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A Method for the Detection of Desmethylbromethalin in Animal Tissue Samples for the Determination of Bromethalin Exposure Michael Filigenzi, Adrienne Bautista, Linda Aston, and Robert Poppenga J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5052706 • Publication Date (Web): 17 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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A Method for the Detection of

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Desmethylbromethalin in Animal Tissue

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Samples for the Determination of Bromethalin

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Exposure

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Michael S. Filigenzi*, Adrienne C. Bautista, Linda S. Aston, Robert H. Poppenga

7 8 9

California Animal Health and Food Safety Laboratory System, Toxicology Laboratory

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University of California, Davis, CA 95616

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TITLE RUNNING HEAD: Desmethylbromethalin Determination by LC-MS

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*To whom correspondence should be addressed: Tel: 530-754-5608; Fax 530-752-3361;

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E-mail: [email protected]

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1

ACS Paragon Plus Environment


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ABSTRACT

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Bromethalin, a potent neurotoxin, is widely available for use as a rodenticide. As access

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to other rodenticides is reduced due to regulatory pressure, the use of bromethalin is

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likely to increase with a concomitant increase in poisonings in non-target animals.

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Analytical methods for the detection of bromethalin residues in animals suspected to have

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been exposed to this rodenticide are needed to support post-mortem diagnosis of

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toxicosis. This article describes a novel method for the analysis of desmethylbromethalin,

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bromethalin’s toxic metabolite, in tissue samples such as liver, brain, and adipose.

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Samples were extracted with 5 % ethanol in ethyl acetate and an aliquot of the extract

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was evaporated dry, reconstituted, and analyzed by reverse phase ultrahigh performance

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liquid chromatograph mass spectrometry. The mass spectrometer utilized electrospray

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ionization in negative ion mode with multiple reaction monitoring. This method was

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qualitatively validated at a level of 1.0 ng/g in liver tissue. The quantitative potential of

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the method was also evaluated and a method detection limit of 0.35 ng/g wet weight was

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determined in fat tissue. Desmethylbromethalin was detected in tissue samples from

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animals suspected to have been poisoned by this compound. To our knowledge, there

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have been no other methods reported for analysis of DMB in tissue samples using LC-

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MS/MS.

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Keywords: Bromethalin, desmethylbromethalin, electrospray, LC-MS/MS, poisoning

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Introduction

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Bromethalin (Figure 1a) is a substituted diphenylamine used for rodent control. It is

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widely available to the general public in a variety of formulations. Bromethalin was

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developed from an analog which was originally considered for use as a fungicide, but

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which was determined to be overly toxic for that purpose. Desmethylbromethalin (DMB,

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Figure 1b) was determined to be an effective rodenticide; however, low palatability in

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wild rats required the addition of a methyl group to eliminate an acidic proton.1 This

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resulted in the commercial product that is used currently.2

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Commercially available as 0.01% bait pellets, bars and place packs, the rodenticide

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bromethalin selectively targets the nervous system. Following ingestion, bromethalin is

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metabolized by the liver to DMB, the active metabolite. Desmethylbromethalin

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uncouples mitochondrial oxidative phosphorylation leading to decreased cellular

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adenosine triphosphate production and disruption of sodium-potassium ATPase pumps.3

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With decreased activity of the ATPase pumps, the central nervous system (CNS) no

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longer maintains a normal sodium-potassium gradient leading to fluid buildup inside the

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CNS and subsequently the development of cerebral edema and increased cerebrospinal

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fluid pressures.

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Clinical signs of intoxication, including muscle tremors, hyperthermia, hyperexcitability

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and focal or generalized seizures which have been observed in rodents, cats, dogs,

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monkeys and other wildlife such as raccoons, usually develop within 24 hours after

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ingestion of bromethalin at or above the median lethal dose.2-3 At concentrations below

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the median lethal dose, lethargy, hind limb weakness and/or paralysis can develop several

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days after exposure.4

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Documented oral median lethal doses range from as low as 1.8 mg/kg in the cat to as high

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as 13 mg/kg in the rabbit with dogs and monkeys falling in between at around 5 mg/kg.2

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Bromethalin poisonings have been documented in non-target animals

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fatality in a human.8

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5,6,7

and in one

Recently, the EPA has canceled the registrations of a number of second generation formulations.9

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anticoagulant

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commercially available rodenticides and it is highly likely that bromethalin use will

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increase along with the attendant non-target poisonings. Therefore, a reliable method of

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determination of bromethalin and/or its metabolites in tissue samples is critical for

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confirmation of bromethalin exposure and toxicosis post-mortem. The distribution and

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accumulation of bromethalin and DMB in adipose tissue is expected based on their

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octanol-water partition coefficients (log P values) of 6.70 and 4.26, respectively.

