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Development and Validation of Quantitative UPLCMS/MS Assay for Anticoagulant Rodenticides in Liver Lori L. Smith, Boying Liang, Marcia C. Booth, Michael Filigenzi, Andriy Tkachenko, and Cynthia L. Gaskill J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02280 • Publication Date (Web): 12 Jul 2017 Downloaded from http://pubs.acs.org on July 12, 2017

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Journal of Agricultural and Food Chemistry

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Development and Validation of Quantitative UPLC-MS/MS Assay for

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Anticoagulant Rodenticides in Liver

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Lori L. Smith*†, Boying Liang‡, Marcia C. Booth§, Michael S. Filigenzi§, Andriy Tkachenko┴

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and Cynthia L. Gaskill†

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University of Kentucky Veterinary Diagnostic Laboratory, Toxicology Laboratory, University of Kentucky, Lexington, Kentucky 40511, United States

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University of California, Davis, California 95616, United States

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California Animal Health and Food Safety Laboratory System, Toxicology Laboratory,

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U.S. Food & Drug Administration, Center for Veterinary Medicine, 8401 Muirkirk Rd, Laurel, Maryland 20708, United States

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*Corresponding Author: Phone: (859) 257-8283; Fax: (859) 255-1624;

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Email: [email protected]

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‡

Present Address: Medical Products Research and Development, Baxter Healthcare Co., Ltd., Suzhou 215028, People’s Republic of China ACS Paragon Plus Environment


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ABSTRACT:

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Anticoagulant rodenticides (ARs) are used to control rodent populations, however exposure to

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non-target animals occurs.

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optimized and validated for eight ARs in liver. Target analytes comprised two chemical classes:

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hydroxycoumarins (warfarin, coumachlor, dicoumarol, bromadiolone, brodifacoum and

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difethialone) and indanediones (diphacinone and chlorophacinone). In this method, liver extracts

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were cleaned up using dispersive solid phase extraction (d-SPE) to remove matrix interferences

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and analyzed by reverse phase ultra-performance liquid chromatography-tandem mass

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spectrometry (UPLC-MS/MS). Electrospray ionization in negative ion mode, combined with

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multiple reaction monitoring (MRM) using a triple quadrupole mass spectrometer, provided

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simultaneous confirmation and quantitation. Detection limits spanned 0.75 to 25 ng/g and lower

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quantitation limits were established as 50 ng/g. Inter-assay method accuracy ranged from 92-

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110% across the analytical range (50-2500 ng/g) using matrix-matched calibrants, with good

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repeatability (RSDs 2-16%).

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Orbitrap mass analyzer, providing high mass accuracy, was assessed by good method

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reproducibility during blinded study analyses (6-29%; Horwitz ratios (HORRAT) < 1.5).

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KEYWORDS:

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indanediones, dispersive SPE

A sensitive and rugged quantitative method was developed,

Successful method transfer to another laboratory utilizing an

rodenticides, electrospray, UPLC-MS/MS, poisoning, hydroxycoumarins,

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ACS Paragon Plus Environment

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INTRODUCTION:

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Anticoagulant rodenticides (ARs) are pesticides used to control rodent populations, acting as

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vitamin K antagonists by inhibiting vitamin K epoxide reductase.1, 2 Consequently, formation of

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prothrombin and related blood-clotting factors (VII, IX and X) is blocked, causing massive

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hemorrhage and mortality. Commercially available in feed formulations as bait packs, pellets,

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bars and other products, ARs have been in use since the early 1950s in agricultural, urban and

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suburban settings and credited with revolutionizing vertebrate pest control.3 As a direct result of

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this popularity, a number of accidental poisonings of non-target species (i.e. wildlife, domestic

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farm animals and pets) have been reported.4-8 First generation ARs (FGARs; e.g. warfarin and

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coumachlor) were synthesized based on the hydroxycoumarin chemical structure for naturally

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occurring dicoumarol (see Figure 1), first discovered in improperly cured sweet clover.9-11

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FGARs generally have shorter elimination half-lives and require multiple feeds at higher doses

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to cause rodent death; their use eventually led to genetic resistance in rats and house mice.

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Second generation ARs, referred to as “superwarfarin” or long-acting ARs (SGARs; e.g.

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brodifacoum, difethialone, and bromadiolone), were developed to overcome resistance and are

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more potent than FGARs, often requiring only a single dose to induce death. Indanedione

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derivatives, notably chlorophacinone and diphacinone, are also considered to be SGARs in

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function and potency, but differ by the core chemical structure (see Figure 1).

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Multi-residue analytical methods have been developed for qualitative and quantitative

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determination of ARs in biological specimens to confirm exposure in post-mortem cases. A

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comprehensive review article has been recently published in which several analytical methods

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for ARs are summarized.12 Most methods currently in use rely on liquid chromatography (LC)

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due to the complexity of the prepared sample and the non-volatile nature of the AR analytes.12

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Coupling LC systems to either fluorescence or UV detectors is common and allows detection and

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quantitation of these analytes. Unfortunately, AR methods based on fluorescence spectroscopy

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are limited to hydroxycoumarin-based ARs while analysis of indanedione ARs requires

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additional instrumentation to detect absorption of UV radiation. Increasingly, reported methods

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incorporate the use of mass spectrometry (MS)-based detection schemes, primarily using

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electrospray ionization to produce negatively charged ions and monitoring production of unique

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fragment ions for each AR.13-18 Enhanced selectivity and sensitivity, as well as detection of

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multiple ARs in a single analysis, are primary advantages for using MS-based detection.

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Extensive sample clean-up techniques are often required to minimize matrix effects and

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suppress elevated baseline noise due to co-extracted interferences. Sample clean-up techniques

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for biological tissue extracts often rely on solid phase extraction (SPE) strategies, in which

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column conditioning, washing, sample elution and evaporative reconstitution steps are

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employed. While SPE methods can produce relatively clean chromatograms, the cartridges are

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susceptible to clogging and there are factors that must be thoroughly characterized for the

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technique to be robust (i.e. control of temperature, flow rate, pH, solvent compatibility, and

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matrix variations).19 Dispersive SPE (d-SPE) is a sample clean-up technique first introduced in

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2003 for pesticide analyses in fruits and vegetables20 in which matrix interferences in the sample

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extract adsorb onto solid sorbent materials. Analytes of interest remain in the liquid phase that

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may be subsequently filtered and directly analyzed, providing the advantages of decreased

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sample preparation time in a single step, decreased solvent use and an easily reproducible

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process. The successful use of d-SPE sample clean-up in conjunction with HPLC-fluorescence

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and UV detection schemes for AR analysis in biological specimens has been previously

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reported.21

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The aim of this research was to develop and validate a multi-residue quantitative method

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for hydroxycoumarin- and indanedione-based ARs in liver, using ultra-high performance liquid

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chromatography-tandem mass spectrometry (UPLC-MS/MS) in combination with d-SPE sample

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clean-up. The developed method was extensively validated in a multi-laboratory collaborative

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study to address several limitations of previously published methods adopted by the diagnostic

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community with varying degrees of success. Many methods have been designed for use in a

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single laboratory, using a single instrument platform with conditions optimized for a limited set

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of ARs in a matrix, often narrowed to a single source species. The result is a wide body of

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published methods developed and validated for research studies primarily focused on academic

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endeavors, which may not extend beyond their envisioned application. Successful adoption of

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methods for routine use in diagnostic settings requires compatibility with samples from various

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source species, use on a range of instrument platforms, and detection limits compatible with

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toxicologically relevant AR concentrations. The developed method met these requirements and

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was confirmed to be suitably rugged through extensive validation, producing consistent

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analytical results in blinded studies when successfully transferred to a collaborating laboratory.

