Positive and negative electrospray LC–MS–MS methods for quantitation of the antiparasitic endectocide drugs, abamectin, doramectin, emamectin, eprinomectin, ivermectin, moxidectin and selamectin in milk

Abstract

Avermectin endectocides are used for the treatment of cattle against a variety of nematode and arthropod parasites, and consequently may appear in milk after normal or off-label use. The compounds abamectin, doramectin, and ivermectin, contain only C, H and O and may be expected to be detected by LC–MS in negative ion mode. The others contain nitrogen in addition and would be expected to be preferentially ionized in positive mode. The use of positive ion and negative ion methods with electrospray LC–MS–MS were compared. Using negative ion the compounds abamectin, doramectin, ivermectin, emamectin, eprinomectin, and moxidectin gave a curvilinear response and were quantified in raw milk by LC–MS–MS with a triethylamine–acetonitrile buffer over the concentration range 1–60 ppb (μg/kg) using selamectin as the internal standard. The limits of detection (LOD) were between 0.19 ppb (doramectin) and 0.38 ppb (emamectin). The compounds gave maximum sensitivity with positive ionisation from a formic acid–ammonium formate–acetonitrile buffer and were detected in milk (LC–MS–MS) also with a curvilinear response over the range 0.5–60 ppb. Although the positive ion signals were larger, with somewhat lower limits of detection (LOD between 0.06 ppb (doramectin) and 0.32 ppb (moxidectin) the negative ion procedure gave a more linear response and more consistent results. Comparison of spiked samples in the range 2–50 ppb showed a high degree of correlation between the two methods.

Introduction

Macrolide endectocides, Fig. 1, are active against a wide variety of mammalian internal and external (endo- and ecto-) parasites such as nematodes, heart, lung, round, and intestinal worms, arthropods, mites, lice, flees, ticks etc [1]. They have very high potencies and consequently are convenient for treatment of agricultural animals. These macrocyclic lactone compounds consist of two subgroups: the avermectins, which include abamectin, doramectin, ivermectin, emamectin, eprinomectin and selamectin, and which have saccharide substituents at position C13, and the milbemycins (also called nemodectins), of which moxidectin is the representative here, and which do not have saccharide substituents. Avermectins were originally isolated from cultures of Streptomyces avermitilis and the milbemycins were isolated from S. cyanogrise or S. hygroscopicus. Both groups were found to have potent insecticidal properties. Abamectin is the commercialised major pair of isomers of avermectin itself, and ivermectin is the cis-hydrogenated product of abamectin (at the 22,23 position). In both compounds, the major isomer, B1a, contains a 2-butyl group, and the minor isomer (B1b) an isopropyl group attached to the carbon at the 25 position [2]. Many new avermectins have been synthesised to improve both the anti-parasitic efficacy and to change their pharmacokinetic profiles. Doramectin was biosynthesised from mutant S. avermitilis with a cyclohexyl group at the 25 position [3]. These three avermectins contain only C, H and O atoms (Fig. 1). Further modifications of the avermectin and nemodectin structures have been obtained by introducing groups containing nitrogen atoms to obtain different insecticidal properties, and reduce meat and milk withdrawal times. Thus various OH groups were replaced by: in emamectin, a methylamino group, in eprinomectin, an acetylamino group, in selamectin an oxime and in moxidectin a methoxime group. The compounds are registered for use in cattle and other food animals, for fish farming or for pet animals only. For example, in Canada, abamectin, doramectin, eprinomectin, ivermectin and moxidectin are used to treat food animals: cattle, swine, sheep, bison, deer and reindeer against nematodes and arthropods and for mastitis; emamectin is used to control sea lice in fish farms and selamectin is used for the treatment of pets against heart and round worms. Of this group, only eprinomectin and moxidectin are permitted for use with dairy cattle, with no milk withholding time. Eprinomectin was designed to exhibit a low milk/plasma ratio [4], and moxidectin is less toxic with a larger acceptable daily intake (ADI). Eprinomectin has an (administrative) MRL of 20 ppb in Canada, and MRLs of 20 ppb in the EU and 12 ppb in the USA. The marker residue is the B1a isomer. Moxidectin has a MRL value of 40 ppb for the USA and EU. The avermectins are also used for control of mites in fruits and vegetables. Although they are considered safe with low toxicity towards mammals, misuse or extra-label use may produce food levels in violation of zero tolerance or MRL levels. Thus a number of methods for quantitation and verification of avermectin and milbemycin endectocides have been developed.

In many laboratories the macrolide endectocides are screened in a variety of fluids and tissues using HPLC with fluorescence detection. The fluorophor is formed by dehydration of the fused hexahydrobenzofuran ring system to a conjugated benzofuran system with a strong acid such as trifluoroacetic acid in the presence of an organic base such as methylimidazole [2], [5], [6], [7], [8], [9].

Mass spectrometry, with either particle beam negative ion chemical ionization or thermospray, was originally used to confirm the presence of ivermectin [10] or moxidectin [11], after quantitation by the LC-fluorescence method since the MS response was not linear [10]. Use of positive ion electrospray ionization (ESI) for quantitation has been plagued by the tendency of the avermectins to form sodium adduct ions [M + Na]⁺ [12], [13], [14], [15]. Monitoring of the [M + Na]⁺ ion tended to produce a very non linear response, which was attributed to traces of sodium present in the extracts from the matrix or the method [12], [15]. Using positive electrospray ionization and the [M + Na]⁺ ion it was possible to get a linear relative response when abamectin was used as the internal standard for quantitation of ivermectin. Presumably these close analogues respond equally to the presence of sodium in the extracts and the non linear responses cancel. However, this method was limited to only one analyte, and presumed the absence of abamectin in the samples. To overcome the sodium adduct and linearity problems, several methods have been reported using LC–MS with atmospheric pressure chemical ionization (APCI) with positive ion detection in which either the [M + H]⁺ [12], the [M + NH₄]⁺ [16], or fragment [17] ions were monitored. Linear calibration curves were obtained using APCI with negative ion [M − H]⁻ monitoring [15], [18], [19]. Second order polynomial calibration curves were found to be more appropriate for quantitation using the [M − H]⁻ ions formed by negative ion APCI with a wide range of analyte concentrations.

The aim of this work was twofold. Firstly to examine the different conditions for analysis of a wide variety of avermectins using electrospray ionization with both positive and negative ions, and secondly compare the results obtained when quantifying the avermectins in milk. Although eprinomectin and moxidectin are the only avermectins permitted for use with dairy cattle, the other compounds were included since off-label use of various avermectins with dairy cattle does occur [20]. Selamectin was chosen as the internal standard as it is unlikely to be used in food producing animals.