ESI⁺MS/MS confirmation of canine ivermectin toxicity

Abstract

Ivermectin is a semisynthetic macrocyclic lactone anthelmintic of the avermectin family derived from Streptomyces fermentation products. Avermectins are used as antiparasitic agents in domestic animals; although considered relatively safe, one must consider animal species, breed, weight, and age in dosage determinations.In January 2006, two canines were presented to the UK Livestock Disease Diagnostic Center after dying from suspected ivermectin overdoses [30-50 mg/kg body weight]. To confirm this clinical diagnosis we developed a rapid, sensitive semiquantitative ElectroSpray Ionization-Mass Spectrometry (ESI/MS) method for ivermectin in canine tissue samples. Pharmaceutical ivermectin contains two ivermectins differing by a single methyl group, and each compound forms interpretation-confounding adducts with tissue Na(+) and K(+) ions. We now report that ivermectin administration was clearly confirmed by comparison with standard and dosage forms of ivermectin, and simple proportionalities based on mass spectral intensity of respective molecular ions allowed semiquantitative estimates of injection site tissue concentrations of 20 and 40 microg/g tissue (wet weight) in these animals, consistent with the history of ivermectin administration and the clinical signs observed.There is a distinct need for both rapid detection and confirmation of toxic exposures in veterinary diagnostics, whether for interpretation of clinical cases antemortem or for forensic reasons postmortem. It is vital that interpreters of analytical results have appropriate guidance in the scientific literature and elsewhere so as to enable clear-cut answers. The method presented here is suitable for routine diagnostic work in that it allows rapid extraction of ivermectin from tissue samples, avoids the need for high-performance liquid chromatography and allows ready interpretation of the multiple ivermectin species seen by ESI(+) MS/MS in samples originating from veterinary dosage forms.

Structural characterization of product ions by electrospray ionization and quadrupole time‐of‐flight mass spectrometry to support regulatory analysis of veterinary drug residues in foods. Part 2: Benzimidazoles, nitromidazoles, phenothiazines, and mectins

“…The structures shown in Table (#3–4) are in agreement with the product ions reported for mectins by Alberts‐Shönberg et al . Saccharide product ions were also selected for avermectin ( m/z 145 and 113, #1), doramectin ( m/z 145 and 113, #2), ivermectin ( m/z 183, #5) and selamectin ( m/z 145 and 113, #7). In the case of selamectin, the side‐chain ion with m/z 95 (Product Ion III, #7) was also selected.…”

“…[13] Moxidectin loses the aliphatic side chain (-C 7 H 12 O) from the protonated molecule thereby producing the ion at m/z 528 (Product Ion I, #6), as described by Awasthi et al [14] Further fragmentation led to the ion at m/z 498 (Product Ion II, # 6), and the ion at m/z 199 resulted from fragmentation of the macrocyclic lactone. [14] For emamectin and eprinomectin, the three selected product ions are from the saccharide portion of the molecule, observed at m/z 302, 158, and 126 ( Table 4 (#3-4) are in agreement with the product ions reported for mectins by Alberts-Shönberg et al [15] Saccharide product ions were also selected for avermectin (m/z 145 and 113, #1), doramectin (m/z 145 and 113, #2), ivermectin [16] (m/z 183, #5) and selamectin (m/z 145 and 113, #7). In the case of selamectin, the side-chain ion with m/z 95 (Product Ion III, #7) was also selected.…”

“…A Na‐adduct product ion was observed for all the mectins, including moxidectin, and the ion does not appear in any of the MS/MS spectra of the [M + NH 4 ] + or [M + H] + forms. Lehner et al also observed the Na‐adduct product ion in an ivermectin spectrum, but its structure was not proposed . Sodiated OE structures are very uncommon in ESI, and our literature search only found a report describing that the benzene carboxylate sodium salt can form a cation radical by elimination of Na • .…”

“…Lehner et al also observed the Na-adduct product ion in an ivermectin spectrum, but its structure was not proposed. [16] Sodiated OE structures are very uncommon in ESI, and our literature search only found a report describing that the benzene carboxylate sodium salt can form a cation radical by elimination of Na • . [23] However, there is no evidence in the mass spectrum of ivermectin for the addition of more than one sodium to justify the ejection of Na • .…”

“…Thus, few reports have described fragmentation pathways or proposed product ion structures for the compounds of interest, and we revise structures published for benzimidazoles, [7] nitroimidazoles, [8,9] phenothiazines, [10][11][12] and mectins. [13][14][15][16] High-resolution mass spectrometry (HRMS) is a valuable approach to the characterization of product ions for the screening and identification of pesticides, veterinary drugs, and other analytes. [17,18] To characterize the product ions of interest, it is necessary to calculate ion formulae from the experimental data collected, and hence HRMS is useful.…”