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Consequently, adipose tissue may serve as the best diagnostic sample7 and could easily

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be harvested ante- or post-mortem.

rodenticide

There

are

relatively

few

alternative

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Bromethalin rapidly metabolizes to DMB2. Thus, the ability to detect DMB would

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serve as confirmation of exposure to the neurotoxic rodenticide, bromethalin. This is

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particularly important in cases in which there is no history of exposure to the rodenticide.

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MATERIALS AND METHODS

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HPLC grade methanol, water, acetonitrile, ethyl acetate, and formic acid were obtained

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from Fisher Scientific (Pittsburgh, PA). Anhydrous USP grade ethanol (Koptec brand)

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was obtained from VWR (Radnor, PA).

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Analytical standards. Bromethalin (99.3%) was obtained from the U.S. Environmental

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Protection Agency National Pesticide Standard Repository (Fort Meade, MD). DMB

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(98%) was obtained from Toronto Research Chemicals (Toronto, Ontario).

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Control Matrices. Fresh control bovine adipose tissue was obtained from animals

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submitted to the California Animal Health and Food Safety Laboratory for necropsy.

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Fresh control liver tissue was purchased from a local market. Control samples were tested

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to insure that no detectible DMB was present and were stored at -20°C.

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Extraction of DMB from tissue. One g. of tissue (adipose or liver) was weighed into a

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250 mL French square homogenization jar. Fifty mL of 5% ethanol in ethyl acetate were

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added and the sample was homogenized using an Ultra-Turrax homogenizer (IKA

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Works, Inc., Wilmington, NC). The homogenate was centrifuged for 5 minutes at 52 g

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and a 5 mL aliquot of the extract was transferred to a clean 15 mL glass tube. The

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solution was dried under nitrogen using an N-Evap nitrogen evaporator (Organomation,

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Berlin, MA). The dried extract was reconstituted in 200 µL of methanol and filtered

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through a 0.45 µm Millex-HV PVDF syringe filter (Millipore, Billerica, MA) into a 2 mL

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autosampler vial.

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Instrument Calibration Standards. Five-point calibration curves for quantification of

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DMB in adipose tissue samples were prepared by fortifying control bovine adipose tissue

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and extracting as per the method listed above. Calibrators were prepared at levels of 1.0,

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5.0, 10, 25, and 50 ng/g. 5



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Quality control samples. Each batch of 3-12 diagnostic samples included an appropriate

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negative control tissue sample and a positive control sample consisting of negative

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control tissue fortified at a level of 1 ng/g of DMB. Fortified samples were prepared by

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adding an appropriate level of standard solution in methanol to the negative control

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matrix and allowing it to equilibrate at least 15 min prior to extraction. Each batch of

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validation samples included an appropriate negative control tissue sample.

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LC-MS/MS Analysis. A model 1290 high performance liquid chromatograph coupled

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to a model 6460 triple stage quadrupole mass spectrometer equipped with a Jetstream

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electrospray interface (Agilent Corp, Santa Clara, CA) was used for all reported analyses.

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The chromatograph was fitted with a 100 mm x 2.1 mm i.d. 1.7 µm Zorbax Eclipse Plus

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C 18 column fitted with an Eclipse Plus guard column (Agilent Corp, Santa Clara, CA).

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The mobile phases consisted of 0.1% aqueous formic acid (channel A), and 0.1% formic

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acid in acetonitrile (channel B). The flow rate was 0.350 mL/min throughout. Gradient

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elution was used beginning at 20% B which was held for 1 min and then ramped to 95%

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B over 8 min. It was held at 95% B for 4 min and then returned to the initial conditions

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and held for 4 min for re-equilibration. The column was maintained at 35 °C. DMB

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eluted at approximately 8.5 min under these conditions. Chromatographic flow was

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diverted to waste for the first 2 min and after 15 min. The injection volume was 10 µL.

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For diode array detection, an Agilent Model 1290 diode array detector was placed in line

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between the HPLC column and the mass spectrometer. The detector was set to acquire

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the UV spectrum in the range of 190 – 400 nm. Agilent’s Masshunter software was used

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for all data acquisition and processing.

