J Vet Diagn Invest 4:441-446 (1992)

Confirmation of indandione rodenticide toxicoses by mass spectrometry/mass spectrometry W. Emmett Braselton, Jr., Regg D. Neiger, Robert H. Poppenga Abstract. Mass spectrometry/mass spectrometry (MS/MS) with collision-activated dissociation (CAD) was utilized to unequivocally distinguish 1,3-indandione rodenticides in 2 cases of anticoagulant toxicosis. Anecdotal evidence provided by the veterinarian in a case involving feedlot cows and physical evidence at the site of occurrence in a similar case involving lambs strongly implicated diphenadione (diphacinone; DP) in both instances. However, high performance liquid chromatography indicated chlorophacinone (CP), not DP, was present in the blood samples obtained from both cows and lambs. Intact 1,3-indandiones exhibit poor gas chromatographic properties, so procedures were developed for analysis by MS/MS using a direct exposure probe for sample introduction. The EI mass spectra of DP and CP contained a base peak at m/z 173, with molecular ions (M+) at m/z 340 and m/z 374 (Cl isotope cluster), respectively. Corresponding MS/MS CAD parent ion spectra of m/z 173 showed an ion of m/z 340 for DP and 374 (Cl cluster) for CP. CAD analysis of the blood extracts showed a parent ion scan of m/z 173 identical to that of CP, with the m/z 374 (Cl cluster). (Additional evidence was obtained by MS/MS examination of the CAD daughter ion spectrum of m/z 374.) Blood extracts from the affected animals revealed CAD daughter ion spectra for m/z 374 identical to that of reference CP. Positive confirmation of CP in both cases led to identification of the source of the toxicant and prevention of further animal exposures.

From the Animal Health Diagnostic Laboratory, Michigan State University, East Lansing, MI 48824 (Braselton, Poppenga), and the Animal Disease Research and Diagnostic Laboratory, South Dakota State University, Brookings, SD 57007 (Neiger). Received for publication October 31, 1991.

The determination of trace levels of anticoagulant rodenticides in tissues such as blood, liver, and kidney is often confounded by the complexity of the background material in these matrices. Traditional methods involving determination by thin-layer chroma-

Figure 1. High performance liquid chromatography on a PRP-1 column with the basic solvent system and UV detection at 280 nm. A. Reference chlorophacinone (10 ng). B. Extract of pooled blood from affected lambs (equivalent to 50 mg blood). 441 Downloaded from vdi.sagepub.com by guest on April 13, 2015

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Figure 2. Direct exposure probe electron impact mass spectrum (70 eV) of reference standard. A. Chlorophacinone. B. Diphenadione.

tography (TLC) and high performance liquid chromatography (HPLC) have required extensive cleanup procedures to remove interferences, although recent applications of solid phase extraction techniques have greatly simplified these approaches and reduced detection limits.2 Methods utilizing gas chromatography with electron-capture detection (GC/ECD) and mass spectrometry (GUMS) have greatly reduced detection limits but at the expense of specificity, because the parent compounds must be oxidized to more volatile fragments to achieve desirable gas chromatographic characteristics. 1,4 Recent developments in technology to interface the HPLC and mass spectrometer should allow mass spectral identification of the compounds in reverse-phase liquid chromatography effluents; several hydroxycoumarin-type anticoagulants have now been determined by thermospray and particle beam LC/MS.3 Mass spectrometry/mass spectrometry (MS/ MS) is an emerging technique with particular advan-

