Development and validation of a multi-residue liquid chromatography–tandem mass spectrometry confirmatory method for eleven coccidiostats in eggs

Abstract

A confirmatory method for the determination of residues of eleven coccidiostats including ionophore antibiotics: lasalocid, maduramycin, monensin, narasin, salinomycin, semduramycin and chemical coccidiostats: decoquinate, diclazuril, halofuginone, nicarbazin and robenidine in poultry eggs was developed and validated. The sample was extracted with acetonitrile, defatted with hexane and cleaned-up on a silica SPE cartridge. The analytes were identified and quantified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). The method performance characteristics required by Commission Decision 2002/657/EC were estimated adopting a more flexible and simple validation design. In this alternative approach the experimental study focuses on a larger dynamic range with progressively increasing validation levels. Each of them presents equal concentrations of all the analytes. On the contrary the classical Decision plan investigates a restricted concentration range necessarily different for each analyte being determined by the individual permitted limit (0.5–1.5 times the permitted limit). As a consequence each validation level involves the simultaneous fortification with complex mixtures containing different concentrations of each analyte. Adopting this alternative strategy the validation of several substances with significantly different permitted limits is considerably simplified and a deeper knowledge of the method is achieved. The results proved that the method enables the confirmation of regulated coccidiostats in eggs at the levels required in the official control of residues (CCα in the range of 2.2–174 μg kg⁻¹, depending on the coccidiostat). The repeatability (CV_r in the range of 1.1–19%) and within-laboratory reproducibility (CV_Rw in the range of 1.8–30%) are also acceptable. The procedure was successfully verified in the proficiency test and implemented in the national residue control plan.

Introduction

Coccidiostats are feed additives widely used for the prevention of coccidiosis, a severe poultry infection caused by genus Eimeria. Particularly in warm, humid environments it causes intestinal lesions, which result in diarrhea and related animal health problems. In its acute form, coccidiosis causes high mortalities. In its sub-acute form, small numbers of oocysts can cause poor weight gain, poor feed conversion and poor egg production. Therefore, especially in large intensive farms, much attention is paid to the prevention and the treatment of this disease. Practically all poultry farms have resorted to feeding coccidiostats as feed additives to pullets and broiler breeders for 12–16 weeks and to broiler chickens for almost their entire life [1], [2], [3].

The occurrence of unavoidable carry-over of feed additives in non-target feed may result in the presence of residues of coccidiostats in animal products like meat and eggs. Recently the European Commission has set maximum levels (MLs) for eleven coccidiostats in various foods from non target animals [4]. The regulated drugs are six ionophore antibiotics (lasalocid (LAS), maduramycin (MAD), monensin (MON), narasin (NAR), salinomycin (SAL), semduramycin (SMD)) and five chemical coccidiostats (decoquinate (DEC), diclazuril (DIC), halofuginone (HFG), nicarbazin (DNC) and robenidine (ROB)). Only for lasalocid and monensin Maximum Residue Limits (MRLs) were established respectively in poultry and bovine matrices [5]. On the whole, the permitted limits (PLs) for the eleven regulated anticoccidials in eggs are: 20 μg kg⁻¹ (DEC), 2 μg kg⁻¹ (DIC), 6 μg kg⁻¹ (HFG), 150 μg kg⁻¹ (LAS), 2 μg kg⁻¹ (MAD), 150 μg kg⁻¹ (MON), 2 μg kg⁻¹ (NAR), 100 μg kg⁻¹ (DNC), 3 μg kg⁻¹ (SAL), 2 μg kg⁻¹ (SMD) and 25 μg kg⁻¹ (ROB).

The availability of analytical methods suitable for the simultaneous determination of these molecules is a very important target for laboratories involved in official controls. However the concurrent analysis of ionophore antibiotics and chemical coccidiostats poses serious difficulties since coccidiostats, despite their common pharmacological effects, differ greatly in their chemical structures and properties. Several analytical approaches have been used to determine one or more coccidiostats in different biological matrixes. As screening methods, immunoassay techniques are the most popular and highly sensitive [2], [6], [7], [8], [9], however they can normally detect only one, or occasionally two coccidiostats. Actually, just considering their very different chemical properties, also high-performance liquid chromatography (HPLC) when coupled to conventional detection (UV or fluorescence) fails in the simultaneous determination of several of these drugs [10], [11], [12], [13], [14], [15]. One of the difficulties concerns the ionophore group because, except lasalocid, these compounds do not have suitable UV or fluorescent chromophores and therefore a derivation step is necessary. The coupling of high-performance liquid chromatography with a detector like triple quadrupole mass spectrometer (LC–MS/MS) has only recently led to the development of satisfactory multiresidue methods [13], [14], [15], [16], [17], [18] involving up to fourteen coccidiostats in foods and animal feeds [19], [20], [21], [22]. LC–MS/MS provides better specificity and sensitivity compared to HPLC with conventional detection or LC–MS. In addition a significant reduction in sample preparation and chromatographic development is achievable. By monitoring two parent-daughter transitions, LC–MS/MS is also specific enough to confirm the presence of presumptive positive residues as required by the EC Legislation.

Besides these analytical aspects, an additional problem is represented by the validation strategy. Commission Decision 2002/657/EC (CD 2002/657/EC) indicates not only the performance criteria for analytical methods used in official drug residue controls [23], but also the validation experimental plan. As stated in the CD, during method validation the spiking experiments for permitted substances must be carried out around their permitted level (0.5, 1 and 1.5 times the PL). Since the PLs fixed in eggs both in Commission Regulation 124/2009 and in Commission Regulation 37/2010 are different, this should involve the preparation of complex calibrated solutions enabling suitable fortification levels around the PL of each analyte. Furthermore some drugs (lasalocid, nicarbazin and robenidine) present PLs one or two orders of magnitude greater than the others. Due to the lack of a wide enough linearity range for LC–MS/MS technique, the implementation of simultaneous experiments comprehensive for all eleven coccidiostats is quite impossible. Hence the classical validation scheme as reported in the CD cannot be easily implemented for this multiresidue coccidiostat procedure. For these reasons, the alternative strategy suggested by Kaufmann et al., based on a large validation dynamic range, was applied [24], [25]. The experiments were carried out spiking all the analytes at the same concentration with progressive increasing levels covering a wide interval (1, 3.2, 10, 32, 100 and 320 μg kg⁻¹). Furthermore the more extended validation study provides extensive knowledge of the method and greater flexibility, permitting the re-evaluation of fundamental performance characteristics, especially CCα, also when a legislative limit was changed.