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The mass spectrometer was tuned and calibrated in negative ion mode using its

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automated tuning procedure and the provided tuning solution (Agilent Corp.), a

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proprietary mixture of substituted phosphazines and triazines. DMB was monitored by

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multiple reaction monitoring (MRM) using a precursor ion of m/z 562 and product ions of

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m/z 278 and 254. Fragmentation voltage was 135 V and cell accelerating voltage was 7

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V. Collision energies were 25 eV for the m/z 562 m/z 278 transition and 35 eV for the

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m/z 562 m/z 254 transition. Other ion source parameters were as follows: drying gas

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temp = 350 °C, drying gas flow = 10 L/min, nebulizer pressure = 45 psi, sheath gas temp

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= 300 °C, sheath gas flow = 11 L/min, capillary = 4500 V.

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Infusion of bromethalin and DMB were performed by connecting a syringe pump inline

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with the HPLC, directly ahead of the MS ion source. Separate standards for the two

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compounds at 1 µg/mL were infused into the MS at 20 µL/minute.

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Each analytical sequence for qualitative screening began and ended with a 1 ng/mL

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standard of DMB in methanol. Each sequence for quantitative analysis for method

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validation began and ended with a five-point calibration curve extracted from fortified

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negative control matrix.

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A sample was considered positive when a peak was detected at a signal to noise ratio

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greater than 3 within 5% of the retention time established for DMB with the ratio of

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response between the two product ions being within +/- 15% of that determined by

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analysis of the standard. These identification criteria are consistent with those defined in

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the European Commission Health and Consumer Protection Directorate-General

7



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guidance document for analytical quality control and validation procedures for pesticides

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in food and feed (SANCO/12571/2013).10

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Full Scan LC-MS Analysis. A Model 1200 Rapid Resolution HPLC system coupled

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with an Exactive high resolution accurate mass spectrometer (Thermo Corp.) was used

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for full scan LC-MS analysis. The column and HPLC conditions were similar to those

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used for LC-MS/MS analysis. The mass spectrometer was run in negative ion mode with

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electrospray ionization. Mass calibration and instrument tuning for negative ion mode

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were performed less than 24 hours prior to sample analysis. The instrument was tuned

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and calibrated using the solution provided by the manufacturer which included sodium

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dodecyl sulfate, sodium taurocholate, and Ultramark 1621. Spray voltage was 3000 V,

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capillary temperature was 250 °C, and sheath gas and auxiliary gas were set at 50 and 10,

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respectively (arbitrary units). Mass resolution was set at 100,000 and the AGC target was

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1x106.

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Method Validation. The method was validated using criteria for qualitative screening

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methods established in the SANCO/12571/2013 document. Twenty samples each of

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homogenized liver and homogenized brain tissue were fortified with 1 ng/g of DMB (the

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screening detection limit) and analyzed. The method was also evaluated by analyzing six

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replicate control adipose tissue samples fortified at each level of 1.0, 5.0, and 50 ng/g and

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quantifying the detected DMB against the calibration curve determined by analysis of the

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extracted calibrators.

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RESULTS AND DISCUSSION

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Few methods for the analysis of bromethalin and/or DMB in tissue samples have been

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reported. These methods have included thin layer chromatography11, gas chromatography

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with electron capture detection8, and gas chromatography with negative ion chemical

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ionization.8 High pressure liquid chromatography with diode array and atmospheric

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pressure chemical ionization mass spectrometry (HPLC-negative ion-APCI-MS)12 was

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used for the identification of bromethalin in rodenticide bait formulations. The authors

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reported that bromethalin gave a broad and intense UV absorbance band between 300 and

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415 nm. They also reported that negative ion-APCI-MS did not yield pseudomolecular

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ions but instead showed extensive fragmentation with a variety of isotopic clusters related

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to molecular species such as [M-Br]-, [M-HBr]-, and [M-HBr-NO2]-.

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Infusion of a DMB standard solution gave a predominant ion cluster consisting of

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nominal m/z 560, 562, 564, and 566 in ratios consistent with those expected from its [M-

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H]- ions (Figure 2) in negative ion mode. No identifiable signal was detected using

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positive ion mode. Product ions at m/z 278 and 254 from fragmentation of the precursor

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at m/z 562 were chosen for MRM acquisition. Analysis by HPLC-MS/MS showed high

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sensitivity and adequate retention on the reverse phase column.