tages in analysis of biological mixtures, especially for compounds with poor chromatographic properties. MS/ MS could be used as a final confirmation procedure for anticoagulants in biological tissues, particularly where unequivocal identification is required in cases that may involve litigation. The results of MS/MS studies on two indandione rodenticides, chlorophacinone (CP) and diphenadione (diphacinone; DP) are herein reported. In the winter of 1990, a producer lost 7 animals from a pen of 200 crossbred beef cull cows being fed out for slaughter. The owner found the animals down or dead. The cows died within 12 hours of being found down. Diarrhea was present in several cows before death. Postmortem examination of 1 cow 1 hour after death revealed unclotted blood, a pale, friable liver, and subcutaneous hemorrhage. No hemorrhage was present in body orifices. The veterinarian suspected poisoning and questioned the feedlot owner about possible toxin exposure. The owner recalled finding a packet of rodenticide in corn he had purchased to feed the animals. The attending veterinarian collected and submitted specimens for anticoagulant analysis. The veterinarian had the impression that diphacinone was in the packets of rodenticide found in the corn. Therefore, diphacinone analysis was specifically requested. A similar incident occurred in January 1991, when 4 3-week-old lambs were found dead after being moved out of the lambing barn. Affected lambs came out of a pen that contained 10 ewes and 20 lambs. None of the other animals were sick. The lambing barn was completely enclosed and contained aproximately 100 ewes and 100 lambs. Only half of the ewes had lambed at that time. Grossly, affected lambs had large amounts of hemorrhage. There was major hemorrhage into the peritoneal cavity in 2 of the lambs and in the thoracic cavity and subcutaneous tissue of the neck in the other 2 lambs. Hemorrhage was noted around the tail docking wound of all 4 lambs. A pest exterminator had been on the farm for a rodent problem during the week the lambs were born. The farm manager noted that diphacinone bait packets had been placed in and around the lambing barn. Samples from lambs were submitted for anticoagulant analysis with a specific request for diphacinone determination. Materials and methods Chemicals. Organic solvents were UV grade,a and 18 megohm organic-free water was obtained from a 4-bowl Milli-Q System.b A stock mixture of reference standard anticoagulants contained 1 ml each of 1 mg/ml pindone,c coumafuryl, diphenadione,d and chlorophacinonee in CH3CN; 1 ml of 1 mg/ml dicoumarolf in THF; 0.5 ml each of 1 mg/ml warfarin, coumachlor,g and bromadialoned in CH3CN; and 10 µ1 of 1 mg/ml brodifacoumh in CH3CN diluted to 10 ml with CH3CN.

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Figure 3. Collision-activated dissociation (CAD) parent ion spectrum (parents of m/z 173) of various compounds. A. Reference chlorophacinone. B. Reference diphenadione. C. Blood extract from affected lambs. D. Blood extract from affected cow. CAD was performed with argon at 1 mtorr and a collision energy of - 10 eV.

Blood extraction. Two milliliters of whole blood were mixed with 8 ml of CH3CN, shaken vigorously for 5 min, and centrifuged. An additional 2-ml sample of control blood was fortified with 20 µ1 of the mixed standard and treated as above. The supernatant fraction was transferred to a stoppered 50-ml tube and mixed with 1 ml 1 N H2SO4 and checked for pH < 1. The mixture was extracted twice with 8 ml CH2CL2. The CH2Cl2 phase was dried through anhydrous Na2SO4 and evaporated under N2. The extract was resuspended in 800 µ1 CH 3CN and filtered through a 0.2-µm spin filter for HPLC and TLC analysis. Tissue extraction. Two 10-g samples were measured into small blenders. One was fortified with 100 µ1 of mixed standard. The samples were blended with 40 ml CH3CN and suction filtered through Whatman #4 paper. An additional 40 ml CH3CN was added to the residue, blended, filtered, and combined with the first extract. The volume recovered was recorded, and the extract was transferred to a 250-ml separator-y funnel. The extract was mixed with 80 ml water and 8 ml 4% KC1 and shaken with 100 ml CH2Cl2. The CH2Cl2 fraction was filtered through anhydrous Na2SO4 into a 500-ml round bottom flask. The aqueous phase was adjusted to pH < 1 with about 20 ml 5 N H2SO4 and reextracted twice with 100 ml of CH2Cl2, and the CH2Cl2 fractions were filtered through anhydrous Na2SO2. The combined extracts

