-agonists.2,10−16,18−20,22,23 One of the difficulties that occurs when residues of a banned substance such as clenbuterol are found in animals, however, is the fact that animals are frequently traded. The owner can thus claim that a previous owner was responsible for the illegal administration of the detected substances. Current analysis methods are not sensitive enough to be used for estimated time of drug administration. To achieve this, human hair forensic techniques may be equally applicable in veterinary control and agriforensics. Hair is a very interesting sample matrix because many compounds accumulate in hair via the bloodstream. Hair pigmentation can have an influence on the incorporation of compounds such as clenbuterol into the hair. Gleixner et al. found that more clenbuterol was accumulated into black hair compared to hair with less pigmentation; however, clenbuterol was detectable in hair samples regardless of the level of © 2014 American Chemical Society
Received: Revised: Accepted: Published: 493
September 5, 2014 December 19, 2014 December 24, 2014 December 24, 2014 DOI: 10.1021/jf5056437 J. Agric. Food Chem. 2015, 63, 493−499
Article
Journal of Agricultural and Food Chemistry
analysis, hair samples were divided into 1 cm segments, starting from the proximal end. Assessment of Hair Growth Rate. The average growth rate of calf tail hair was determined both for continued growth of existing hairs (length at start = 4.1 ± 0.5 cm), as well as for newly grown hair. Six female calves (black-and-white Holstein Friesian) were used, 1−3 weeks old (around 50 kg) at the start of the study. Continued hair growth was monitored by measuring the hair length of three locks of clearly marked tail hair during 8 weeks. To determine the average growth rate of newly grown hair, three other locks of tail hair from the same calves were cut as close to the skin as possible at the start of the study. The length of newly grown hair was also monitored during 8 weeks (one data point per week). From these results, average tail hair growth rates were determined per calf, which were then used to calculate the overall growth rate and standard deviation for the six calves. Finally, the average of the data from the first 26 days was used for all calculations regarding the theoretical position of the clenbuterol residues (see Results and Discussion). Sample Preparation. The sample preparation of the hair samples was adopted from that of Nielen et al.18 and adjusted to much smaller sample sizes. In short, 25 mg of hair were cut into small pieces (
