Analysis of β2-agonists in cattle hair samples using a rapid UHPLC–ESI–MS/MS method

Abstract A simple and efficient method was developed for simultaneous analysis of five illegal residual β2-agonists in cattle hair. β2-Agonists were quantified by ultra high performance liquid chromatography coupled with electrospray ionisation tandem mass spectrometry operating in positive multiple-reaction monitoring mode. The method was validated as quantitative confirmatory method according to the EU Decision 2002/657/EC: instrumental linearity, specificity, precision, recovery, decision limit (CCα) and detection capability (CCβ) were evaluated. The recovery were greater than 90% and the method appeared suitable for the control of these β2-agonists in cattle hair samples with LOQ values between 4.9 and 5.5 μg/kg. This method could represent a simple and cheap approach to confirm β2-agonists contamination of cattle for feeding in a not invasive way and before slaughter operations.


Introduction
Beta 2 -adrenergic agonists, or β 2 -agonists, are molecules related to catecholamines, the oldest and most commonly prescribed therapeutic agents for asthma. These compounds are phenyl β-ethanolamines with different substituents on the aromatic ring and on the terminal amino group. These molecules have a powerful pharmacological activity and in human and veterinary medicine are used as tocolytic and bronchodilator agents in chronic obstructive pulmonary disease and numerous other respiratory diseases (Tashkin & Fabbri 2010). However, a possible problem related to β 2 -agonists is their illegal use at higher doses (10-100 times the clinically active dose) as growth-promoting agents in cattle, due to pharmacological lipolytic and protein synthesis stimulating side effects (Mazzanti et al. 2003;Estrada-Montoya et al. 2008;Reig & Toldrá 2008). The prohibition of the use of growth promoters, including β 2 -agonists, is laid down in two Council Directives (Council Directive 96/22/EC; European Commission 96/23/EC) containing guidelines for control of veterinary drug residues in animals and their products with all the necessary information to set up national monitoring plans. In addition, Commission Decision 2002/657/EC (European Commission 2002/657/EC) describes performance criteria for the analytical residue methods to be used in testing and official samples. Hair represents a particularly suited matrix to detect the illegal use of veterinary drugs (Hernández-Carrasquilla 2000;Gratacós-Cubarsí et al. 2006). Over the years, many analytical methods to detect β 2 -agonists in several tissues have been developed including accurate quantification and confirmation that require the sensitivity and specificity of mass spectrometry coupled with chromatographic procedure (Marcos et al. 2004;Salquebre et al. 2007;Juan et al. 2010;Dusi et al. 2011;Leporati et al. 2014). UHPLC methods with a fast-switching triple-quadrupole MS/MS instrument and using multiple reaction monitoring (MRM) transitions in real sample matrices demonstrate that a new versatile tool has been achieved for veterinary control of β 2 -agonists (Nielen et al. 2008). For monitoring purposes plasma and urine may be used in live animals, but only for low-level contaminations characterised by short withdrawal periods.
Earlier studies have drawn attention on the differences in accumulation and persistence patterns of certain β 2 -adrenergic agonists in different tissues and have shown that in farm animals β-agonists are typically eliminated from body fluids and tissues in the following order: plasma < urine < liver < lungs < muscle tissue < hair < eyes (Pleadin et al. 2014).
In the present study, we developed an ultra high performance liquid chromatography method coupled with electrospray tandem mass spectrometry (UHPLC-ESI-MS/MS) in MRM mode, to determine quickly the presence of the five most used β 2 -agonists in veterinary field (mapenterol, mabuterol, clenpenterol, clenbuterol and brombuterol) in bovine hair and to monitoring their illegal use in livestock production (Figure 1).

Results and discussion
The UHPLC-ESI-MS/MS method was developed according to the performance criteria for mass spectrometric detection suggested in Commission Decision 20002/657/EC. Due to its high sensitivity and specificity, the analysis was performed in MRM mode. For β-agonists included in group A of Annex, Council Directive 96/23/EC, a minimum of four identification points are required. In this experiment, four identification points were obtained by monitoring one parent ion (1 point) and two transitions (1.5 points). The selected transitions for the β-agonists and the optimal MS-MS conditions are reported in Table 1.
Many tests of sample digestion of cattle hair samples and extraction have been previously carried out considering four variables: the concentration of the solution of NaOH (0.5, 1, 1.5 N), time (4 h, 8 h, overnight) and temperature of incubation (25, 45, 70 °C) and at last the solvent used for the extraction (hexane/dichloromethane 1:1, 3:7, 7:3 v/v). During the optimisation of UHPLC-ESI-MS/MS method, ion suppression phenomenon was also taken into account and studied, since difficulties for quantitative analysis are usually present. Internal standard method using labelled standard has been applied to compensate signal irreproducibility, matrix interference and loss of recovery. For this purpose, internal standard deuterated with 11 deuteriums MPT-d 11 was used as ISTD for calibration curves of MPT, MBT, CPT, CBT and BBT.
The method was validated as a quantitative confirmatory method according to the EU Decision 2002/657/EC: instrumental linearity, specificity, precision, recovery, decision limit (CCα) and detection capability (CCβ) were evaluated for all the analytes. This method is suitable for quantification and confirmation of illegal residues in living animals to prevent foodstuff contamination.

Conclusion
A robust UHPLC-MS/MS residue analytical method has been developed and validated for five β 2 -agonists in cattle hair samples. This method not require any SPE step and allows the confirmatory (quantitative) determination of five beta agonists, reducing costs and increasing efficiency, according to the EU Decision 2002/657/EC. This method could be useful for a fast and simple preliminary screening of many living animals intended for human consumption before slaughter operations, thanks to its very easy matrix collection, simple and cheap extraction procedure, fast LC-MS/MS analyses useful to survey living animals and to have long-term information of the exposure to these substances. The cattle hair is considered an important tissue in veterinary control, because the active drug compounds are incorporated in a strong and rigid hair structure and could be retained for a longer time respect to other matrices as urine or blood.