were concentrated on a rotary evaporator, transferred to conical centrifuge tubes, and evaporated under N2. Gel permeation chromatography (GPC) clean-up. Tissue samples were resuspended in 200 µ1 CH 3CN and 800 µ1 toluene/CH2Cl2 (85: 15). Each sample was transferred to the top of a 1.5. x 68-cm column of Biobeads SX-3 in toluene/ CH2Cl2 (85: 15) and processed by chromatography at approximately 1 ml/min with the mobile phase toluene/CH2Cl2 (85:15).i The fraction eluting at 55-75 ml (calibrated with reference standards) was collected in a 50-ml tube and evaporated to dryness under N2. Thin-layer chromatography. Samples were resuspended in CH3CN/THF (90: 10) at a concentration of 1 g equivalent sample/20 µ1. The samples were applied to the left comer of a 20- x 20-cm Analtech Uniplate Silica Gel HLF TLC plate, and 1 µg each of the 9 reference standards was applied in individual lanes to the lower right and the upper left comers for 2-dimensional development.j The plates were developed 2 x in “Runner Upper,” ethyl acetate/CH3OH/glacial acetic acid (80: 18:2), to a distance of 1 cm above the sample, with thorough drying between runs. The plates were then developed in ethyl acetate/CH3OH/NH4OH (85:10:5) and dried thoroughly. Plates were turned 45° and developed 2 x in “Runner Upper,” dried thoroughly, and developed in cyclohexane/ethyl acetate/glacial acetic acid (80:18:2). The plates

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Figure 4. Collision-activated dissociation (CAD) parent ion spectrum (parents of m/z 173) of reference chlorophacinone (A) and liver extract from an affected cow (B). CAD conditions were as in the legend to Fig. 3.

were dried thoroughly and viewed under short and long wavelength UV light. High performance liquid chromatography. An acidic reverse-phase HPLC system5 was used to confirm and quantify the hydroxycoumarin anticoagulants and to screen for brodifacoum. A basic system5 was used to confirm brodifacoum and the indandione anticoagulants. Equipment consisted of a binary gradient pumping system, variable wavelength fluorescence detector (ex. 280 nm, em. 410 nm), UV detector (280 nm), Water’s 8 mm x 10 cm Nova-Pak C18 cartridge in RCM with Guard-Pak precolumn (acidic system), and PRP-1 Hamilton MPLC Analytical column, 4.6 mm x 10 cm, with PRP-1 MPLC Microbore Guard Column (basic system).k,l Mass spectrometry. Electron impact (EI) mass spectra were recorded on a Finnigan TSQ-70 triple-stage quadrupole mass spectrometer.m Typical operating conditions for confirmation of indandione residues were as follows: electron energy, 70 eV; electron current, 200 µA; electron multiplier, - 1,200 V; conversion dynode, - 5.0 kV; source, 150 C. Collisionactivated dissociation (CAD) studies were conducted using

Figure 5. Collision-activated dissociation (CAD) daughter ion spectrum (daughters of m/z 374) of reference chlorophacinone (A) and extract of pooled blood from affected lambs (B). CAD conditions were as in the legend to Fig. 3.

Ar as the collision gas at a pressure of 1.0 mtorr with the collision energy at - 10 eV.

