Prerace venous blood gases and acid-base values in Standardbred horses: effects of geography, season, prerace furosemide, gender, age, and trainer using big data analytics.

OBJECTIVE: A retrospective study was conducted to establish the prerace venous acid-base and blood gas values of Standardbred horses at rest using big data analytics. SAMPLES: Venous blood samples (73,382) were collected during seven racing seasons from 3 regional tracks in the Commonwealth of Pennsylvania. Horses were detained 2 hours prior to race time. PROCEDURES: A mixed-effects linear regression model was used for estimating the marginal model adjusted mean (marginal mean) for all major outcomes. The interaction between age and gender, track, and the interaction between month, treatment (furosemide), and year were the major confounders included in the model. Random effects were set on individual animal nested within trainer. Partial pressure of venous carbon dioxide (PVCO2), partial pressure of oxygen (PVO2), and pH were measured, and base excess (BE), total carbon dioxide (TCO2), and bicarbonate (HCO3-) were calculated. RESULTS: Significant (P < .001) geographical differences in track locations were seen. Seasonal reductions in acid-base values started in January with significant (P < .001) decreases from adjacent months seen in June, July, and August followed by a gradual return. There were significant increases (P < .001) in BE and TCO2 and decreases in PVO2 with age. Significant differences (P < .001) in acid-base values were seen when comparing genders. A population of trainers were significantly different (P < .001) from the marginal mean and considered outliers. CLINICAL RELEVANCE: In a population of horses, big data analytics was used to confirm the effects of geography, season, prerace furosemide, gender, age, and trainer influence on blood gases and the acid-base profile.

Pharmacokinetics of glaucine after intravenous and oral administrations and detection of systemic aporphine alkaloids after ingestion of tulip poplar shavings in horses.

Glaucine, an aporphine alkaloid with anti-tussive, anti-inflammatory, and anti-nociceptive properties, has been identified in post-race samples from racehorses. To investigate pharmacokinetics of glaucine in horses, a three-way crossover study of intravenous and oral glaucine (0.1 mg/kg) and orally administered tulip poplar shavings (50 g shavings = 0.001 mg/kg glaucine) was performed in six horses. A two-compartment model best described IV administration with alpha ( t 1 / 2 α ) and beta ( t 1 / 2 ß ) half-life lives of 0.3 (0.1-0.7) and 3.1 (2.4-7.8) h, respectively. The area under the curve ( AUC 0 ∞ iv ) was 45.4 (34.7-52.3) h*ng/ml, and the volume of distribution of the central (Vdc ) and peripheral (Vdp ) compartments was 2.7 (1.3-4.6) and 4.9 (4.3-8.2) L/kg, respectively. A one compartment model best described the oral administration of glaucine with absorption ( t 1 / 2 ka ) and elimination ( t 1 / 2 kel ) half-lives of 0.09 (0.05-0.15) and 0.7 (0.6-0.8) h, respectively. The area under the curve ( AUC 0 ∞ PO ) was 15.1 (8.0-19.5) h·ng/ml. Bioavailability following oral administration was 17%-48%. Following ingestion of shavings, glaucine and liriodenine were detectable in plasma for up to 16 and 48 h, respectively. Glaucine was quantifiable briefly in the urine from two horses. Liriodenine was quantifiable in urine for 12-20 h in four horses and for 48 h in two horses. The presence of liriodenine indicates ingestion of tulip poplar tree parts, however, does not rule out co-administration of purified glaucine in horses.

Gene transcripts expressed in equine white blood cells are potential biomarkers of extracorporeal shock wave therapy.

Extracorporeal shockwave therapy (ESWT) is a treatment applied to musculoskeletal injuries in equine athletes to alleviate pain and accelerate healing. ESWT also causes acute tissue damage. Therefore, its ability to act as an analgesic and cause tissue damage potentially increases the risk of a catastrophic event if used shortly before a strenuous competition such as horseracing. While ESWT is prohibited by many racing jurisdictions within 10 days prior to competition, a test to detect whether a horse has received ESWT is needed. ESWT changes the protein levels of inflammatory mediators in blood, and white blood cells (WBC) typically produce these proteins. Changes in gene expression precede changes in protein production; thus, it was hypothesized that WBC gene transcripts might serve as biomarkers of ESWT. To test this hypothesis, six thoroughbred horses received a single administration of ESWT to the distal limb, and WBC RNA was extracted from blood samples collected before (0 h) and after ESWT (2, 4, 6, 24, 48, and 72 h). Targeted and untargeted analyses evaluated the transcriptome using quantitative PCR (qPCR) and microarray. The expression of IL-1α, IL-1ß, TNF-α, IL-1Ra1, IL-1Ra2 and TGF-ß1, and BMPR1A in circulating WBCs was significantly up-regulated, while IFN-γ, ZNF483, TMEM80, CAH6, ENPP, and S8723 were significantly down-regulated at various time points following ESWT. These data support the hypothesis that changes in WBC gene transcripts could serve as biomarkers for ESWT.

