Development and Validation of Quantitative Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry Assay for Anticoagulant Rodenticides in Liver.

Anticoagulant rodenticides (ARs) are used to control rodent populations; however, exposure to nontarget animals occurs. A sensitive and rugged quantitative method was developed, optimized, and validated for eight ARs in liver. Target analytes comprised two chemical classes: hydroxycoumarins (warfarin, coumachlor, dicoumarol, bromadiolone, brodifacoum, and difethialone) and indanediones (diphacinone and chlorophacinone). In this method, liver extracts were cleaned using dispersive solid phase extraction (d-SPE) to remove matrix interferences and analyzed by reverse phase ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Electrospray ionization in negative ion mode combined with multiple reaction monitoring (MRM) using a triple quadrupole mass spectrometer provided simultaneous confirmation and quantitation. Detection limits spanned 0.75-25 ng/g, and lower quantitation limits were established as 50 ng/g. Interassay method accuracy ranged from 92 to 110% across the analytical range (50-2500 ng/g) using matrix-matched calibrants with good repeatability (relative standard deviations 2-16%). Successful method transfer to another laboratory utilizing an Orbitrap mass analyzer, providing high mass accuracy, was assessed by good method reproducibility during blinded study analyses (6-29%; Horwitz ratios (HORRAT) ≤ 1.5).

Determination of strophanthidin in ingesta and plant material by LC-MS/MS.

An LC-MS/MS method was developed for the semiquantitative determination of strophanthidin glycosides in ingesta from animals. Strophanthidin glycosides were simultaneously extracted and hydrolyzed to the strophanthidin aglycone using aqueous methanolic hydrochloric acid and the extracts cleaned up using solid-phase extraction. Extracts were analyzed using reverse-phase HPLC coupled with positive ion electrospray mass spectrometry. Characteristic product ion spectra were produced by fragmentation of the [M + H](+) precursor ion for each analyte. Quantitation was performed using the internal standard method with digitoxigenin serving as the internal standard. The method detection limit was calculated to be 0.075 microg/g, and the limit of quantitation was calculated to be 0.24 microg/g for strophanthidin in control rumen samples. This method was used in diagnostic investigations to confirm fatal strophanthidin glycoside poisonings in horses.

Diterpenoid alkaloid toxicosis in cattle in the Swiss Alps.

Between 1995 and 1999, several cattle of a group of 80 heifers died acutely on a pasture in the Swiss Alps. The animals were Found dead between July 9th and 15th eachyear. Only 1 animal was examined on post-mortem, and no significant lesions were found. Aconitum vulpera, A napellus, and Delphinium elatum were identified in the pasture. The presence of diterpenoid alkaloid-containing plants in the pasture, the rapid death of the animals, and the lack of pathologic lesions suggested diterpenoid alkaloid toxicosis as a cause of death. A multiresidue alkaloid screen using gas chromatography with a mass spectrometric detector was employed on rumen, abomasal, small intestine, and cecal contents from the I heifer. Deltaline, deltamine, and lycoctonine were identified. Aconitine was found in all gastrointestinal samples using a sensitive and highly specific liquid chromatography/mass spectrometry methodology for aconitine analysis. The findings ofditerpenoid alkaloids in the gastrointestinal contents confirmed exposure to Delphinium and Aconitum spp, possibly resulting in sudden death.