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MATERIALS AND METHODS:

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Chemicals and Materials. Coumachlor and dicoumarol standard reference materials were

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purchased from Sigma-Aldrich (St. Louis, MO).

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bromadiolone, brodifacoum and difethialone standard reference materials were provided by the

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U.S. Environmental Protection Agency National Pesticide Standard Repository (Fort Meade,

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MD). HPLC-grade methanol, acetonitrile, acetone, chloroform and ammonium acetate were

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obtained from Fisher Scientific (Pittsburgh, PA). Distilled, deionized water was produced in-

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Warfarin, diphacinone, chlorophacinone,



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house using a Barnstead MEGA-PURE 6A water distillation unit (Barnstead-Thermolyne,

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Dubuque, IA) and a Barnstead EASYpure II RF water purification system (Thermo Scientific,

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Dubuque, IA). Dispersive SPE tubes, each containing 175 mg magnesium sulfate, 100 mg

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florisil, 50 mg alumina basic and 50 mg primary secondary amine (PSA), were custom-ordered

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from United Chemical Technologies, Inc. (Bristol, PA).

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Calibration Standards. Equine liver was obtained from animals submitted to the University of

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Kentucky Veterinary Diagnostic Laboratory for necropsy, homogenized in bulk by mechanical

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blending, tested to ensure no detectable ARs were present and stored at -20°C until use.

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Calibration curves comprised of seven, non-zero points for AR quantitation were prepared by

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fortifying control liver at increasing concentrations and processing them as unknown samples

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(i.e. matrix-matched calibrants). These calibrants were prepared at 25, 50, 75, 100, 500, 1000

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and 2500 ng/g by adding the appropriate volume of a standard reference solution containing a

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mixture of ARs (10 µg/mL each) directly onto the control liver and vortex-mixing. Quality

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control samples were prepared at three concentration levels: blank (no ARs added), low (50

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ng/g) and high (1000 ng/g).

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concurrently with each analytical batch.

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Sample Extraction and d-SPE Clean-Up. One gram of liver was weighed into a 50-mL

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disposable centrifuge tube. Six milliliters of 10% (v/v) methanol in acetonitrile and a 9.5-mm

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stainless steel grinding ball was added to the tube and vortexed. The sample was homogenized

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for 5 min at 650 rpm using an impact grinder (2010 Geno/Grinder, SPEX SamplePrep,

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Metuchen, NJ). A second homogenization cycle was repeated as needed to achieve thorough

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sample consistency. Sample extraction occurred by agitating the homogenates horizontally on a

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reciprocating shaker table for 30 minutes, followed by centrifugation at 829g for 5 min.

Calibrants and control samples were prepared and analyzed

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The extract supernatant was then transferred to a d-SPE tube and vortex mixed for 10 s to

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completely wet the SPE sorbents. Sample extracts were then placed on a tube rotator at 30 rpm

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for 30 min, followed by centrifugation at 829g for 5 min.

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The cleaned extract supernatant was transferred to an empty 15-mL disposable centrifuge

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tube and evaporated to dryness under nitrogen at 45°C (XcelVap evaporation system; Horizon

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Technology, Salem, NH). The dried residue was reconstituted in 1 mL methanol, sonicated

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briefly to aid complete dissolution (up to 5 min, depending on sample characteristics) and

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filtered through a 0.22 µm PTFE syringe filter (MicroSolv Technologies, Eatontown, NJ)

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directly into a silanized, amber autosampler vial for UPLC-MS/MS analysis.

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UPLC-MS/MS Analysis. All reported analyses from the method-originating laboratory were

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performed on a Thermo Scientific Dionex UltiMate 3000 ultra-performance liquid

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chromatography (UPLC) system comprised of a solvent organizer, temperature-controlled

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autosampler, binary gradient pump, and temperature-controlled column compartment.

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eluate from the UPLC system was introduced by direct coupling to a Thermo Scientific TSQ

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Quantum Access Max triple quadrupole mass spectrometer (Waltham, MA) with a heated

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electrospray ionization (HESI-II) source operated in negative ion mode.

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separation was carried out at 25ºC using an Accucore C18 LC analytical column (2.1 mm i.d. ×

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100 mm; 2.6 µm) preceded by a guard column of the same material (2.1 mm i.d. x 10 mm;

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Thermo Scientific, Waltham, MA). Mobile phase was delivered at a constant flowrate of 0.300

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mL/min. The binary mobile phase consisted of 10 mM ammonium acetate, pH 9 (mobile phase

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A) and methanol (mobile phase B), each of which was degassed by vacuum filtration through

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0.22 µm PTFE membrane filters prior to use. Extracts from matrix-matched calibrants, control

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and unknown samples were injected (1 µL) into the initial gradient conditions of 60% mobile

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The

Chromatographic


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phase A / 40% mobile phase B, which were maintained for 1 min following injection. Mobile

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phase B was increased linearly to 57% over the next 8 min and increased further to 77% over the

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next 6 min. From 15 to 18 min post-injection, mobile phase B was increased to 81% and finally

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to 90% in one minute. Mobile phase B was held at 90% for 5 min to rinse the column of any

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residual non-polar matrix components and minimize carryover. At 24 min post-injection, the

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initial mobile phase conditions were resumed to allow re-equilibration of the column in

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preparation for the next analysis. The total run time for one injection was 34 min, with all target

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ARs eluting within the first 18 min.

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Chromatograms produced by multiple reaction monitoring (MRM) of specific

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fragmentation pathways for deprotonated molecular ions ([M-H+]-) were used for confirmation

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and quantitation of the target ARs. The mass spectrometer was tuned and calibrated in negative

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ion mode using its automated tuning procedure and the corresponding tuning solution (Thermo

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Scientific), a mixture of polytyrosines. Tune parameters were further refined by direct infusion

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of a methanolic mixture of ARs (approximately 10 µg/mL each) into the UPLC eluate flow at

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initial gradient conditions (60% mobile phase A / 40% mobile phase B; 0.300 mL/min; 25ºC).