Results and discussion Two-dimensional (2D) TLC and HPLC in the acidic system of blood extracts from the affected cow and lambs were negative for the presence of hydroxycoumar-in-based anticoagulants. However, 2D TLC indicated the presence of an indandione rodenticide. Because the Rf values for CP and DP were close in both solvent systems (DP = 0.34 and CP = 0.38 in the first direction; DP = 0.05 and CP = 0.03 in the second direction), HPLC utilizing the basic system was employed to differentiate between the 2 compounds. In the basic system, DP and CP had retention times of 17.85 and 19.44 min, respectively, whereas the unknowns in the blood samples chromatographed at 19.41 min (cow) and 19.40 min (lambs) (Fig. 1), indicating the presence of CP. The area of the chromatogram

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MS/MS confirmation of indandiones

around the retention time of DP was clean in the samples from both species. Quantification by HPLC indicated 2.8 ppm CP in the blood of the cow and 0.14 ppm in the lambs. Because physical evidence at the sites of the poisonings indicated that DP, not CP, may have been involved, confirmation of the HPLC results with mass spectrometry was necessary. Both CP and DP exhibit poor gas chromatographic properties as intact molecules, so experiments were conducted on reference standard indandiones employing volatilization immediately within the ion source of the mass spectrometer using a direct exposure probe for sample introduction. The resulting EI mass spectra (Fig. 2) revealed molecular ions (M+) at m/z 340 and m/z 374 (Cl cluster) for DP and CP, respectively, with a major fragment ion at m/z 173 common to both molecules. This information was utilized to set up MS/MS CAD experiments because direct probe EI MS is not usually clean enough to provide spectra in samples from biological matrices such as blood and liver. CAD spectra of the parent ions of m/z 173 were obtained of the reference CP and DP and compared with those obtained from the blood extracts (Fig. 3). Parent scans of 173 for DP revealed the expected M+ at m/z 340 and for CP the expected M+ at m/z 374 with an isotope cluster characteristic of a single chlorine atom. Samples of blood extracts from both cows and sheep revealed parent ion spectra identical to that of CP. An extract of liver from an affected cow was purified by GPC and was also examined by parent ion scans. A spectrum similar to that of CP, with an M+ at m/z 374 (Cl cluster) was obtained (Fig. 4). Cholesterol was not completely removed by the GPC and contributed low levels of extraneous ions to the spectrum. Additional information was obtained by MS/MS examination of CAD daughter ion spectra of the indandiones. The daughter ion spectrum of the molecular ion (m/z 374) of reference CP contained a base peak at m/z 173, with small but characteristic ions at m/z 202 and m/z 167 (Fig. 5). Daughter ion spectra of m/z 374 obtained on the blood extract from affected lambs (Fig. 5) and liver from an affected cow (Fig. 6) were similar to that of reference CP. The small ion at m/z 167 was not present in the spectrum from the liver extract, possibly because CP was only present at a low concentration. Impurities in the liver extract contributed small amounts of extraneous ions to the spectrum in this case also. The MS/MS spectra provided unequivocal evidence for the presence of CP, not DP. In the case involving feedlot cows, further discussion with the owner revealed that the rodenticide throw packets indeed contained CP and not DP. The owner screened his corn closely and removed rodenticide packets, and deaths

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Figure 6. Collision-activated dissociation (CAD) daughter ion spectrum (daughters of m/z 374) of reference chlorophacinone (A) and extract of liver from an affected cow (B). CAD conditions were as in the legend to Fig. 3.

ceased. In the the case involving lambs, the farm manager was informed of analytical results and asked to look for another source of anticoagulant other than DP. A small hole was discovered in the bottom of the inside wall of the lambing barn in the pen where lambs had been housed. The hole was large enough for the lambs to get their muzzle into but too small for the ewes. Traces of a white powder were present in the hole. Upon questioning, the pest exterminator revealed he had used tracking powder containing CP in the walls of the lambing barn. Differentiating chlorophacinone from other anticoagulants, especially diphacinone in these cases, is needed for a conclusive diagnosis. Discovery of the true source of anticoagulant in the case of the lambs prevented exposure of new lambs going to contaminated pens. Because of the ability to analyze biological mixtures directly,6 MS/MS is a powerful and rapid

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approach to the confirmation of suspected toxicants in the field of veterinary toxicology. Investigations are currently in progress to characterize the MS/MS spectra of additional indandione as well as hydroxycoumarin rodenticides. Acknowledgements We are grateful for the expert technical assistance of Patrick C. Rumler, Michael J. Hauer, and Susan M. Stahl.