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For qualitative screening method validation, 20 replicate samples of homogenized liver

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tissue were fortified with DMB at a level of 1 ng/g (the screening detection limit). DMB

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met detection criteria in all 20 samples, demonstrating that it may be detected reliably at

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that level. The primary goal of this work was to develop a method suitable for support of

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post-mortem diagnosis of bromethalin intoxication. Accordingly, it is usually performed

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on a qualitative, “presence/absence” basis. In order to provide further support for the 9



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screening detection limit and evaluation of the method’s quantitative potential we

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performed a quantitative method evaluation in fat tissue. Figures of merit for the fat

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validation data are shown in Table 1. A quadratic model best fit the calibration curve

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giving a correlation coefficient (r2) of 0.996. The method detection limit, calculated using

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Student’s t-value for five degrees of freedom and the 99% confidence level was

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determined to be 0.35 ng/g, supporting the 1 ng/g screening detection limit used for

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qualitative validation. These data indicate that this method is likely to be useful for

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researchers interested in quantitative analysis for DMB. Figure 3 shows extracted ion

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chromatograms for a single injection of an extract from an adipose tissue sample fortified

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with 1 ng/g of DMB. The validation results for fat and liver indicate that this method will

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provide relevant detection limits for analysis of a variety of tissue types.

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This method has been used successfully to establish bromethalin exposure in several

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animals suspected of suffering fatal bromethalin poisoning.7 Figure 4 shows

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chromatograms from a single analysis of fat tissue taken from a fox. This animal had

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displayed signs of severe neurological impairment and was initially suspected of having

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suffered head trauma. No history of bromethalin exposure or use of the rodenticide in the

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area inhabited by the animal was noted on the submission form. Upon necropsy, there

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was no indication of head trauma and the samples taken from the animal were negative

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for canine distemper and rabies. Results of DMB analysis, along with other case

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information including clinical signs consistent with bromethalin toxicosis, were

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considered sufficient to diagnose bromethalin poisoning in this animal. The response of

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detectible DMB in samples from affected animals has ranged from slightly below that of

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the associated 1 ng/g spiked negative control sample to approximately 200 times that of

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the associated spike. 10


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It should be noted that the original goal of this project was the development of an

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analytical procedure to detect bromethalin rather than the demethylated form DMB.

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When a standard of bromethalin was infused into the mass spectrometer, the ions

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detected were inconsistent with those expected from the [M-H]- ions for bromethalin (m/z

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574, 576, 578, and 580). Instead, the dominant signals were consistent with the cluster of

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brominated ions at m/z 560, 562, 564, etc. expected for DMB, albeit with poor sensitivity.

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Initially, it was difficult to determine whether the dominant ion cluster was due to DMB

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in the standard (as a contaminant or a breakdown product of bromethalin) or to

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demethylation of bromethalin in the ion source as there was no DMB standard available

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at the time. LC-MS/MS conditions were developed based on MS/MS of the ions at m/z

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562 and m/z 564 from the bromethalin standard, and analysis of liver and fat from some

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animals suspected of exposure to bromethalin gave positive results. When a DMB

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standard became available and was analyzed using LC-MS/MS, it was determined that

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the retention time and ions measured for DMB were identical to those from the

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bromethalin standard. This raised questions as to the reliability of the bromethalin

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standard material and/or the ability to detect bromethalin itself by electrospray LC-MS.

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In order to resolve these issues, a highly concentrated bromethalin standard (1000

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µg/mL) was analyzed on a system in which a diode array detector was placed ahead of

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the mass spectrometer set to full scan Q1 MS mode, with no MS/MS fragmentation.

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Chromatograms from this analysis are shown in Figure 5. The dominant signal on the

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MS system was that of DMB at m/z 564 (Fig. 5a), corresponding to 3) when plotting the TIC from the m/z 100 - 600 mass range. This 12


12

and also was

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finding is in agreement with the detection of bromethalin by APCI because the retention

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times for diode array and APCI-MS detected coincided. Based on the differences

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between infusion data from our work and that of Mesmer and Flurer, it appears that

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bromethalin responds differently to the APCI and electrospray ionization processes.

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In summary, injection of a 1000 µg/mL bromethalin standard gave a strong response by

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LC-UV but a barely detectible corresponding response by LC-MS/MS for its [M-H]- and

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[M-CH3-H]- ions. Injection of a 1.0 ng/mL standard of DMB consistently gave a peak

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with > 3:1 S:N under the same conditions. A 1000 µg/mL bromethalin standard gave no

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detectible peak in the TIC of a full scan LC-MS analysis while a 100 ng/mL DMB

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standard was clearly detectible under those conditions. Together, these results indicate

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that bromethalin responds very poorly by electrospray LC-MS and that this technique is

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not suitable for its low level detection in tissue samples and that diagnostic analysis of

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tissue samples using electrospray LC-MS should rely on DMB analysis.

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To our knowledge, this is the first detailed report of an LC-MS/MS method for analysis

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of DMB. The method is fast, efficient, and sufficiently sensitive to detect low, clinically

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relevant levels of DMB in complex tissue samples and allows for accurate post mortem

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diagnosis of bromethalin exposure and intoxication.

