Lawrence R. Soma

Renal Papillary Necrosis in Horses after Phenylbutazone and Water Deprivation

Acute renal papillary necrosis occurred in five horses given normal therapeutic doses of phenylbutazone and deprived of water for 36 to 48 hours prior to euthanasia. Five horses given phenylbutazone alone and four horses subjected to water deprivation alone did not develop papillary necrosis. Urinalyses were normal prior to water deprivation, and also after water deprivation in the horses that did not receive phenylbutazone, but the water-deprived, phenylbutazone-treated horses had many red blood cells, transitional epithelial cells, and large numbers of oxalate crystals in their urine. Ulceration of the alimentary tract was seen in more than 50% of these horses. Tongue ulceration was present in one of five horses given phenylbutazone and one of five horses which had phenylbutazone and water deprivation. Ulceration of the gastric mucosa was seen in two of the five phenylbutazone-treated horses, four of five horses with phenylbutazone treatment and water deprivation, and one of four horses with water deprivation alone. Severe colonic ulceration with perforation and peritonitis was present in one horse given phenyl-butazone for three months. No other significant changes in the small or large intestine were seen in the other 13 horses.

Differentiation and identification of recombinant human erythropoietin and darbepoetin Alfa in equine plasma by LC-MS/MS for doping control.

Recombinant human erythropoietin (rhEPO) and darbepoetin alfa (DPO) are protein-based drugs for the treatment of anemia in humans by stimulating erythrocyte production. However, these agents are abused in human and equine sports due to their potential to enhance performance. This paper describes the first method for differentiation and identification of rhEPO and DPO in equine plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method comprised analyte extraction and enrichment by immunoaffinity separation with anti-rhEPO antibodies, dual digestion by trypsin and peptide-N-glycosidase F (PNGase F), and analysis by LC-MS/MS. Two unique deglycosylated tryptic peptides, (21)EAENITTGCAEHCSLNENITVPDTK (45) (T 5) from rhEPO and (77)GQALLVNSSQVNETLQLHVDK (97) (T 9) from DPO, were employed for differentiation and identification of rhEPO and DPO via LC retention times and major product ions. The limit of identification was 0.1 ng/mL for DPO and 0.2 ng/mL for rhEPO in equine plasma, and the limit of detection was 0.05 ng/mL for DPO and 0.1 ng/mL for rhEPO. Analyte carryover problem encountered was solved by adding 20% acetonitrile to the solvent of the sample digest to increase solubility of the peptides. This method was successfully applied to identification of DPO in plasma samples collected from a research horse following DPO administration and from racehorses out of competition in North America. Thus, it provides a powerful tool in the fight against blood doping with rhEPO and DPO in the horse racing industry.

Collision-induced dissociation pathways of anabolic steroids by electrospray ionization tandem mass spectrometry

Anabolic steroids are structurally similar compounds, and their product-ion spectra obtained by tandem mass spectrometry under electrospray ionization conditions are quite difficult to interpret because of poly-ring structures and lack of a charge-retaining center in their chemical structures. In the present study, the fragmentation of nine anabolic steroids of interest to the racing industry was investigated by using triple quadrupole mass spectrometer, Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, and a linear ion trap instrument. With the aid of an expert system software (Mass Frontier version 3.0), accurate mass measurements, and multiple stage tandem mass spectrometric (MSn) experiments, fragmentation pathways were elucidated for boldenone, methandrostenolone, tetrahydrogestrinone (THG), trenbolone, normethandrolone and mibolerone. Small differences in the chemical structures of the steroids, such as an additional double-bond or a methyl group, result in significantly different fragmentation pathways. The fragmentation pathways proposed in this paper allow interpretation of major product ions of other anabolic steroids reported by other researchers in a recent publication [19]. The proposed fragmentation pathways are helpful for characterization of new steroids. The approach used in this study for elucidation of the fragmentation pathways is helpful in interpretation of complicated product-ion spectra of other compounds, drugs and their metabolites.