Sources and manufacturers a. b. C.

d. e. f.

g. h. i. j.

Burdick and Jackson, Baxter Diagnostics, McGraw Park, IL. Millipore Corp., Bedford, MA. PolyScience Corp., Niles, IL. Chem Service, West Chester, PA. Chempar Chemical Co., New York, NY. ICN Biomedicals, Irvine, CA. Aldrich Chemical Co., Milwaukee, WI. ICI Americas, Goldsboro, NY. Bio-Rad Laboratories, Richmond, CA. Analtech, Newark, DE.

Waters Chromatography Division, Milford, MA. Brownlee Labs, The Anspec Co., Ann Arbor, MI. m. Finnigan MAT, San Jose, CA.

k. l.

References 1. Bullard RW, Holguin G, Peterson JE: 1975, Determination of chlorophacinone and diphenadione residues in biological materials. J Agr Food Chem 23:72-74. 2. Chalermchaikit T, Felice LJ, Murphy MJ: 1992, Simultaneous determination of eight anticoagulant rodenticides in blood serum and liver. J Anal Toxicol (in press). 3. DeVries JX, Kymber KA: 1991, Thermospray and particle beam liquid chromatographic-mass spectrometric analysis of coumarin anticoagulants. J Chromatogr 562:31-38. 4. Ray AC, Murphy MJ, DuVall MD, Reagor JC: 1989, Determination of brodifacoum and bromadiolone residues in rodent and canine liver. Am J Vet Res 50546-550. 5. Reynolds JD: 1980, Extraction and identification of ten anticoagulant rodenticides from baits and stomach contents by high pressure liquid chromatography. Proc Annu Meet Am Assoc Vet Lab Diagn 23: 187-194. 6. Yost RA, Enke CG: 1979, Triple quadrupole mass spectrometry for direct mixture analysis and structure elucidation. Anal Chem 51:1251A-1264A.

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JVDXXX10.1177/1040638712450578

Erratum Journal of Veterinary Diagnostic Investigation 24(4) 813 © 2012 The Author(s) Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1040638712450578 http://jvdi.sagepub.com

Corrigendum

Stegelmeier, BL, et al.: 2010, Experimental rayless goldenrod (Isocoma pluriflora) toxicosis in goats. J Vet Diagn Invest. 22: 570–577

In the article “Experimental rayless goldenrod (Isocoma pluriflora) toxicosis in goats” by Bryan L. Stegelmeier et al., the published mean body weight and the means and statistics of serum biochemistries were carried out on groups of 4 animals, not 3, as described in the Material and Methods section. The additional animal in each group was part of an auxiliary physiologic study and though the animals were dosed and treated the same, they were not necropsied and were not included in the histologic study. To correct this oversight, the corrected weight and chemistry table (shaded cells indicate corrected numbers) are listed below. The differences are minimal and do not alter the conclusions. In addition, reference 7 has been deleted. Material and Methods: “Fifteen, yearling, female Spanish goats weighing 29.4 ± 3.4 kg (mean ± standard deviation) were randomly divided into 5 groups with 3 animals per group.”

References: Reference 7 should be deleted Corrected Table 1. Selected mean serum biochemical data from groups of 3 goats dosed with rayless goldenrod (Isocoma pluriflora) to obtain benzofuran ketone doses of 0, 10, 20, 40, and 60 mg/kg body weight for 7 days.* Serum result (mean ± standard deviation) Serum test (reference range†) Creatinine kinase (< 350 U/l)         Cardiac troponin-I (

mass spectrometry.

Mass spectrometry/mass spectrometry (MS/MS) with collision-activated dissociation (CAD) was utilized to unequivocally distinguish 1,3-indandione roden...
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