Screening and mass spectral confirmation of beta-lactam antibiotic residues in milk using LC-MS/MS.

Milk is typically screened for beta-lactam antibiotics by nonspecific methods. Although these methods are rapid and sensitive, they are not quantitative and can yield false positive findings. A sensitive and specific method for the quantitation and mass spectral confirmation of five beta-lactam and two cephalosporin antibiotics commonly or potentially used in the dairy industry is described using high-performance liquid chromatography with tandem mass spectrometry. The antibiotics studied were ampicillin, amoxicillin, penicillin G, penicillin V, cloxacillin, cephapirin, and ceftiofur. The antibiotics were extracted from milk with acetonitrile, followed by reversed-phase column cleanup. The extract was analyzed by liquid chromatography coupled with a mass spectrometer, using a water/methanol gradient containing 1% acetic acid on a C-18 reversed-phase column. Determination was by positive ion electrospray ionization and ion trap tandem mass spectrometry. Quantitation was based on the most abundant product ions from fragmentation of the protonated ion for amoxicillin, cephapirin, ampicillin, and ceftiofur and on the fragmentation of the sodium adduct for penicillin G, penicillin V, and cloxacillin. The method was validated at the U.S. FDA tolerance or safe level and at 5 or 2.5 ng/mL for these compounds in bovine milk. Theoretical method detection limits in milk based on a 10:1 signal to noise ratio were 0.2 ng/mL (ampicillin), 0.4 ng/mL (ceftiofur), 0.8 ng/mL (cephapirin), 1 ng/mL (amoxicillin and penicillin G), and 2 ng/mL (cloxacillin and penicillin V) using a nominal sample size of 5 mL.

Determination of grayanotoxins in biological samples by LC-MS/MS.

A rapid LC-MS/MS method was developed for the quantitative determination of grayanotoxins I, II, and III in rumen contents, feces, and urine. The grayanotoxins were extracted from solid samples with methanol. The methanol extract was diluted with water and cleaned up using a reversed phase solid phase extraction column. HPLC separation was performed by reversed phase HPLC using a gradient of water and methanol containing 1% acetic acid. Determination was by positive ion electrospray ionization and ion trap tandem mass spectrometry. Grayanotoxin I quantitation was based on fragmentation of the sodium adduct ion at m/z 435 to a product ion at m/z 375. Grayanotoxins II and III were quantitated on the basis of fragmentation of the ion at m/z 335 to the product ion at m/z 299. The method detection limits were 0.2 microg/g in rumen contents and feces and 0.05 microg/g in urine. Fortifications at the detection limits and 10 times the detection limits of bovine rumen contents, caprine feces, and ovine urine were recovered in the range 80-114%. The diagnostic utility of the method was tested by analyzing samples submitted to the veterinary toxicology laboratory.

Grayanotoxin poisoning in three goats.

Six Nubian goats were exposed to an azalea branch (Rhododendron indica) at the Riverbanks Zoological Park in Columbia, South Carolina. The following day, 3 of the 6 goats were referred to the veterinary hospital of the Riverbanks Zoological Park with bloat, profuseregurgitation, and signs of depression, intermittent head pressing, and fine muscle tremors in the hind limbs. The goats were treated with magnesium hydroxide, activated charcoal, and lactated Ringer's solution and recovered within 24 hours. Definitive diagnosis of grayanotoxin exposure was accomplished by use of liquid chromatography-mass spectrometry analysis of urine and fecal samples. Rhododendron spp are members of the Ericaceae (Heath) family and may contain grayanotoxins, which exert toxic effects by binding to sodium channels in cell membranes and increasing the permeability of sodium ions in excitable membranes. Rhododendron poisoning should be considered in animals with clinical evidence of gastrointestinal tract irritation, cardiac arrhythmias, and neurologic signs.

Multiresidue screen for cardiotoxins by two-dimensional thin-layer chromatography.

A two-dimensional thin-layer chromatographic method was developed for the qualitative determination of the cardiotoxins oleandrin, gitoxin, digitoxin, gitoxigenin, and grayanotoxins I, II, and III in gastrointestinal contents (stomach, rumen, colon, and cecum contents), feces, and plant material. The cardiotoxins were extracted with dichloromethane. The extract was cleaned up by charcoal and reverse phase solid-phase extraction columns. Analysis was performed by two-dimensional thin-layer chromatography on silica gel plates and visualized by aluminum chloride followed by chloramine T spray. The method detection limits were 0.05 microg/g for oleandrin, 0.1 microg/g for gitoxin, and 0.2 microg/g for the other toxicants in gastrointestinal contents and feces and were 5 times higher in plant material. Four replicate fortifications of bovine rumen contents, bovine feces, and alfalfa at these levels were all well recovered. The diagnostic utility of the method was tested by analyzing samples submitted to the veterinary toxicology laboratory.