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Source vaporizer and capillary temperatures were set to 380ºC and 300ºC, respectively, with an

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ESI spray voltage at 4000 V. Sheath, auxillary, and ion sweep gases were set at 50, 45, and 0

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(arbitrary units), respectively, sourced from either compressed ultra-high purity (UHP) nitrogen

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or a nitrogen generator (NitroFlowLab; Parker Balston, Haverhill, MA). The collision-induced

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dissociation gas (UHP argon) was maintained at a constant pressure of 1.7 mTorr in the collision

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cell throughout analysis. Multiple scan channels were acquired simultaneously, corresponding to

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each of the monitored fragmentation transitions established for either confirmation or

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quantitation of the target ARs within 2 min of the expected retention time (see Table 1). Tube

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lens offset potentials and collision energies for individual fragmentation transitions were

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optimized for maximum sensitivity and are listed in Table 1 as well.

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Data Analysis.

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Qualitative Assessment. The respective AR was positively identified in the unknown sample if

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the following criteria were met: i). The quantifying ion and the corresponding confirming ion

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co-eluted within 0.1 min of one another, each with a signal-to-noise ratio (S/N) > 3, ii). The

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retention times of the quantifying and confirming ions were within 0.25 min of the mean

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retention time for the corresponding AR in all calibrants and overspiked control samples

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acquired within the same analysis batch, and iii). The ratio of signals for the quantifying and

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confirming ions (ion ratio) was consistent with those observed in the calibrants acquired

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concurrently (e.g. within 20% of the average ion ratio).

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Quantitation. Quantitative results for method recovery experiments were determined using

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external calibration unless otherwise stated. Chromatographic peak area for the quantifying ion

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transition was plotted versus concentration of the corresponding AR for a set of matrix-matched

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calibrants, carried through the extraction and d-SPE clean-up steps.

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concentrations were interpolated from the resulting calibration curves.

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Analyte Stability. Stability of ARs in liver was investigated by analyzing control liver spiked

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at either low or high levels (50 and 2000 ng/g) after storage in various environments. The

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prepared liver was held in three different storage conditions (benchtop at ambient temperature,

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refrigerated storage at 4°C, and freezer storage at -20°C) over varied lengths of time. Benchtop

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storage was investigated over four days, refrigerated storage was investigated for one week and

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freezer storage continued for up to one month. Results were compared to initial values obtained

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immediately after control liver was fortified (Day 0, no storage).

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Unknown sample


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Method Validation. Validation of method performance was carried out according to U.S. FDA

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Office of Foods and Veterinary Medicine guidelines22, but adapted to non-food diagnostic

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matrices. Validation comprised four stages. First, determination of the following performance

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characteristics was performed using fortified liver in the method-originating laboratory:

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selectivity, limits of detection and quantitation, detector response characteristics, accuracy and

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precision (i.e. repeatability (RSDr)). Additionally, matrix effects and any potential analyte losses

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from sample preparation were assessed.

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originating laboratory analyzed samples during a blinded study organized by an independent

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laboratory. Analysts remained blinded to AR concentrations, number of spike levels, number of

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replicates at each level and any additional challenges scheduled by the organizers. The method

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was then transferred to a collaborating laboratory and original method performance

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characteristics (i.e. sensitivity, accuracy and precision) were verified. In the final validation

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stage, inter-laboratory reproducibility (RSDR) and ruggedness/robustness were assessed by

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concurrent analysis of blinded samples (also prepared at the independent laboratory) at both the

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method-originating and collaborating laboratories.

During the second validation stage, the method-

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Signal intensity changes (i.e. suppression or enhancement) based on the influence of

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residual matrix in processed sample extracts on ionization efficiency were studied. Subsamples

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of blank control liver were processed as unknown samples (i.e. extracted and submitted to d-SPE

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clean-up). Sufficient volumes of AR standard solution (10 µg/mL) were then added to these

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prepared blank extracts to mimic complete recovery of 50, 500 and 2000 ng/g. Matrix effects

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were determined by comparing mean peak areas of ARs prepared in extracted control liver

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samples to those prepared in neat solvent (i.e. methanol), expressed as a percentage. This

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determination was made over three days for four replicates at each concentration level.

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The extent to which AR loss may have occurred during sample preparation was

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evaluated, combining extraction and d-SPE clean-up recovery. An appropriate volume of AR

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standard solution (10 µg/mL) was added to blank control liver to achieve 50, 500 or 2000 ng/g

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prior to treatment as an unknown sample (“Pre-Process Spike”). For comparison, blank control

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liver was extracted and submitted to d-SPE clean-up first, followed by enrichment at the same

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concentrations (“Post-Process Spike”).

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quantifying ion transition for each AR in the Pre-Process Spike was normalized relative to the

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corresponding Post-Process Spike, expressed as a percentage. The recovery percentages for

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sample preparation were determined in this manner over three days for a total of three replicates

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at each concentration level.

The integrated chromatographic peak area of the

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Method accuracy and inter-assay precision (i.e. repeatability within a single laboratory)

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were determined by analyzing fortified control liver at five levels (e.g. 25, 50, 500, 1000 and

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2000 ng/g) against matrix-matched calibrants across multiple days (n = 3 or 5, depending on

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spike level).

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Blinded samples were prepared with control liver spiked with ARs by an independent

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laboratory and submitted to the method-originating laboratory for analysis to further evaluate

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method performance. Scheduled challenges to the validated method were incorporated into the

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blinded sample set and included introduction of another source species (i.e. porcine) and the liver

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was treated more rigorously prior to fortification in an attempt to induce cell lysis (i.e. additional

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homogenization after mechanical blending, followed by sonication and repeated freeze/thaw

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cycles).

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Following completion of the blinded study, the method was transferred to and verified by

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an external collaborating laboratory, allowing assessment of method reproducibility and

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ruggedness/robustness.

Method implementation was performed with a Thermo Scientific

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QExactive MS system equipped with a Dionex Ultimate 3000 UPLC.

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monitoring (PRM) was used for detection of all panel analytes. All chromatographic and ion

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source parameters were consistent with the previous descriptions. Mass spectrometer settings for

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the PRM scans included mass resolution (FWHM at m/z 200) of 17,500, AGC target of 2x105,

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maximum injection time of 50 msec and an isolation window of 4.0 u.

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corresponding fragment ions, as well as normalized collision energies (NCE) are listed in Table

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1.

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precursor ions. Therefore, evaluation of full scan spectra rather than product ion ratios was used

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for analyte identification.

