What’s going on in a What’s going on in a Veterinary Toxicology Veterinary Toxicology

Laboratory?Laboratory?

Birgit PuschnerBirgit PuschnerVeterinary ToxicologistVeterinary ToxicologistDVM, PhD, Dipl. ABVTDVM, PhD, Dipl. ABVT

bpuschner@ucdavis.edubpuschner@ucdavis.edu

Toxicology Laboratory Toxicology Laboratory CapabilitiesCapabilities

Providing informationProviding information Diagnosis of intoxications and Diagnosis of intoxications and

nutritional imbalancesnutritional imbalances Prevention of intoxicationsPrevention of intoxications Ensuring food safetyEnsuring food safety

Prevention/identification of Prevention/identification of chemical agroterrorismchemical agroterrorism

Laboratory Laboratory Set-upSet-up

2 ABVT certified toxicologists2 ABVT certified toxicologists 1 resident/PhD student1 resident/PhD student 1 Senior analytical chemist 1 Senior analytical chemist

(PhD)(PhD) 10 analytical chemists (M.S., 10 analytical chemists (M.S.,

B.S)B.S) Sample receiving areaSample receiving area

Toxicology TestingToxicology Testing

450 to 500 case submissions 450 to 500 case submissions per monthper month

~35,000 tests per year~35,000 tests per year Chain-of-custodyChain-of-custody QC/QA in placeQC/QA in place Over 80 validated analytical Over 80 validated analytical

methodsmethods Established turn-around-timesEstablished turn-around-times Method developmentMethod development

Toxicology Toxicology InstrumentationInstrumentation

Triple quadrupole LC-MS/MS Triple quadrupole LC-MS/MS (2) (2)

Ion-trap LC-MS/MS (1)Ion-trap LC-MS/MS (1) HPLC and GC/MSHPLC and GC/MS ECD, FPD, FID, NPD, UV, and ECD, FPD, FID, NPD, UV, and

fluorescence detectorsfluorescence detectors ICP-MS (1)ICP-MS (1) ICP/AES (1)ICP/AES (1) AA (1)AA (1) Elisa plate reader (2)Elisa plate reader (2)

Case DiscussionsCase Discussions

OleanderOleander ParaquatParaquat Zinc phosphideZinc phosphide Anatoxin-aAnatoxin-a

OleanderOleander

Highly toxicHighly toxic Dried and Dried and

freshfresh ~7 leaves can ~7 leaves can

kill a horse or kill a horse or cowcow

~1 leaf can ~1 leaf can kill a humankill a human

Oleander Method by LC-Oleander Method by LC-MSMS

Serum, urine and tissuesSerum, urine and tissues MDL (serum, urine): 1 ppbMDL (serum, urine): 1 ppb MDL (liver): 5 ppbMDL (liver): 5 ppb

Mass spec identification and Mass spec identification and quantitationquantitation

C:\Xcalibur\...\OLE\0105-19_010107153044 01/07/01 03:30:44 PM D0013527-D458 Heart Blood, 20 ul

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RT: 5.57MA: 4 70 5834

NL: 2 .24E5

m/z= 354 .5-355.5+372 .5-373 .5 F: + c ESI Full ms3 577.0 0@19 .50 43 3 .00@18 .00 [ 115.00 -6 00 .0 0 ] MS 0105-19_01010 715304 4

0105-19_010107153044#215-237 RT: 5.24-5.69 AV: 23 SB: 42 4.78-5.20, 5.93-6.37 NL: 1.30E5F: + c ESI Full ms3 577.00@19.50 433.00@18.00 [ 115.00-600.00]

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432.7354.8 373.9 404.2 434.0337.0 464.7 495.0295.5

Oleandrin

Oleandrin in Human Blood – Oleandrin in Human Blood – 9 ppb9 ppb

Tests for Plant ToxinsTests for Plant Toxins Cardiotoxin screen: oleandrin, Cardiotoxin screen: oleandrin,

gitoxin, GTXsgitoxin, GTXs StrophanthidinStrophanthidin Alkaloids: Alkaloids:

coniine, nicotine, anabasine, lupanineconiine, nicotine, anabasine, lupanine atropine, scopolamineatropine, scopolamine taxinestaxines senecionine, seneciophylline, retrorsinesenecionine, seneciophylline, retrorsine

Nitrate/nitriteNitrate/nitrite CyanideCyanide GossypolGossypol

ParaquatParaquat History:History:

OR in 2003 and 2004: more than 20 dogs OR in 2003 and 2004: more than 20 dogs become ill after visiting an off-leash park become ill after visiting an off-leash park and a beach area. Most of them died. and a beach area. Most of them died.

Acute onset of vomiting and diarrhea Acute onset of vomiting and diarrhea after park or beach visit.after park or beach visit.

Many dogs were not presented until 2-3 Many dogs were not presented until 2-3 days.days.