Blue-green algae toxicosis in cattle.

Twenty-four of 175 heifers died after ingesting water from a stock pond containing blue-green algae (genus Microcystis) in southern Colorado. Affected cattle were found dead or had signs of nervousness, and were recumbent, weak, anorectic, and hypersensitive to noise when first examined. All cattle died within 3 days after the onset of signs. At necropsy, the rumen contained blue-green algae, and the liver was larger than normal, friable, and dark red. The most important histologic lesion was hepatocyte degeneration and necrosis. Intraperitoneal administration of lyophilized cell material from the bloom caused hepatic necrosis and death in mice, and water from the pond had clumps of cells surrounded by a clear calyx, consistent with the appearance of organisms of the genus Microcystis. Samples of pond water were examined by means of high-pressure liquid chromatography; microcystin-LR, one of the hepatotoxins produced by Microcystis spp, was found. Chromatography may be useful in the diagnosis of blue-green algae toxicosis.

Sweet clover poisoning in dairy cattle in California.

Eight of 600 Holstein heifers and cows died after ingestion of sweet clover silage (Melilotus sp) that contained excessive concentrations of dicumarol caused by mold infestation. The cattle developed subcutaneous hemorrhages and bled from the vagina, became weak, were unable to move, and died. To the best of our knowledge, this is the first report of sweet clover poisoning in cattle from California and is discussed in light of previous findings in the Midwest and Canada. Sweet clover poisoning is caused by dicumarol, a fungal metabolite produced from substrates in sweet clover, and is a common livestock problem in the Northern Plains and Canada. Sweet clover poisoning should be considered in livestock animals with clinical evidence of hemostatic dysfunction, prolonged coagulation times, subcutaneous hemorrhages, and hemorrhagic abortions. Definite diagnosis of moldy sweet clover poisoning can be accomplished by analysis of serum and feed samples for dicumarol concentrations.

Diagnosis of oleander poisoning in livestock.

Since mid-1989, 37 cases of oleander poisoning in livestock have been diagnosed at the California Veterinary Diagnostic Laboratory System. The most frequent source for oleander exposure was plant clippings. Sudden death was the most common presenting complaint. Other signs reported included diarrhea, pulmonary edema, tachycardia, cardiac arrhythmias, colic, and lethargy. In the past, a presumptive diagnosis of oleander poisoning could be based only on matching clinical signs with evidence of consumption of oleander. A new 2 dimensional Thin-layer chromatography analysis of ingesta for oleandrin and an awareness of lesions in heart muscle have greatly improved the ability to diagnose oleander toxicosis.

A rapid multiresidue screen for organophosphorus, organochlorine, and N-methyl carbamate insecticides in plant and animal tissues.

A multiresidue screen for the quantitative determination of 43 organophosphorus, 17 organochlorine, and 11 N-methyl carbamate insecticides in 10 g of plant or animal tissues is described. The insecticides are extracted with 5% ethanol in ethyl acetate (v/v). Samples with high lipid content are cleaned up by automated gel permeation chromatography with a 30% ethyl acetate in hexane (v/v) eluant and in-line silica gel minicolumns. Highly pigmented samples are cleaned up with class-specific solid-phase extraction columns. The concentrated extracts are analyzed by selective detection with gas chromatography or liquid chromatography. Recovery of 71 insecticides ranged from 77 to 113%. Analysis of fortified bovine liver (n = 5) resulted in an average recovery of 96 +/- 4% at the 0.5 to 0.05 micrograms/g level. Analysis of fortified alfalfa hay (n = 5) resulted in a mean recovery of 94 +/- 4% at the 0.06 to 0.5 micrograms/g level, and analysis of fortified fresh tomatoes (n = 5) resulted in an average recovery of 97 +/- 3% at the 0.06 to 0.5 micrograms/g level. Method detection limits ranged from 0.02 to 0.5 micrograms/g for the compounds studied with a nominal 10 g sample.

Determination of cholinesterase activity in brain and blood samples using a plate reader.

A rapid method is described for the quantitative determination of cholinesterase activity in large batches of blood and brain samples. The technique is an adaptation of the Ellman procedure for a 96-well microtiter plate reader. Ten samples can be analyzed simultaneously in 5 min, with all calculations, including statistical analysis, done automatically. The method detection limit is 0.1 microM/mL/min for blood and 0.1 microM/g/min for brain samples. The procedure has been applied to the routine analyses of samples presented to the veterinary diagnostic laboratory. Method performance, quality control, and normal ranges of cholinesterase activity in livestock and other animals are described.

