A Validated LC-MS-MS Method for Simultaneous Identification and Quantitation of Rodenticides in Blood

Abstract

A rapid, highly sensitive and specific analytical method for the extraction, identification and quantification of nine rodenticides from whole blood has been developed and validated. Commercially available rodenticides in Australia include coumatetralyl, warfarin, brodifacoum, bromadiolone, difenacoum, flocoumafen, difethialone, diphacinone and chlorophacinone. A Waters ACQUITY UPLC TQD system operating in multiple reaction monitoring mode was used to conduct the analysis. Two different ionization techniques, ES+ and ES-, were examined to achieve optimal sensitivity and selectivity resulting in detection by MS-MS using electrospray ionization in positive mode for difenacoum and brodifacoum and in negative mode for all other analytes. All analytes were extracted from 200 µL of whole blood with ethylacetate and separated on a Waters ACQUITY UPLC BEH-C18 column using gradient elution. Ammonium acetate (10 mM, pH 7.5) and methanol were used as mobile phases with a total run time of 8 min. Recoveries were between 70 and 105% with limits of detection ranging from 0.5 to 1 ng/mL. The limit of quantitation was 2 ng/mL for all analytes. Calibration curves were linear within the range 2-200 ng/mL for all analytes with the coefficient of determination ≥0.98. The application of the proposed method using liquid-liquid extraction in a series of clinical investigations and forensic toxicological analyses was successful.

Core-shell LC–MS/MS method for quantification of second generation anticoagulant rodenticides diastereoisomers in rat liver in relationship with exposure of wild rats

“…More basic conditions (ammonium bicarbonate 10mM, pH=7) did not allow chromatographic separation for whichever SGARs diastereoisomers but only BROMA with the poroshell column. Porous particles C18 columns did not even separate BROMA diastereoisomers in basic conditions [19,27], and did not separate diastereoisomers of the other SGARs with LC-MS/MS buffer modifiers [18][19][20][21][22][23]26], even with sub-2µm UHPLC columns [2,23].…”

“…They had hence to be treated as separate species and analytical methods were developped to characterize their dissociated physiological properties [17]. Numerous Liquid Chromatography on line with two dimensional Mass Spectrometry (LC-MS/MS) multi-residue methods have been published to quantify ARs in biological or environmental matrices [18][19][20][21][22][23]. Imran et al [24] published a review of analytical methods for the determination of anticoagulant rodenticides in biological samples; none of them reported the quantification of SGARs diastereoisomers, even less SGARs enantiomers.…”

“…Hunter [25] examined the chromatographic properties of BROMA diastereoisomers in normal phase, reversed phase, ion pair and ion exchange chromatography. Some authors reported two resolved chromatographic peaks for BROMA diastereoisomers on C8 or C18 reversed phase columns in acidic conditions with acetate or formate ammonium ions in the mobile phase [18,20,21,23,26] using only the first and major peak for the quantification of bromadiolone residue. But others reported only one chromatographic peak for BROMA on C18 reversed phase column in neutral or basic conditions [19,27].…”

“…But others reported only one chromatographic peak for BROMA on C18 reversed phase column in neutral or basic conditions [19,27]. No multi-residue quantification method was produced for SGARs diastereoisomers even with Ultra-Performance Liquid Chromatography (UPLC) columns [22,23].…”

First evidence of anticoagulant rodenticides in fish and suspended particulate matter: spatial and temporal distribution in German freshwater aquatic systems

“…Furthermore, there are numerous AR substances that may enter the environment and so a comprehensive assessment of the presence of AR requires very sensitive and accurate multi-methods, covering a wide range of different ARs. Several analytical approaches for multi-methods for the quantitative determination of AR in biological samples have been developed, such as liquid chromatography (LC) and also ion chromatography (IC) coupled to tandem mass spectrometry (MS/MS) (Bidny et al 2015 , Chen et al 2009 , Jin et al 2009 , Jin et al 2008 , Marek and Koskinen 2007 ), two-dimensional LC coupled to MS/MS (Marsalek et al 2015 ), IC coupled to fluorescence detection (Jin et al 2007 ), methods using high resolution MS (Schaff and Montgomery 2013 ), and some other strategies that are presented in a review by Imran et al ( 2015 ). Available analytical methods are so far hampered by the number of captured AR, as well as high limits of detection caused by complex biological and environmental matrices that are in contrast to low relevant environmental concentrations.…”