Bayesian-based withdrawal estimates using pharmacokinetic parameters for two capsaicinoid-containing products administered to horses.

Capsaicinoids deter horses from chewing on bandages and are applied topically to provide analgesia to musculoskeletal injuries. They are banned during competition due to their nerve blocking properties. The pharmacokinetics of oral (PO) and direct gastric administration via nasogastric tube (NG), or topical (TOP) administration of two capsaicinoid-containing products were investigated, and the withdrawal times required prior to competition were estimated. Capsaicin (CAP) and dihydrocapsaicin (DCAP) were quantified in plasma, and both compounds were best described by a delayed absorption two compartment elimination model following PO administration and by a first order absorption one compartment elimination model following TOP administration. Capsaicin and DCAP could not be quantified in most samples following NG administration. Following PO administration, the time to maximum plasma concentration (Tmax ) for CAP and DCAP was 0.25 (0.08-0.50) hr. Following TOP application, the Tmax for CAP and DCAP was 4 (2-6) and 5 (3-12) hr, respectively. By 8 hr post-PO administration and 36 hr post-TOP application, CAP and DCAP were below the lower limit of quantification. Capsaicin and DCAP were not detected in urine samples. Withdrawal times were predicted using the 99.99% credibility interval limits of the pharmacokinetic parameters calculated with Bayesian estimation.

Pharmacokinetics of xylazine after 2-, 4-, and 6-hr durations of continuous rate infusions in horses.

Intravenous (i.v.) bolus administration of xylazine (XYL) (0.5 mg/kg) immediately followed by a continuous rate infusion (CRI) of 1 mg kg-1  hr-1 for 2, 4, and 6 hr produced immediate sedation, which lasted throughout the duration of the CRI. Heart rate decreased and blood pressure increased significantly (p > .05) in all horses during the first 15 min of infusion, both returned to and then remained at baseline during the duration of the infusion. Compartmental models were used to investigate the pharmacokinetics of XYL administration. Plasma concentration-time curves following bolus and CRI were best described by a one-compartment model. No differences were found between pharmacokinetic estimates of the CRIs for the fractional elimination rate constant (Ke ), half-life (t1/2e ), volume of distribution (Vd ), and clearance (Cl). Median and range were 0.42 (0.15-0.97)/hr, 1.68 (0.87-4.52) hr, 5.85 (2.10-19.34) L/kg, and 28.7 (19.6-39.5) ml min-1  kg-1 , respectively. Significant differences were seen for area under the curve ( AUC 0 ∞ ) (p < .0002) and maximum concentration (Cmax ) (p < .04). This indicates that with increasing duration of infusion, XYL may not accumulate in a clinically relevant way and hence no adjustments are required in a longer XYL CRI to maintain a constant level of sedation and a rapid recovery.

High-throughput doping control analysis of 28 amphetamine-type stimulants in equine plasma using hydrophilic interaction liquid chromatography-tandem mass spectrometry.

A hydrophilic interaction liquid chromatography-tandem mass spectrometry method (HILIC-MS/MS) was developed for the simultaneous determination of 28 amphetamine-type stimulants (ATSs) in equine plasma for doping control analysis. In this method, stimulants were recovered from equine plasma by liquid-liquid extraction (LLE) at pH 9.5 using methyl tert-butyl ether and detected on a Thermo Finnigan triple quadrupole mass spectrometer operating in positive-ion mode electrospray ionization. All stimulants were eluted within 7 minutes and baseline separation was achieved for isomeric and isobaric compounds using HILIC chromatography. Extraction efficiency was greater than 80% and matrix effect was acceptable for most stimulants. The limit of detection (LOD) was in the range of 10-50 pg/mL and the lower limit of quantification (LLOQ) was in the range of 50-100 pg/mL. Quadratic regression was employed for quantification and the dynamic range of quantification was 50-10000 pg/mL. Confirmatory analysis criteria were established using product ion ratios and retention time. The limit of confirmation (LOC) was in the range of 20-100 pg/mL. Stability study results indicated that some stimulants were unstable in equine plasma at room temperature and 4°C. However, all the stimulants studied were stable at -20°C and - 80°C for the 6 month study period.

Pharmacokinetics of intravenous, subcutaneous, and topical administration of lidocaine hydrochloride and metabolites 3-hydroxylidocaine, monoethylglycinexylidide, and 4-hydroxylidocaine in horse.

