Rapid quantification of ionophores in feeds by liquid chromatography-tandem mass spectrometry.

Ionophores are widely used in veterinary medicine as coccidiostats and for improving nutrient utilization in livestock production. Because of widespread use, ionophores sometimes cause poisoning in livestock. Quantifying concentration of these compounds in feeds for diagnostic purposes is needed. A method with a single step of solvent extraction was developed for rapid simultaneous quantification of monensin, lasalocid, salinomycin, and narasin in feeds by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The ionophores were extracted using methanol:water (90:10). With the high specificity and high sensitivity of tandem mass spectrometry, the extract was introduced for measurement without further processing. The effect of particle size of feeds on extraction efficiency was also investigated. It was found that feeds passing through a 1-mm filter or sieve show better quantitative extraction. Nigericin was used as internal standard for the measurement. The method was validated by fortification of the selected ionophore compounds in horse feed at different concentrations. The typical recovery rate was 69-122%. Meanwhile, various interlaboratory proficiency test samples of different matrices were also quantified as part of the procedure for method validation. A good agreement was found between results and the suggested values. The method is very sensitive, with detection limits between 0.018 µg/g and 0.056 µg/g for the compounds tested. Results showed that the lower limit of quantification was 0.2 µg/g for the ionophore compounds, which is much lower than the contents of the ionophores in medicated feeds, which is generally approximately 10-100 µg/g feed.

Rapid and reliable identification of ionophore antibiotics in feeds by liquid chromatography-tandem mass spectrometry.

Veterinary diagnostic laboratories are constantly required to be innovative to develop quick, reliable, and cost-effective methods for use in diagnosis of intoxications. Livestock intoxication with ionophore antibiotics is sometimes encountered. A rapid qualitative method based on liquid chromatography-tandem mass spectrometry was developed to screen for the presence of monensin, lasalocid, salinomycin, and narasin antibiotics in feeds. This new method has high specificity and high sensitivity, and yet minimum sample preparation was employed. The analytes were extracted in methanol-water (9010) and analyzed without further purification. Because the test uses a single extraction procedure and no cleanup is necessary, its turnaround time is 4 hr. The method applies the concept of "identification points" (IP) score to identify suspected ionophore antibiotics. The total IP score used to identify each of the ionophore antibiotics by this method was 7.0, which exceeds the minimum score of 4.0 required for acceptable identification of unknown compounds. The specificity of the method was achieved using retention time and multiple reaction monitoring with a total IP score of 7.0.

Standardized protocols for plasma clearance of iohexol are not appropriate for determination of glomerular filtration rates in anesthetized California sea lions (Zalophus californianus).

Abstract: Plasma clearance of iohexol was evaluated in eight anesthetized California sea lions (Zalophus californianus), without evidence of renal dysfunction, to determine if the one-compartment model and the sample protocol used in dogs and cats could be applied to this species. Nonlinearity between samples in 75% (6/8) of sea lions voided those results. An additional two anesthetized sea lions were sampled at 5, 30, 45, 60, 120, 180, 240, and 360 min post iohexol injection and semi-logarithmic curves calculated. Plasma iohexol clearance values calculated by one-, two-, and noncompartment models were in poor agreement, suggesting that the standardized protocol described for dogs and cats cannot simply be applied to California sea lions, probably due to the effects of the dive reflex induced during anesthesia.

Health evaluation of free-ranging Humboldt penguins (Spheniscus humboldti) in Peru.

As part of ongoing ecological studies of Humboldt penguins (Spheniscus humboldti) at Punta San Juan, Ica Department, Peru, health surveys were conducted in November 1992, 1993, and 1994. In the three surveys, 98 birds in total were handled for examination, and blood was collected for laboratory analysis from 90 of these birds. All birds seemed to be in good condition. Body weights of females were significantly lower in 1994 than in the other years. Fleas (Parapsyllus humboldti) and ticks (Ornithodoros amblus) were found on the penguins and in their nests. Females had significantly higher plasma calcium and phosphorus levels, and they had lower weights than males. No other differences were found between the sexes. Hematology, plasma chemistries, and plasma mineral levels varied between years. Positive antibody titers for Chlamydophila psittaci (62%), avian adenovirus (7%; 1994 only), paramyxovirus-2 (7%; 1993 only), and Salmonella Pullorum (7%) were found. Plasma chemistry and mineral levels differed between individuals testing positive vs. negative on serologic tests for avian adenovirus and Salmonella Pullorum. Serologic tests for antibodies to avian influenza A virus, avian encephalomyelitis virus, infectious bronchitis virus, avian reovirus, duck viral enteritis virus, equine encephalitis (eastern, western, and Venezuelan) viruses, infectious bursal disease virus, infectious laryngotracheitis virus, Aspergillus sp., and paramyxovirus-1 and -3 were negative. All chlorinated pesticide and polychlorinated biphenyl analyses were below detectable limits.

Utility of high-performance liquid chromatography in the characterization of iatrogenic sodium diatrizoate-induced seizures in a horse.

Diagnostic evaluation of a mildly ataxic young gelding Quarterhorse included a contrast myelogram. Unfortunately, sodium diatrizoate was inappropriately administered by intrathecal injection resulting in severe unrelenting seizure activity and necessitated the destruction of the animal. The contrast agent was identified by analyzing cerebrospinal fluid by high-performance liquid chromatography and confirmed with probe mass spectrometric analysis. This contrast agent is highly ionic, has high osmolarity, and also blocks neurotransmission at specific receptors; for these reasons it is contraindicated for use in myelography. This is the first report of intrathecal administration of this agent in a horse, although there has been a previous report of similar untoward reactions in a dog, and numerous cases have been reported in humans.

Evaluation of iohexol clearance used to estimate glomerular filtration rate in clinically normal foals.