291 292 293 294

Figure 1 – Structures of a) bromethalin and b) DMB 13



295 296 297

Figure 2 – Negative ion electrospray mass spectrum obtained via infusion of a 10 µg/mL standard solution of DMB.

298 299 300

Figure 3 – MRM chromatograms from a single analysis of adipose tissue spiked with 1 ng/g DMB

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Figure 4 – MRM chromatograms from a single analysis of adipose tissue taken from a fox suspected of having been intoxicated by bromethalin

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Figure 5 – Chromatograms from diode array and MS analysis of a 1 mg/mL bromethalin standard. a) and b) are selected ion chromatograms while c) is a selected wavelength chromatogram from the diode array detector.

308 309

Figure 6 – Diode array spectrum of bromethalin

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Table 1 – Figures of merit for the quantitative method validation

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ACKNOWLEDGEMENTS

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The authors wish to thank Elizabeth Tor and Dr. Birgit Puschner for their invaluable

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assistance in reviewing the data and in preparing the manuscript. We also wish to thank

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Paul Zavitsanos, Steve Royce, and Dr. Jerry Zweigenbaum from Agilent Corporation for

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their kind assistance in obtaining and operating the LC-MS system used for this work.

318 319 320 321 322

Supporting Information Available:

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Literature Cited

324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359

1. Dreikorn, B. A.; Odoherty, G. O. P., The Discovery and Development of Bromethalin, an Acute Rodenticide with a Unique Mode of Action. Acs Sym Ser 1984, 255, 45-63. 2. van Lier, R. B. L.; Cherry, L. D., The toxicity and mechanism of action of bromethalin: a new single-feeding rodenticide. Fundam Appl Toxicol 1988, 11 (4), 66472. 3. Dorman, D. C.; Parker, A. J.; Buck, W. B., Bromethalin toxicosis in the dog. Part I: clinical effects. Journal of the American Animal Hospital Association 1990, 26 (6), 589-594. 4. Dorman, D. C., Bromethalin. In Small Animal Toxicology, Peterson, M. E.; Talcott, P. A., Eds. Elsevier: St. Louis, MO, 2006; pp 609-618. 5. Martin, T.; Johnson, B., A suspected case of bromethalin toxicity in a domestic cat. Veterinary and human toxicology 1989, 31 (3), 239-40. 6. Gupta, R. C., Non-anticoagulant rodenticides. Veterinary Toxicology: Basic and Clinical Principles, 2nd Edition 2012, 698-711. 7. Bautista, A. C.; Woods, L. W.; Filigenzi, M. S.; Puschner, B., Bromethalin poisoning in a raccoon (Procyon lotor): diagnostic considerations and relevance to nontarget wildlife. Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 2014, 26 (1), 154-7. 8. Pasquale-Styles, M. A.; Sochaski, M. A.; Dorman, D. C.; Krell, W. S.; Shah, A. K.; Schmidt, C. J., Fatal bromethalin poisoning. Journal of forensic sciences 2006, 51 (5), 1154-7. 9. U.S.E.P.A. Canceling Some d-CON Mouse and Rat Control Products. http://www2.epa.gov/rodenticides/canceling-some-d-con-mouse-and-rat-control-products (accessed July 16, 2014). 10. DIRECTORATE, E. C. H. C. P., Method Validation & Quality Control Procedures for Pesticide Residues Analysis in Food & Feed. 2013. 11. Braselton, W. E.; Johnson, M., Thin layer chromatography convulsant screen extended by gas chromatography-mass spectrometry. Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 2003, 15 (1), 42-5. 12. Mesmer, M. Z.; Flurer, R. A., Determination of bromethalin in commercial rodenticides found in consumer product samples by HPLC-UV-vis spectrophotometry and HPLC-negative-ion APCI-MS. Journal of chromatographic science 2001, 39 (2), 4953.

360 361

15



Br

Br

N NO2

Br F3C

NO2

a)

Br

Br

NH NO2

Br F3C

NO2

b)

Figure 1

16


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Figure 2

17



m/z 562 254

m/z 562 278 Ratio m/z 254/278 = 0.46

Figure 3

18


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m/z 562 254

m/z 562 278 Ratio m/z 254/278 = 0.40

Figure 4

19



a)

Full Scan m/z 564

b)

Full Scan m/z 576

c)

DAD @ 340 nm

Figure 5 20


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Figure 6

21



Mean % Recovery

% Relative Standard Deviation

1.0

110

11

5.0

110

15

50

105

24

Spike Level (ppb) n=6

Table 1

22


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For Table of Contents Only

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Method for the Detection of Desmethylbromethalin ... - ACS Publications

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