Parallel reaction

Precursor and

PRM scanning provides full scan high resolution data from fragmentation of selected

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Blinded duplicate sample sets (n=11 per set) were prepared by an independent laboratory

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with control liver at various concentrations and submitted to both laboratories for concurrent

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analyses. Pretreatment of liver tissue to induce cell lysis, as described for the single-laboratory

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blinded method performance test, remained a scheduled challenge for this event as well. Horwitz

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ratios (HORRAT)23 were used as a tool to assess method reproducibility and were calculated

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using Eq. 1: HORRAT = %RSDR / %PRSDR

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Eq. 1

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where %RSDR is the reproducibility precision of the pooled results from the participating

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laboratories. The predicted relative reproducibility precision (%PRSDR) was calculated using

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Eq. 2:

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%PRSDR = 2C-0.1505 where C is the known spike level concentration expressed on a g/g basis.

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Eq. 2

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RESULTS AND DISCUSSION:

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Tandem mass spectrometry generally provides highly selective detection with good sensitivity

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due to very low background noise and is ideally suited for multi-residue assays in complex

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biological matrices.24

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fragmentation of deprotonated molecular ions of the target analytes, it is possible to easily

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discern signals of interest from potential matrix interferences (i.e. chemical noise). Incorporating

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chromatographic separation prior to MS/MS analysis provides an additional level of selectivity

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in the form of unique retention times for each analyte. The monitored product ion transitions for

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each AR included in the panel were selected by characterizing the fragmentation patterns of the

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corresponding deprotonated molecular ion in the absence of matrix. As a consequence of

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chemical structure similarity, the quantifying ion transitions for deprotonated warfarin (m/z 307)

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and dicoumarol (m/z 335), as well as the confirming ion transition for deprotonated coumachlor

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(m/z 341), depend on the same product ion, m/z 161 (see Table 1 and Figure 2B-C). However,

281

exclusive retention times and precursor ion masses provide enough information to uniquely

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distinguish each AR during a single analysis.

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transitions were defined for individual ARs, blank equine liver extracts were prepared as

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unknowns and analyzed by UPLC-MS/MS. The resultant MRM chromatograms yielded no

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detectable signals above baseline noise for the combined quantifying and confirming ion

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transitions selected for each AR (see Figure 2A; note the vertical scale is approximately 2 orders

287

of magnitude lower than shown in Figure 2B-C). Results were similarly negative for blank liver

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from other sources as well, including bovine, canine, feline and avian species, indicating high

289

detection selectivity for the target ARs included in this method. Method selectivity was further

290

verified by investigating the rate of false positive results in a blinded collaborative study for

By monitoring unique product ion transitions that arise from

13

Once the quantifying and confirming ion



Page 14 of 33

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spiked test samples in which a single AR was omitted. Co-occurrence of multiple ARs extracted

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from a single sample is unlikely to cause interferences or produce false positive results.

293

Instrument Response Characteristics.

294

quantitation (LOQs) were estimated (data not shown) from replicate injections of serially diluted

295

AR standards in methanol (n = 3 for LODs, n = 6 for LOQs). Instrument LODs were set at the

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lowest level for which confirming ions were detected with a signal-to-noise ratio greater than 3,

297

within 0.25 min of expected retention times. Instrument LOQs were set at the lowest level for

298

which acceptable variance (+/- 20%) from the known concentration was routinely achieved.

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Method quantitation limits (MQLs) were verified by assessing the accuracy and precision of

300

method recovery for control liver spiked at levels near the instrument LOQs (+/- 20%).

Instrument

limits

of

detection

(LODs)

and

301

Detection limits ranged from 0.75 to 25 ng/g, depending upon AR, while MQLs were

302

established uniformly at 50 ng/g based on accuracy requirements for method recovery, rather

303

than low signal-to-noise ratios (see Figure 2B-C). Instrument response was determined by

304

producing multiple calibration curves from 7 matrix-matched calibrants over the course of

305

several days. A direct proportionality to concentration for all ARs in the range of 25 to 2500

306

ng/g was established, in which a coefficient of correlation ((R2; weighted 1/x2) greater than or

307

equal to 0.985 was considered acceptable. Instrument response at AR concentrations greater

308

than 2500 ng/g exhibited non-linearity (i.e. quadratic). There are many proposed hypotheses to

309

attempt explanation of non-linear relationships between ESI-MS instrument response and

310

concentration; however they are generally beyond the scope of this work.25 The upper limit of

311

quantitation (ULOQ) is generally of less consequence than method sensitivity, provided the

312

practical requirements of the method are met. A sample extract that induces an instrument

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response exceeding the ULOQ is immediately indicative of AR exposure at substantial amounts

314

and may be diluted with solvent for re-analysis if an accurate quantitative result is desired.

315

From a practical perspective, the analytical concentration range over which this method

316

was established is appropriate for the intended purpose. Threshold levels of concern are difficult

317

to establish due to a variety of confounding factors. Expected AR concentrations in liver

318

samples from natural exposure are dependent on the initial dosage and the time elapsed between

319

exposure and either death of the animal or sample collection from a live animal. ARs are

320

metabolized and excreted over time, with excretion half-lives varying depending on both the

321

compound and the species exposed.26 Wildlife studies have shown animals with low dose,

322

secondary exposures to ARs through predation (e.g. barn owls consuming rodents) can develop

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low detectable concentrations in tissues, yet show no evidence of coagulopathy.27, 28 Conversely,

324

an animal with direct AR exposure living for an extended period of time (i.e. up to weeks after

325

the initial encounter) can also have very low concentrations of ARs remaining in the liver at the

326

time of eventual death. Therefore, it is difficult to establish reliable threshold levels for ARs in

327

tissue to distinguish intoxication from coincidental exposure background levels. Furthermore,

328

threshold levels for these ARs in liver may remain ambiguous in published literature due to

329

possible method inconsistencies between laboratories as there are currently no formal

330

proficiency testing programs for ARs in liver. As such, diagnoses of AR poisoning require a

331

combination of clinical findings consistent with coagulopathy and detection of ARs in liver.

332

A common feature of ESI-based analyses is susceptibility to signal suppression and/or

333

enhancement in the presence of matrix, especially when the sample originates from a complex

334

system (e.g. tissue extract). The degree to which formation of deprotonated molecular ions and

335

corresponding quantifying and confirming ions of the reported ARs was affected by the presence

15



Page 16 of 33

336

of matrix co-extractants was determined. Percent matrix effects were calculated by comparing

337

chromatographic peak areas for each AR in the presence of residual matrix to solvent-based

338

standard solutions. There were no appreciable changes to detected signals for any of the ARs, at

339

any concentration tested, as noted by percent matrix effects ranging from 90-121% (see Table 2).