OBJECTIVE: To determine whether pharmacokinetic analysis of data derived from a single i.v. dose of iohexol could be used to predict creatinine clearance and evaluate simplified methods for predicting serum clearance of iohexol with data derived from 2 or 3 blood samples in clinically normal foals. ANIMALS: 10 healthy foals. PROCEDURE: Serum disposition of iohexol and exogenous creatinine clearance was determined simultaneously in each foal (5 males and 5 females). A 3-compartment model of iohexol serum disposition was selected via standard methods. Iohexol clearance calculated from the model was compared with creatinine clearance. Separate limited-sample models were created with various combinations of sample times from the terminal slope of the plasma versus time profile for iohexol. Correction factors were determined for the limited-sample models, and iohexol clearance calculated via each method was compared with exogenous creatinine clearance by use of method comparison techniques. RESULTS: Mean exogenous creatinine clearance was 2.17 mL/min/kg. The disposition of iohexol was best described by a 3-compartment open model. Mean clearance value for iohexol was 2.15 mL/min/kg and was not significantly different from mean creatinine clearance. A method for predicting serum iohexol clearance based on a 2-sample protocol (3- and 4-hour samples) was developed. CONCLUSIONS AND CLINICAL RELEVANCE: Iohexol clearance can be used to predict exogenous creatinine clearance and can be determined from 2 blood samples taken after i.v. injection of iohexol. Appropriate correction factors for adult horses and horses with abnormal glomerular filtration rate need to be determined.

Hematology, plasma biochemistry, and serosurvey for selected infectious agents in southern giant petrels from Patagonia, Argentina.

In conjunction with reproductive and feeding ecology studies on southern giant petrels (SGP, Macronectes giganteus) blood samples were collected for baseline health evaluations. Twenty-five adult SGP from a breeding colony in Chubut, Argentina, were sampled during two consecutive breeding seasons, 1999-2000 (n = 15) and 2000-01 (n = 10). Values for hematology, plasma biochemistry, and minerals are described for 20 birds in apparent good physical condition. A serologic survey of exposure to selected infectious agents was also conducted on all 25 birds sampled. Southern giant petrels were serologically negative for evidence of exposure to infectious laryngotracheitis virus, avian encephalomyelitis virus, avian influenza virus, avian reovirus, infectious bursal disease virus, infectious bronchitis virus, paramyxovirus 1, 2, and 3 virus, Chlamydophila, and Aspergillus. Antibodies to avian adenovirus were found in 14% of SGP during the first sampling season, and 60% in the second year. Additionally, all birds were negative for antibodies to Salmonella pullorum at the first sampling date, but 90% had low titers the following breeding season. This study contributes to understanding the health status of South Atlantic seabirds and to establishment of baseline information for SGP. Long-term monitoring of pelagic predator-scavenger seabirds such as SGP should be established for the surveillance of marine ecosystem health.

Thin layer chromatography convulsant screen extended by gas chromatography-mass spectrometry.

Acute onset convulsive disorders in the canine may result from exposure to a variety of toxicants including strychnine, insecticides, metaldehyde, zinc phosphide, methylxanthines, drugs of abuse, bromethalin, and the tremorgenic mycotoxins (roquefortine and penitrem A). Although several of the above can be identified in a single gas chromatography-mass spectrometry (GC-MS) screen most have to be determined by separate tests. This report describes a modification of the strychnine extraction procedure, which allows thin layer chromatographic (TLC) identification of strychnine, bromethalin, roquefortine, and penitrem A in suspect baits, stomach contents or vomitus, and extends the identification to a wide variety of drugs, pesticides, and environmental contaminants by GC-MS. Samples were mixed with base, extracted into CH2Cl2 and the organic fraction back-extracted with acid. The organic fraction (neutrals) was purified by gel permeation chromatography (GPC) and analyzed by TLC to determine penitrem A and bromethalin. The acidic aqueous fraction was adjusted to pH > 9 and extracted into CH2Cl2. The resulting CH2Cl2 layer (bases) was then analyzed by TLC to determine strychnine and roquefortine. The organic basic and neutral fractions were recombined with a late eluting GPC fraction and analyzed by GC-MS. Of 312 samples analyzed by TLC from 1995 to 2001, 35 were positive for strychnine alone, 58 were positive for both roquefortine and penitrem A, 4 were positive for roquefortine alone, and 1 was positive for bromethalin. None of the samples were positive for penitrem A alone. Samples negative by TLC were analyzed by the GC-MS extended procedure since mid-1999, and 14 have shown positive for a wide variety of compounds with convulsant activity.

Earliest cardiac toxicity induced by iron overload selectively inhibits electrical conduction.

Female guinea pigs were injected intraperitoneally with 0.083 g/kg iron dextran (Fe-D) to achieve progressively increasing levels of iron load; controls received dextran. Delayed and blocked cardiac conductivity at the Purkinje fiber-papillary muscle junction was initially observed with Fe-D loads of 0.33 g/kg. Serial magnetic resonance relaxation time measurements obtained from livers of live animals showed a decrease (8.1 +/- 0.86 vs. 14.8 +/- 1.03 ms in controls, P < 0.001) that was first observed in animals loaded with 0.25 g/kg Fe-D. Iron concentrations in hearts and livers were significantly increased (P < 0.001). Left ventricular pressure measurements on 1.5 g/kg Fe-D animals failed to demonstrate a defect in contractility, but 27% (9/33) (P < 0.050) of the animals died without warning signs. We conclude that 1) initial decreases in liver magnetic resonance-relaxation time occur in the same range of iron excess as the threshold of iron load that induces delay or blockade of cardiac conduction and 2) a high incidence of sudden death, presumably from cardiac arrhythmias, was observed with large doses of iron that did not decrease left ventricular contractility.