Equine leukocyte antigens: Relationships with sarcoid tumors and laminitis in two pure breeds

Frequencies of equine leukocyte antigen distribution were determined by complement-mediated cytotoxicity testing among populations of Thoroughbred and Standardbred horses, including animals affected with equine sarcoid and laminitis. A highly significant association is described between the presence or history of sarcoid lesions in Thoroughbreds and the expression of the major histocompatibility complex (MHC)-encoded antigens, W3 and B1. No association was found between antigenic expression frequencies and laminitis in either breed. These findings suggest that a strong relationship exists between the equine MHC and a predisposition to sarcoid.

ANESTHETIC AND ANALGESIC CONSIDERATIONS IN THE EXPERIMENTAL ANIMAL

Many new compounds and combinations of many of the more traditional drugs have been used in a wide range of species. This, plus the rapid development in equipment for inhalational anesthesia, has simplified the restraint, anesthetic, post-surgical, and analgesic management of experimental animals.

Pharmacokinetic profile and behavioral effects of gabapentin in the horse.

Gabapentin is being used in horses although its pharmacokinetic (PK) profile, pharmacodynamic (PD) effects and safety in the equine are not fully investigated. Therefore, we characterized PKs and cardiovascular and behavioral effects of gabapentin in horses. Gabapentin (20 mg/kg) was administered i.v. or p.o. to six horses using a randomized crossover design. Plasma gabapentin concentrations were measured in samples collected 0-48 h postadministration employing liquid chromatography-tandem mass spectrometry. Blood pressures, ECG, and sedation scores were recorded before and for 12 h after gabapentin dosage. Nineteen quantitative measures of behaviors were evaluated. After i.v. gabapentin, the decline in plasma drug concentration over time was best described by a 3-compartment mammillary model. Terminal elimination half-life (t(1/2γ) ) was 8.5 (7.1-13.3) h. After p.o. gabapentin terminal elimination half-life () was 7.7 (6.7-11.9) h. The mean oral bioavailability of gabapentin (± SD) was 16.2 ± 2.8% indicating relatively poor absorption of gabapentin following oral administration in horses. Gabapentin caused a significant increase in sedation scores for 1 h after i.v. dose only (P < 0.05). Among behaviors, drinking frequency was greater and standing rest duration was lower with i.v. gabapentin (P < 0.05). Horses tolerated both i.v. and p.o. gabapentin doses well. There were no significant differences in and . Oral administration yielded much lower plasma concentrations because of low bioavailability.

Identification of racehorse and sample contamination by novel 24-plex STR system

Proper identification of racehorses competing in an official race and maintenance of defensible chain of custody are important in doping control regulations. The purpose of this study was to develop a reliable multiplex PCR method for providing genetic evidence for matching donors to test samples by using short tandem repeat (STR) loci. Amplification of 21 STR loci from blood, urine or hair root was achieved in a single tube and STR length polymorphism was analyzed using fluorescent labeled capillary electrophoresis. This novel approach showed an allele confidence interval of 0.19-0.43 bp and size estimation error of 0-0.48 bp. In 90 thoroughbred (TB) and 171 standardbred (STB) horses, the method was highly discriminating and reproducible with probability of false identification of 1 in 10(11) (TB) and 1 in 10(13) (STB). All loci were highly polymorphic with an average probability of identity of 0.18 (TB) and 0.13 (STB), heterozygosity of 0.65 (TB) and 0.68 (STB), and polymorphism information content (PIC) of 0.62 (TB) and 0.69 (STB). The highest allele frequency also reflected the degree of polymorphism due to high correlation with PIC. To obtain evidence of sample tampering with human material, three human specific STR markers were included in the panel. This method is the first in the horseracing industry, specifically designed for racehorse identification and detection of equine sample contamination by human DNA.

