Development and validation of a multi-residue liquid chromatography–tandem mass spectrometry confirmatory method for eleven coccidiostats in eggs
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.
Section snippets
Chemicals and reagents
Acetonitrile and methanol UpS (Ultra Purity solvent) were supplied by Romil Pure Chemistry (Cambridge, Great Britain). Absolute ethanol, dimethylsulfoxide, n-hexane and ammonia were of HPLC or analytical grade and purchased from Carlo Erba (Milan, Italy). Formic acid 50% was purchased from Fluka (Buchs, Switzerland). Sodium acetate trihydrate and sodium sulphate anhydrous were purchased from Carlo Erba (Milan, Italy). Demineralised water was HPLC grade generated by a Milli-Q purification system
Method development
The simultaneous determination of the eleven coccidiostats is a challenging task due to their very different chemical structures and properties. This also prevents extensive sample purification and frequently the sample treatment is reduced to a simple egg extraction with acetonitrile [17], [18], [28]. However, in spite of the selectivity of LC–MS/MS systems, the ionization efficiency of the samples in the ESI source may be affected by matrix interferences, giving signal suppression or
Conclusions
The reported LC–MS/MS method was found very sensitive and accurate for the screening and the confirmation of the eleven regulated coccidiostats in eggs starting from 1 μg kg−1. Only for halofuginone the quantitative analysis can be carried out starting from 2.5 μg kg−1. In addition the adopted validation approach tries to combine the necessary pragmatism of a EU routine laboratory with rigorous methodology, preventing useless method revalidation. The satisfactory Z-scores obtained in the FAPAS
Acknowledgements
This work was supported by the Italian Health Ministry (Ricerca Corrente IZS UM RC0032009). We thank Gabriella Griffi for the English text revision.
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