Inappetence and lethargy, renal and Inappetence and lethargy, renal and respiratory failurerespiratory failure

Samples collected for toxicology Samples collected for toxicology testing:testing: Urine, lung, vomitusUrine, lung, vomitus

ParaquatParaquat Colorimetric, SPE quick testColorimetric, SPE quick test Development of a new LC/MS methodologyDevelopment of a new LC/MS methodology

SampleSample SPESPE LC/MSLC/MSUrine #1Urine #1 PositivePositive N/AN/A

Urine #2Urine #2 NegativeNegative Positive (10-50 ppb)Positive (10-50 ppb)

Urine #3Urine #3 NegativeNegative Positive (10-50 ppb)Positive (10-50 ppb)

Urine #4Urine #4 NegativeNegative Positive (10-50 ppb)Positive (10-50 ppb)

LungLung NegativeNegative Positive (0.5 ppm)Positive (0.5 ppm)

VomitusVomitus PositivePositive Positive (1-10%)Positive (1-10%)

Paraquat LC/MS – Paraquat LC/MS – ImplicationsImplications

Provides unambiguous identificationProvides unambiguous identification Very fast: analysis within 1-2 hoursVery fast: analysis within 1-2 hours Low detection limits:Low detection limits:

Urine: 50 ppbUrine: 50 ppb Lung: 500 ppbLung: 500 ppb

Quantitation of paraquat in serum or Quantitation of paraquat in serum or plasma of poisoned animals or plasma of poisoned animals or humans:humans: Used to assess the severity of the intoxicationUsed to assess the severity of the intoxication Used as a prognostic indicatorUsed as a prognostic indicator

Zinc PhosphideZinc Phosphide

History:History: Puppy presented in critical condition at vet Puppy presented in critical condition at vet

clinicclinic Owners mentioned “gopher bait” in Owners mentioned “gopher bait” in

backyardbackyard Vets and veterinary technicians developed Vets and veterinary technicians developed

severe headaches, itchy skin and sore severe headaches, itchy skin and sore throatsthroats

Samples collected for toxicology Samples collected for toxicology testing:testing: Stomach contentsStomach contents

Zinc PhosphideZinc Phosphide

Diagnostic testing:Diagnostic testing: SPME extraction, followed by GC/MSSPME extraction, followed by GC/MS Confirmed presence of phosphineConfirmed presence of phosphine

Phosphide-containing gopher baits:Phosphide-containing gopher baits: Grain bait, paste, tablets, tracking powderGrain bait, paste, tablets, tracking powder Concentration from 0.5% to 10%Concentration from 0.5% to 10% Minimum lethal dose for most species:12 – Minimum lethal dose for most species:12 –

50 mg/kg50 mg/kg Precaution when collecting samples!Precaution when collecting samples!

Tests for Tests for Pesticides/Household ToxinsPesticides/Household Toxins Insecticides:Insecticides:

Organophosphates Organophosphates (43)(43)

Carbamates (11)Carbamates (11) Organochlorines (22)Organochlorines (22) Pyrethrins (8)Pyrethrins (8)

Rodenticides:Rodenticides: Anticoagulant Anticoagulant

rodenticides (8)rodenticides (8) StrychnineStrychnine BromethalinBromethalin Zinc phosphideZinc phosphide

Others:Others: AvitrolAvitrol ParaquatParaquat MetaldehydeMetaldehyde Ethylene glycolEthylene glycol CyanideCyanide

Drugs:Drugs: NSAIDsNSAIDs Macrolide Macrolide

endectocidesendectocides

CyanotoxinsCyanotoxins

History:History: 3 dogs developed seizures and died after 3 dogs developed seizures and died after

swimming in the Eel River swimming in the Eel River Samples collected for toxicology Samples collected for toxicology

testing:testing: Stomach contentsStomach contents LiverLiver WaterWater

Initial testing: Strychnine, Initial testing: Strychnine, metaldehyde, Zinc/Al phosphide, OPs metaldehyde, Zinc/Al phosphide, OPs and carbamatesand carbamates

Algae IdentificationAlgae Identification

Water sample from Eel River

? Planktothrix sp.

Lyngbya sp.

Planktothrix spp.

CyanotoxinsCyanotoxins LC/MS analysisLC/MS analysis Confirmed presence of anatoxin-Confirmed presence of anatoxin-

a in stomach contents and watera in stomach contents and waterD:\z-ck\U.C. Davis\Dogs\S42\fsmsms 05/14/2003 08:31:41 PM

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166.00

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165.93165.92165.76

NL:8.87E7

Base Peak m/z= 165.5-166.5 F: + c ESI SIM ms [ 165.50-166.50] MS sim

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NL: 1.92E7

Base Peak m/z= 148.5-149.5 F: + c ESI Full ms2 166.00@38.00 [ 50.00-300.00] MS fsmsms

fsmsms#1001 RT: 8.03 AV: 1 NL: 1.25E7F: + c ESI Full ms2 166.00@38.00 [ 50.00-300.00]

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Tests for Natural ToxinsTests for Natural Toxins

Algal toxins:Algal toxins: MicrocystinsMicrocystins Anatoxin-aAnatoxin-a Domoic acidDomoic acid SaxitoxinsSaxitoxins

Mushroom toxinsMushroom toxins AmanitinsAmanitins

MycotoxinsMycotoxins AflatoxinAflatoxin TricothecenesTricothecenes FumonisinFumonisin OchratoxinOchratoxin ZearalenoneZearalenone Penitrem Penitrem

A/RoquefortineA/Roquefortine

Tests for Metals and Tests for Metals and MineralsMinerals

Metals:Metals: Lead, arsenic, mercury, cadmium, Lead, arsenic, mercury, cadmium,

manganesemanganese Copper, zinc, selenium, molybdenum, ironCopper, zinc, selenium, molybdenum, iron Thallium, barium, and many othersThallium, barium, and many others

Minerals:Minerals: Calcium, magnesium, sodium, sulfur, Calcium, magnesium, sodium, sulfur,

potassiumpotassium Vitamins:Vitamins:

Vitamin A and EVitamin A and E

TOXICOLOGISTSDr. Birgit Puschner

Dr. Robert Poppenga

Dr. Asheesh TiwaryPHONE: (530) 752-

6322http://

cahfs.ucdavis.edu/