Estrogenic activity in forages: diagnostic use of the classical mouse uterine bioassay.

The classical mouse uterine bioassay was evaluated and adapted for routine diagnostic use in response to requests for evaluation of forages suspected of being estrogenic. Forages were extracted in acetone or 10% ethanol in acetone (v/v). Extracts were mixed with ground corn-based mouse feed. Immature female mice (n = 3/group) were fed a total of 100 g of the ground feed for 5 days. Body weights were monitored before and after the trial. After 6 days, the mice were euthanized and uterine weights were determined. Mean uterine weights were compared using 1-way analysis of variance with preselected contrasts for individual means. Selected uteruses were fixed in 10% neutral buffered formalin for histologic examination. Control feeds, diethylstilbestrol (DES), estradiol, coumestrol, feeds with no reported estrogenic properties, and a feed that caused hyperestrogenism in cattle were tested. Moderate levels of estrogenic compounds resulted in dose-responsive uterine enlargements (10-270 ppm coumestrol over 5 days). Extremely high levels of estrogen frequently resulted in feed refusal and lack of uterine enlargement (10 ppm DES, 100 ppm estradiol). Diagnostically significant estrogenic activity was recovered from the feed known to have been estrogenic in cattle. The classical mouse uterine bioassay was relatively inexpensive, quick, repeatable, and capable of detecting clinically relevant coumestrol levels in hay.

Toxicosis in dairy cattle exposed to poison hemlock (Conium maculatum) in hay: isolation of Conium alkaloids in plants, hay, and urine.

Cattle in two herds developed signs of bloating, increased salivation and lacrimation, depression, respiratory distress, ataxia, and death after ingestion of hay that contained large amounts of poison hemlock (Conium maculatum). Twenty of 30 Angus cows and calves were affected in the first herd (2 died). In the second herd, 5 of 30 Holstein heifers were affected (1 died). The Conium alkaloids, coniine and gamma-coniceine, were quantified in the hay, the plants from the responsible hayfield, and the urine of affected animals.

Multiresidue screen for organophosphorus insecticides using gel permeation chromatography--silica gel cleanup.

A multiresidue screen for quantitative determination of 43 organophosphorus insecticides in 5 g of plant and animal tissues is described. The organophosphorus insecticides are extracted with methanol-dichloromethane (10 + 90, v/v) and cleaned up using automated gel permeation chromatography with hexane-ethyl acetate (60 + 40) eluant and in-line silica gel minicolumns. Concentrated extracts are analyzed by gas chromatography with flame photometric detection. The method recovers 43 organophosphorus insecticides in the range of 72 to 115%. Analysis of fortified bovine liver (n = 5) shows an average 95.9 +/- 4.8% recovery at the 0.05 micrograms/g level and 93 +/- 3.8% at the 0.5 micrograms/g level. Analysis of fortified bovine rumen content (n = 5) shows an average 98 +/- 4.2% recovery at the 0.1 micrograms/g level and 98.7 +/- 2.8% at the 1 micrograms/g level. Method detection limits ranged from 0.01 to 0.05 micrograms/g for the compounds studied using a nominal 5 gram sample.

Clinical illness associated with a commercial tick and flea product in dogs and cats.

A commercial flea and tick product containing 9.0% fenvalerate for use in dogs and cats was suspected of causing illness. An acute toxicity study was performed in 10 dogs and 10 cats exposed to the product orally (po) and dermally at differing doses. Samples were obtained for DEET and fenvalerate analysis. Oral dosing of dogs and cats produced severe clinical illness at doses as low as 0.66% of a can (7 ounce spray can)/kg body weight. Dermal application of the product resulted in minor clinical abnormalities in dogs. Oral exposure at 0.5% can/kg body weight resulted in severe illness, and dermal application caused severe illness or death in cats at 20% and 40% of a can/kg body weight. The cats receiving 10% of a can/kg body weight dermally became depressed for several hours but recovered uneventfully. Serum DEET concentrations closely paralleled the clinical signs observed in the animals. Serum concentrations of DEET above 20 ppm were considered diagnostic for intoxication. Urine concentrations of DEET above 1 ppm and tissue (liver, bile, and kidney) concentrations of DEET above 10 ppm were supportive of poisoning; values near 100 ppm were diagnostic for fatal poisoning.