340

Typically, percent matrix effects are of concern when the variation exceeds 20%.29

341

General sample preparation techniques like dispersive solid phase extraction (d-SPE) are

342

principally based on non-specific interactions between adsorbents and mixture components. For

343

broad-spectrum applications like multi-residue analyses, d-SPE provides quick and simple means

344

to reduce potential matrix interferences in complex mixtures (i.e. tissue extracts). However,

345

there is potential for target analytes to interact with sorbent materials, adversely affecting

346

recovery. The extent to which AR recovery was impacted by d-SPE clean-up was investigated

347

by comparing mean chromatographic peak areas for control liver spiked with ARs before sample

348

extraction and clean-up, to blank control liver extract in which ARs were added after sample

349

preparation. Recoveries, with respect to sample preparation, ranged from 51-72% for all ARs

350

across the analytical range (see Table 2); the degree of AR loss was independent of both AR

351

chemical structure and concentration under the validated experimental conditions. However, AR

352

recovery was inconsistent and poor (approximately 10%-20% for non-polar ARs) during

353

preliminary recovery studies in which ARs were diluted with pure extraction solvent to mimic a

354

matrix-free extract. This strongly indicates AR recovery from d-SPE clean-up is dependent on

355

the presence of matrix in the extract solution. Should the validated method ever be extended to

356

another sample type (i.e. bait, stomach contents, etc.), there are aspects of the d-SPE clean-up

357

procedure that need to be optimized and validated.

16


Page 17 of 33

358


Two critical parameters were identified to achieve uniform AR recovery from liver

359

extracts:

i). duration of interaction between d-SPE sorbents and sample extracts and ii).

360

exclusion of C18 sorbent material as a clean-up component. Frequently, d-SPE is promoted as a

361

quick sample clean-up procedure in which extracts are vortex-mixed in the presence of sorbent

362

materials for seconds or a few minutes, prior to separating solid sorbent material from extract

363

solution by centrifugation. Previously published literature for AR quantitation in liver by HPLC

364

with fluorescence and UV detection21 recommended the use of the following d-SPE components

365

for sample clean-up: 50 mg PSA, 100 mg florisil, 175 mg magnesium sulfate, 50 mg basic

366

alumina and 50 mg C18 sorbent.

367

extract/sorbents mixture for 15 s, allowing it to rest for 5 min, followed by centrifugation at 1400

368

rpm for 8 min.

369

recoveries. Less polar ARs (i.e. later-eluting) suffered primarily and were recovered at rates

370

ranging from 25% to 45%.

371

improved recovery for chlorophacinone, bromadiolone, brodifacoum and difethialone by 12% to

372

18%. An additional 18% to 27% gain for these same four ARs was achieved by extending the

373

length of time the sample extract interacted with the sorbents from 9 min (1 min vortex-mix plus

374

8 min centrifugation) to 30 min. Interestingly, diphacinone recovery was low (40%) at short

375

clean-up durations whether or not C18 was included in the sorbent mixture. Instead, diphacinone

376

recovery was increased by approximately 28% solely based on extending the clean-up interaction

377

time to 30 min. All ARs more polar than diphacinone (i.e. expected retention times 7.5 min and

378

earlier) were unaffected by C18 and the duration of exposure to d-SPE sorbents. Calibration

379

curves generated from matrix-matched calibrants that have experienced sample preparation steps

380

(i.e. d-SPE clean-up) are required to accomplish accurate AR quantitation to account for known

The referenced method describes vortex-mixing the

Using similar parameters for sample clean-up produced inconsistent AR

Omission of C18 sorbent from the d-SPE component mixture

17



Page 18 of 33

381

analyte loss. Despite losing an appreciable amount of analyte in sample clean-up, d-SPE remains

382

an attractive matrix removal procedure due to low technical difficulty in implementation and

383

uniform recovery across ARs once optimized.

384

Method Accuracy and Precision. Analysis of spiked control liver at four concentration levels

385

(50, 500, 1000 and 2000 ng/g) against matrix-matched calibrants revealed inter-assay accuracy

386

was 92-110% with corresponding precision ranging from 2-16% (see Table 3). A fifth spike

387

level (25 ng/g) was analyzed concurrently to verify method quantitation limits set at 50 ng/g;

388

accuracy ranged from 63-88% and precision ranged from 7-22%. Results clearly demonstrated

389

that the method is adequately accurate and precise for a multi-residue quantitative assay, based

390

on criteria that method accuracy and RSD at each concentration level is within ± 20%.

391

Furthermore, accuracy and intra-assay precision were assessed by analysis of blinded overspiked

392

liver samples prepared by an independent laboratory. Method results were concluded to be

393

accurate and precise within the same analytical batch regardless of liver source species (i.e.

394

equine versus porcine), low concentration spike level (50 ng/g) and rigorous sample pretreatment

395

to induce cell lyses (see Table 4).

396

Stability. Comparison of means across days was performed using one-way ANOVA at a 95%

397

confidence interval for spiked control liver placed in various storage conditions. There was no

398

significant difference between time points at each storage condition for either low or high

399

concentrations in liver for the following ARs:

400

dicoumarol, difethialone and diphacinone.

401

difference at high concentrations for the investigated storage conditions for bromadiolone.

402

Likewise, there was no statistically significant difference noted for warfarin at low concentration.

brodifacoum, chlorophacinone, coumachlor,

Further, there was no statistically significant

18


Page 19 of 33


403

Liver containing low AR concentrations (e.g. 50 ng/g) can be easily distinguished from

404

liver containing high AR concentrations (e.g 2000 ng/g) at all storage conditions and time points

405

investigated. Further, liver with low concentrations near the method quantitation limit can be

406

reliably analyzed for up to one month when stored frozen (-20°C) or up to one week when

407

refrigerated (4°C). For clinical diagnostic purposes, exposure to these ARs can be determined

408

with a sufficient degree of certainty.

409

Further, prepared calibrant and sample extract solutions were determined to be stable

410

overnight at ambient temperatures. During the blinded method performance study, an unplanned

411

suspension of the analytical batch necessitated restarting the analysis the following morning. All

412

calibrants, controls and blinded sample extract solutions had been stored on the autosampler tray

413

overnight prior to a successful and complete analysis. The final results obtained for the blinded

414

samples were comparable to the overspiked control samples prepared on the day of analysis in an

415

unblinded manner (Sample Types 1, 2, 4 and 5 versus Sample Type 3 in Table 4).

416

Blinded Collaborative Study. Method performance demonstrated in the multiple-laboratory

417

blinded study met validation guidelines for a quantitative method and verified the method was

418

suitable for the intended purposes. There was a 0% false positive rate at both laboratories for the

419

blinded samples in which analytes were intentionally omitted, indicating high selectivity for ARs

420

in liver. Furthermore, Horwitz ratios (HORRAT) were less than or equal to 1.5, an indication

421

quantitative results for a matched sample set from two different laboratories were in good

422

agreement based on method reproducibility (see Table 5). Moreover, method performance

423

remained unaffected by inter-laboratory variations including different analysts, sources of

424

reagents and types of instrumentation (e.g. centrifuges, MS instrumentation, etc.), confirming the

425

method is rugged.