High‐throughput UHPLC–MS/MS method for the detection, quantification and identification of fifty‐five anabolic and androgenic steroids in equine plasma

Anabolic and androgenic steroids (AASs) are synthetic substances related to the primary male sex hormone, testosterone. AASs can be abused in both human and equine sports and, thus, are banned by the International Olympic Committee and the Association of Racing Commissioners International (ARCI). Enforcement of the ban on the use of AASs in racehorses during competition requires a defensible and robust method of analysis. To address this requirement, a high-throughput ultra high-performance liquid chromatography-mass spectrometric (UHPLC-MS) method was developed for the detection, quantification and confirmation of 55 AASs in equine plasma. AASs were recovered from equine plasma samples by liquid-liquid extraction with methyl tert-butyl ether (MTBE). Analytes were chromatographically separated on a sub-2 µm particle size C(18) column with a mobile phase gradient elution and detected by selected-reaction monitoring (SRM) on a triple quadrupole mass spectrometer. AASs with isobaric precursor ions were either chromatographically resolved or mass spectrometrically differentiated by unique precursor-to-product ion transitions. A few of them that could not be resolved by both approaches were differentiated by intensity ratios of three major product ions. All the epimer pairs, testosterone and epitestosterone, boldenone and epiboldenone, nandrolone and epinandrolone, were chromatographically base-line separated. The limit of detection and that of quantification was 50 pg/ml for most of the AASs, and the limit of confirmation was 100-500 pg/ml. Full product ion spectra of AASs at concentrations as low as 100-500 pg/ml in equine plasma were obtained using the triple quadrupole instrument, to provide complementary evidentiary data for confirmation. The method is sensitive and selective for the detection, quantification and confirmation of multiple AASs in a single analysis and will be useful in the fight against doping of racehorses with AASs.

Efficient Use of Retention Time for the Analysis of 302 Drugs in Equine Plasma by Liquid Chromatography-MS/MS with Scheduled Multiple Reaction Monitoring and Instant Library Searching for Doping Control

Multiple drug target analysis (MDTA) used in doping control is more efficient than single drug target analysis (SDTA). The number of drugs with the potential for abuse is so extensive that full coverage is not possible with SDTA. To address this problem, a liquid chromatography tandem mass spectrometric method was developed for simultaneous analysis of 302 drugs using a scheduled multiple reaction monitoring (s-MRM) algorithm. With a known retention time of an analyte, the s-MRM algorithm monitors each MRM transition only around its expected retention time. Analytes were recovered from plasma by liquid-liquid extraction. Information-dependent acquisition (IDA) functionality was used to combine s-MRM with enhanced product ion (EPI) scans within the same chromatographic analysis. An EPI spectrum library was also generated for rapid identification of analytes. Analysis time for the 302 drugs was 7 min. Scheduled MRM improved the quality of the chromatograms, signal response, reproducibility, and enhanced signal-to-noise ratio (S/N), resulting in more data points. Reduction in total cycle time from 2.4 s in conventional MRM (c-MRM) to 1 s in s-MRM allowed completion of the EPI scan at the same time. The speed for screening and identification of multiple drugs in equine plasma for doping control analysis was greatly improved by this method.

Effects of cromolyn in horses with chronic obstructive pulmonary disease.

The effect of disodium cromoglycate (cromolyn) in preventing the pulmonary dysfunction caused by the inhalation of barn and hay dust was studied in 5 horses with confirmed chronic obstructive pulmonary disease (COPD). The horses were studied before (Con) and after exposure to hay and dust allergens (Expos) and after pretreatment with cromolyn followed by exposure (Cr-Expos).There was a significant reduction in PaO2 from 86.8±8.3 to 73.1±8.8 when the horses were exposed to hay and dust allergens. The PaO2 after pretreatment with cromolyn and exposures was 78.1±5.5. There were no significant changes in PaCO2, FRC, pH and A-aDO2 when the Con, Expos, and Cr-Expos periods were compared.There were significant increases in VE from a control value of 77.9±18.2 to 128.7±55.1 and 133.7±17.1 L/min during the exposures, which was due primarily to increases in respiratory frequency. Respiratory dead space (VD/VT) increased from 0.55±0.10 to 0.71±0.07 and 0.65±0.05, and alveolar ventilation (VE) remained constant. Pulmonary resistance (RL) and transpulmonary pressure (Ptr) increased from a control of 0.77±0.28 cm H2O/L/sec and 7.73±3.38 cm H2O to 2.93±1.01 and 20.17±4.81 during the Expos period and tidal volume (VT) fell from 7.5±1.0 to 5.7±1.3 L. The pre-treatment with cromolyn before exposures significantly reduced the increase in RL and Ptr and returned VT to Con levels.