19



426

Routine Use of Method. Since completion of the validation, one of the two laboratories has

427

employed this method for the analysis of over 1000 liver tissue samples submitted from various

428

sources. The method has proven to be efficient and rugged, consistently meeting quality control

429

requirements (e.g. coefficients of determination (r2 > 0.99), overspike recoveries within 80 –

430

120%). Naturally incurred AR residues were detected and quantified in many of the submitted

431

samples.

Page 20 of 33

432

In summary, based on data obtained during all four stages of validation including (i) in-

433

house method validation in method-originating laboratory, (ii) single laboratory blinded study,

434

(iii) method verification in collaborating laboratory, and (iv) multiple laboratory blinded study,

435

as well as analysis of actual diagnostic samples in the collaborating laboratory, the method was

436

concluded to be reproducible and rugged to quantify ARs in liver samples. Such extensive

437

method validation provides a high degree of confidence the method will perform as described if

438

adopted by other laboratories for routine analysis of diagnostic samples.

439 440

ABBREVIATIONS:

441

AGC, ARs, d-SPE, (-)ESI, FGARs, FWHM, HORRAT, NCE, PRM, %PRSDR, % RSDR,

442

SGARs, UHP, ULOQ, UPLC-MS/MS

443 444

ACKNOWLEDGEMENT:

445

The authors acknowledge valuable input from Dr. Renate Reimschuessel and Ms. Sarah Nemser

446

from the Veterinary Laboratory Investigation and Response Network (Vet-LIRN), Center for

447

Veterinary Medicine, U.S. Food and Drug Administration.

448

20


Page 21 of 33


449

FUNDING:

450

This study was funded (FOA PA-13-244) and performed in collaboration with the U.S. Food and

451

Drug Administration’s Veterinary Laboratory Investigation and Response Network (FDA Vet-

452

LIRN) under Grant No. 1U18FD005015.

453 454

DISCLAIMER:

455

The views expressed in this article are those of the authors and do not necessarily reflect the

456

official policy of the Department of Health and Human Services, the U.S. Food and Drug

457

Administration, or the U.S. Government.

458 459

21



Page 22 of 33

460

REFERENCES:

461

1.

462

Review of the Pharmacology, Metabolism and Toxicology of Warfarn and Congeners. Drug

463

Metabolism and Drug Interactions 1987, 5, 225-271.

464

2.

465

Vitamin K, Litwack, G., Ed. Elsevier Academic Press Inc: San Diego, 2008; Vol. 78, pp 103-130.

466

3.

467

Outcome Pathway and Risks of Anticoagulant Rodenticides to Predatory Wildlife. Environ. Sci.

468

Technol. 2014, 48, 8433-8445.

469

4.

470

Exposure and Toxicosis in Coyotes (Canis latrans) in the Denver Metropolitan Area. J. Wildl.

471

Dis. 2015, 51, 265-268.

472

5.

473

Intoxication of Nontarget Wildlife with Rodenticides in Northwestern Kansas. J. Wildl. Dis.

474

2011, 47, 212-216.

475

6.

476

Poppenga, R.; Crooks, K. R.; Wayne, R. K.; Riley, S. P. D., Anticoagulant rodenticides in urban

477

bobcats: exposure, risk factors and potential effects based on a 16-year study. Ecotoxicology

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2015, 24, 844-862.

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7.

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in Red-Tailed Hawks, Buteo jamaicensis, and Great Horned Owls, Bubo virginianus, from New

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Jersey, USA, 2008-2010. Bull. Environ. Contam. Toxicol. 2014, 92, 6-9.

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E. P., Ed. Washington, D.C., 1998.

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9.

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Isolation and Crystallization of the Hemorrhagic Agent. Journal of Biological Chemistry 1941,

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138, 21-33.

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10.

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Disease. V. Identification and Synthesis of the Hemorrhagic Agent. Journal of Biological

489

Chemistry 1941, 138, 513-527.

Sutcliffe, F. A.; MacNicoll, A. D.; Gibson, G. G., Aspects of Anticoagulant Action: A

Tie, J. K.; Stafford, D. W., Structure and function of vitamin K epoxide reductase. In Rattner, B. A.; Lazarus, R. S.; Elliott, J. E.; Shore, R. F.; van den Brink, N., Adverse

Poessel, S. A.; Breck, S. W.; Fox, K. A.; Gese, E. M., Anticoagulant Rodenticide

Ruder, M. G.; Poppenga, R. H.; Bryan, J. A.; Bain, M.; Pitman, J.; Keel, M. K.,

Serieys, L. E. K.; Armenta, T. C.; Moriarty, J. G.; Boydston, E. E.; Lyren, L. M.;

Stansley, W.; Cummings, M.; Vudathala, D.; Murphy, L. A., Anticoagulant Rodenticides

Reregistration Eligibility Decision (R.E.D.) Facts: Rodenticide Cluster. In Agency, U. S. Campbell, H. A.; Link, K. P., Studies on the Hemorrhagic Sweet Clover Disease. IV. The

Stahmann, M. A.; Huebner, C. F.; Link, K. P., Studies on the Hemorrhagic Sweet Clover

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11.

Overman, R. S.; Stahmann, M. A.; Huebner, C. F.; Sullivan, W. R.; Spero, L.; Doherty,

491

D. G.; Ikawa, M.; Graf, L.; Roseman, S.; Link, K. P., Studies on the Hemorrhagic Sweet Clover

492

Disease. XIII. Anticoagulant Activity and Structure in the 4-Hydroxycoumarin Group. Journal of

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Biological Chemistry 1944, 153, 5-24.

494

12.

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for determination of anticoagulant rodenticides in biological samples. Forensic Sci.Int. 2015,

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253, 94-102.

497

13.

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anticoagulant rodenticides in tissues by column-switching UHPLC-ESI-MS/MS. Anal. Bioanal.

499

Chem. 2015, 407, 7849-7854.

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14.

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by high-performance liquid chromatography/electrospray/mass spectrometry. J. Agric. Food

502

Chem. 2007, 55, 571-576.

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15.

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Chromatography-Tandem Mass Spectrometry Ion-Trap Technique for the Simultaneous

505

Determination of Thirteen Anticoagulant Rodenticides, Drugs, or Natural Products. J. Anal.

506

Toxicol. 2010, 34, 95-102.

507

16.

508

eight anticoagulant rodenticides in animal plasma and liver using liquid chromatography

509

combined with heated electrospray ionization tandem mass spectrometry. J. Chromatogr. B

510

2008, 869, 101-110.

511

17.

512

quantification of second generation anticoagulant rodenticides diastereoisomers in rat liver in

513

relationship with exposure of wild rats. J. Chromatogr. B 2017, 1041, 120-132.

514

18.

515

Analysis of anticoagulant rodenticide residues in Microtus arvalis tissues by liquid

516

chromatography with diode array, fluorescence and mass spectrometry detection. J. Chromatogr.

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B 2013, 925, 76-85.

518

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519

Milford, MA, 2012.

Imran, M.; Shafi, H.; Wattoo, S. A.; Chaudhary, M. T.; Usman, H. F., Analytical methods

Marsalek, P.; Modra, H.; Doubkova, V.; Vecerek, V., Simultaneous determination of ten

Marek, L. J.; Koskinen, W. C., Multiresidue analysis of seven anticoagulant rodenticides

Fourel, I.; Hugnet, C.; Goy-Thollot, I.; Berny, P., Validation of a New liquid

Vandenbroucke, V.; Desmet, N.; De Backer, P.; Croubels, S., Multi-residue analysis of

Fourel, I.; Damin-Pernik, M.; Benoit, E.; Lattard, V., Core-shell LC-MS/MS method for

Hernandez, A. M.; Bernal, J.; Bernal, J. L.; Martin, M. T.; Caminero, C.; Nozal, M. J.,

Arsenault, J. C., Beginner's Guide to Solid-Phase Extraction. Waters Corporation:

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Anastassiades, M.; Lehotay, S. J.; Stajnbaher, D.; Schenck, F. J., Fast and easy

521

multiresidue method employing acetonitrile extraction/partitioning and "dispersive solid-phase

522

extraction" for the determination of pesticide residues in produce. J. AOAC Int. 2003, 86, 412-

523

431.

524

21.

525

Rodenticides in Animal Blood and Liver Tissue Using Principles of QuEChERS Method. J.

526

Anal. Toxicol. 2010, 34, 273-279.

527

22.

528

Guidelines for the FDA FVM Program, 2nd ed. In 2nd ed.; on-line, 2015.

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23.

530

1056-1058.

531

24.

532

Mass Spectrometry Applications in Endocrinology. Mass Spectrometry Reviews 2010, 29, 480-

533

502.

534

25.

535

of non-linearity in liquid chromatography/tandem mass spectrometry bioanalytical assays and

536

strategy to predict and extend the linear standard curve range. Rapid Commun. Mass Spectrom.

537

2012, 26, 1465-1474.

538

26.

539

Mammals: A Comparative Approach. In U.S. Environmental Protection Agency, O. o. P.,

540

Pesticides and Toxic Substances, Office of Pesticide Programs, Ed. U.S. Government Printing

541

Office: Washington, DC, 2004.

542

27.

543

FOUR SPECIES OF BIRDS OF PREY PRESENTED TO A WILDLIFE CLINIC IN

544

MASSACHUSETTS, 2006-2010. J. Zoo Wildl. Med. 2011, 42, 88-97.

545

28.

546

Anticoagulant exposure and notoedric mange in bobcats and mountain lions in urban southern

547

California. Journal of Wildlife Management 2007, 71, 1874-1884.

548

29.

549

of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal. Chem.

550

2003, 75, 3019-3030.

Vudathala, D.; Cummings, M.; Murphy, L., Analysis of Multiple Anticoagulant

US Food & Drug Administration, O. o. F. a. V. M., Chemical Method Validation McClure, F. D.; Lee, J. K., Computation of HORRAT values. J. AOAC Int. 2003, 86, Kushnir, M. M.; Rockwood, A. L.; Bergquist, B. J., Liquid Chromatography-Tandem

Yuan, L.; Zhang, D. X.; Jemal, M.; Aubry, A. F., Systematic evaluation of the root cause

Erickson, W.; Urban, D., Potential Risks of Nine Rodenticides to Birds and Nontarget

Murray, M., ANTICOAGULANT RODENTICIDE EXPOSURE AND TOXICOSIS IN

Riley, S. P. D.; Bromley, C.; Poppenga, R. H.; Uzal, F. A.; Whited, L.; Sauvajot, R. M.,

Matuszewski, B. K.; Constanzer, M. L.; Chavez-Eng, C. M., Strategies for the assessment

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551

30.

Lodal, J.; Hansen, O.C. Human and environmental exposure for rodenticides - Focus on

552

the Nordic countries; Nordic Council of Ministers, TemaNord: Copenhagen, Denmark, 2002.

553

31.

554

Steric Constants, Washington, D.C.: American Chemical Society, 1995.

Hansch, C.; Leo, A.; Hoekman, D. Exploring QSAR - Hydrophobic, Electronic, and

555 556

FIGURE CAPTIONS:

557

Figure 1. Chemical structures for anticoagulant rodenticides

558

Figure 2. Typical MRM chromatograms of (I) warfarin, (II) coumachlor, (III) diphacinone, (IV)

559

dicoumarol, (V) chlorophacinone, (VI) bromadiolone, (VII) brodifacoum and (VIII) difethialone.

560

(A) Blank liver; (B) SRM of quantifying ion transition for liver spiked with ARs (25 ng/g); (C)

561

SRM of confirming ion transition for spiked liver. Note the vertical scale for (A) is

562

approximately 2 orders of magnitude lower than (B) and (C).

25



Page 26 of 33

Table 1. MS/MS acquisition parameters for eight anticoagulant rodenticides Thermo Scientific TSQ Quantum Access Max retention time

rodenticide warfarin

(min)

2.00

coumachlor

4.87

diphacinone

7.48

dicoumarol

7.68

chlorophacinone

11.08

bromadiolone

13.76

b

brodifacoum

16.28

difethialone

16.53

a b

precursor ion ((M-H+)-; u) a

307 307 341 341 339 339 335 335 373 373 525 525 521 521 537 537

Thermo Scientific Q Exactive

fragment ion

collision energy

tube lens

precursor ion

fragment ion

(u)

(eV)

(V)

((M-H+)-; u)

normalized collision energy

(u)

(NCE; eV)

161 250 284 161 167 165 161 117 201 145 250 273 135 143 151 371

22 25 26 23 28 48 21 47 24 25 38 40 40 57 41 35

70 70 71 71 77 77 47 47 76 76 97 97 101 101 100 100

307.098

161.024

30

341.059

161.024

30

339.103

167.086

35

335.055

161.024

50

373.064

201.047

30

525.071

250.063

40

521.076

78.918

40

537.053

78.918

35

Transitions in bold were used for quantitation Two isomers are present for bromadiolone; the earliest eluting (and most abundant) isomer was used for detection and quantitation.

26


Page 27 of 33


Table 2. Overview of AR recovery with respect to matrix effects and sample preparation (e.g. homogenization, liquid-solid extraction and d-SPE clean-up processing) matrix effectsa sample preparation recoveryb (mean ± SD; %) (mean ± SD; %) rodenticide 50 ng/g 500 ng/g 2000 ng/g 50 ng/g 500 ng/g 2000 ng/g warfarin 90 ± 17 90 ± 17 101 ± 9 63 ± 2 65 ± 3 65 ± 5 coumachlor 99 ± 5 98 ± 6 103 ± 8 65 ± 4 68 ± 2 72 ± 2 diphacinone 99 ± 7 100 ± 11 105 ± 7 51 ± 8 59 ± 1 65 ± 5 dicoumarol 99 ± 7 100 ± 11 105 ± 6 63 ± 9 65 ± 3 69 ± 2 chlorophacinone 92 ± 5 103 ± 7 120 ± 37 65 ± 5 59 ± 2 68 ± 2 bromadiolone 100 ± 26 110 ± 10 110 ± 35 70 ± 9 66 ± 4 70 ± 2 brodifacoum 90 ± 22 106 ± 4 121 ± 3 65 ± 7 66 ± 2 68 ± 9 difethialone 115 ± 8 107 ± 7 116 ± 7 61 ± 7 61 ± 3 63 ± 7 a b

n = 4 across 3 different days n = 3 across 3 different days

27



Table 3. Inter-assay accuracy and precision of AR method at the methodoriginating laboratory

rodenticide warfarin coumachlor diphacinone dicoumarol chlorophacinone bromadiolone brodifacoum difethialone a b

25 ng/g 63 (11) 69 (13) 84 (7) 75 (8) 70 (10) 71 (13) 80 (9) 88 (22)

method accuracya,b (mean (RSDr); %) 50 500 1000 ng/g ng/g ng/g 106 (8) 104 (5) 103 (5) 104 (8) 104 (3) 99 (5) 104 (8) 96 (7) 102 (15) 104 (6) 98 (6) 98 (9) 102 (6) 100 (8) 105 (3) 110 (5) 98 (2) 100 (10) 104 (10) 96 (7) 107 (16) 100 (12) 92 (8) 98 (9)

2000 ng/g 105 (8) 108 (2) 106 (4) 107 (5) 100 (10) 99 (3) 105 (7) 105 (5)

n = 3 across 3 days for 25 and 1000 ng/g n = 5 across 5 days for 50, 500 and 2000 ng/g

28


Page 28 of 33

Page 29 of 33


Table 4. Quantitative results obtained at the method-originating laboratory on blinded samples prepared by an independent laboratory rodenticide (mean ± SD; ng/g)c

sample type 1

source species Equine

sample pretreatmenta untreated

spike levelb (ng/g) 50

warfarin

coumachlor

diphacinone

dicoumarol

chlorophacinone

bromadiolone

brodifacoum

difethialone

57.2 ± 1.1

53.7 ± 10.6

43.8 ±.3

46.2 ± 1.1

48.1 ± 1.9

50.3 ± 2.6

50.6 ± 2.6

42.9 ± 2.8

2

Equine

lysed cells

50

52.4 ± 8.4

48.1 ± 4.4

42.2 ± 1.9

43.2 ± 1.7

47.1 ± 3.2

44.9 ± 2.3

48.8 ± 0.1

46.2 ± 3.7

3

Equine

lysed cells

50

57.7 ± 0.9

51.6 ± 1.3

44.4 ± 1.4

44.5 ± 0.4

50.0 ± 1.5

50.0 ± 1.1

50.2 ± 3.0

40.4 ± 1.5

4

Porcine

untreated

50

56.5 ± 1.4

54.3 ± 2.5

50.1 ± 3.3

49.1 ± 2.8

52.0 ± 2.8

55.9 ± 1.6

58.7 ± 1.2

54.1 ± 9.8

5

Porcine

lysed cells

50

56.8 ± 3.0

51.1 ± 1.8

49.0 ± 2.7

48.0 ± 1.7

53.7 ± 5.5

49.0 ± 3.6

53.0 ± 3.9

53.0 ± 1.5

Untreated tissue was homogenized to uniform consistency prior to pre-weighing and spiking; Lysed tissue was homogenized, sonicated and frozen/thawed repeatedly prior to pre-weighing and spiking. b Sample type 3 was spiked at the method-originating laboratory (not blinded) for quality control purposes; all other test samples were pre-weighed, spiked and randomized by an independent laboratory in blinded manner. c n=2 a

29



Table 5. Results obtained at the method-originating and collaborating laboratories on blinded samples prepared by an independent laboratory rodenticide warfarin

coumachlor

diphacinone

dicoumarol

chlorophacinone

bromadiolone

brodifacoum

difethialone

spike level

pooled meana

SDRb

RSDR

PRSDR

(ng/g)

(ng/g)

(ng/g)

(%)

(%)

HORRAT

50

66

4

6

25

0.2

1100

1256

206

16

16

1.0

1800

2065

372

18

15

1.2

50

74

5

6

25

0.3

900

1062

121

11

16

0.7

1900

2133

245

11

15

0.8

50

59

17

29

25

1.2

1000

1171

221

19

16

1.2

1950

2513

425

17

15

1.2

50

94

11

12

25

0.5

800

1232

262

21

16

1.3

1900

1869

156

8

15

0.6

50

68

6

9

25

0.4

1100

1204

130

11

16

0.7

1850

1911

106

6

15

0.4

50

60

12

20

25

0.8

800

716

144

20

16

1.3

1900

1812

393

22

15

1.5

50

68

5

8

25

0.3

1000

900

64

7

16

0.4

1800

1635

179

11

15

0.7

50

57

14

24

25

0.9

1150

929

139

15

16

0.9

1800

1881

397

21

15

1.4

n = 6; 3 samples were analyzed at each laboratory b standard deviation for pooled data set a

30


Page 30 of 33

Page 31 of 33


Figure 1.

General Hydroxycoumarin Structure Category Hydroxycoumarin

Indanedione

General Indanedione Structure

Warfarin (2.6)a

Rodenticide Substituent (R) Dicoumarol (2.07)b Coumachlor

Bromadiolone (4.27)

Brodifacoum (est. 8.5)

Diphacinone (4.27)

Chlorophacinone (4.22)

a

Partition coefficients (log Kow) from Ref. 30 Partition coefficient (log Kow) from Ref. 31 c Complete structure shown.

b


Difethialonec (5.17)


Figure 2: Typical MRM chromatograms of (I) warfarin, (II) coumachlor, (III) diphacinone, (IV) dicoumarol, (V) chlorophacinone, (VI) bromadiolone, (VII) brodifacoum and (VIII) difethialone. (A) Blank liver; (B) SRM of quantifying ion transition for liver spiked with ARs (25 ng/g); (C) SRM of confirming ion transition for spiked liver. Note the vertical scale for (A) is ~2 orders of magnitude lower than (B) and (C). 226x273mm (300 x 300 DPI)


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N/A (This is the Table of Contents Graphic) 254x190mm (300 x 300 DPI)


MS Assay for

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