Intravenous (iv), subcutaneous (sq), and topical (tp) lidocaine was administered to six horses in a cross-over, randomized design study. Samples were collected for up to 72 hr. Compartmental models were used to investigate the pharmacokinetics of (LD) and its metabolites 3-hydroxylidocaine (3-OH), 4-hydroxylidocaine (4-OH), and monoethylglycinexylidide (MEGX). Metabolites 3-OH and 4-OH were present in conjugated forms, whereas LD and metabolite MEXG were present primarily in the un-conjugated form. Plasma concentrations of LD after iv administration (100 mg) were described by three-compartment model with an additional three compartments to describe the elimination of metabolites. Median (range) elimination micro-constants (Ke ) for LD, 3-OH, 4-OH, and MEXG were 4.12 (2.62-6.23), 1.25 (1.10-2.15), 1.79 (1.22-2.39), and 1.69 (1.03-1.99)/hr, respectively. Median (range) values of alpha (t½α ), beta (t½ß ), and gamma (t½Î³ ) half-lives were 0.08 (0.07-0.13), 0.57 (0.15-1.25), and 4.11 (0.52-7.36) hr. Plasma concentrations of LD after sq (200 mg) administration were described by absorption and two-compartment elimination model. The median (range) of the LD absorption half-life (t½ab ) was 0.47 (0.29-0.61) hr. The Ke for LD, 3-OH, 4-OH, and MEXG was 3.91 (1.48-9.25), 1.00 (0.78-1.08), 1.76 (0.96-2.11), and 1.13 (0.69-1.33)/hr. The median (range) of t½α and t½ß was 0.15 (0.06-0.27) and 3.04 (2.53-6.39) hr. Plasma concentrations of LD after tp (400 mg) application were described by one-compartment model with a t½ab of 8.49 (5.16-11.80) hr. The Ke for LD, 3-OH, and MEXG was 0.24 (0.10-0.81), 0.41 (0.08-0.93), and 0.38 (0.26-1.14)/hr.

Evaluation of the analgesic and pharmacokinetic properties of transdermally administered fentanyl in goats.

OBJECTIVE: Evaluate the analgesic properties and pharmacokinetics of transdermal fentanyl patches (TFPs) in goats. DESIGN: Prospective, randomized study. SETTING: Preclinical Testing Facility at a University Teaching Hospital. ANIMALS: Thirty-four adult female Boer-cross goats. INTERVENTIONS: Goats underwent surgery as part of a concurrent orthopedic research study. Twelve hours prior to surgery, each goat received a TFP (target dosage of 2.5 µg/kg/h), or a placebo patch with analgesia provided by buprenorphine (0.01 mg/kg, IM, q 6 h). Patches were removed after 72 hours. Blood was sampled at specified intervals, up to 84 hours following TFP placement. Plasma concentrations of fentanyl (FEN) were determined using liquid chromatography-mass spectrometry. Postoperative pain assessments were performed by two independent blinded observers. MEASUREMENTS AND MAIN RESULTS: TFPs were applied at a mean (± standard deviation, SD) dose of 2.54 ± 0.36 µg/kg/h. No adverse events occurred. Pain scores between TFP and BUP groups were not significantly different at any time point. Mean plasma FEN concentration (± SD) 2 hours following patch application was 1.06 ± 0.85 ng/mL, and remained above 0.5 ng/mL for 40 hours. Maximum mean plasma FEN concentration (Cmax ) was 1.84 (ranging from 0.81 to 3.35) ng/mL with average time to maximum concentration (Tmax ) of 12 hours after patch application. CONCLUSIONS: TFP resulted in consistent FEN absorption and plasma concentrations within the human and ovine therapeutic ranges. Pain scores for goats administered TFP were not different than those administered buprenorphine. Ease of administration, duration of analgesia, and decreased dosing frequency make TFPs an attractive option for pain management in goats.

Validated LC-MS-MS Method for Simultaneous Analysis of 17 Barbiturates in Horse Plasma for Doping Control.

A rapid and sensitive method for simultaneous screening, quantification and confirmation of 17 barbiturates in horse plasma using liquid chromatography-tandem mass spectrometry is described. Analytes were recovered from plasma by liquid-liquid extraction using methyl tert-butyl ether, separated on a C18 column, and analyzed in negative electrospray ionization mode. Multiple-reaction monitoring was employed for screening and quantification. Confirmation for the presence of the analytes was achieved by comparing ion intensity ratio. The ranges for limits of detection, quantification and confirmation were 0.003-1 ng/mL (S/N ≥ 3), 0.01-2.5 ng/mL and 0.02-5 ng/mL, respectively. The linear dynamic range of the method was 0.1-100 ng/mL. The precision and accuracy at 0.5, 5 and 50 ng/mL of all 17 barbiturates during intra-day assay were 1.6-8.6% and 96-106%, respectively. For inter-day assay, precision and accuracy at the same three concentrations were 2.6-8.9% and 96-106%, respectively. Analysis of all 17 analytes was completed within 7 min. Thus, the present method is fast, simple, sensitive and reproducibly reliable.

Confirmatory analysis of etanercept in equine plasma by LC-MS for doping control.

Etanercept is a protein-based medication for the treatment of human patients with rheumatoid arthritis and other autoimmune-based diseases; its pharmacological action is to inhibit and antagonize tumour necrosis factor alpha. Etanercept was rumoured to be used in horse racing in North America. To detect such use, the aim of this study was to develop a liquid chromatography-mass spectrometry (LC-MS) method for confirmation of etanercept in equine plasma. Etanercept was extracted from plasma by anti-human IgG antibody linked to magnetic beads. The analyte was reduced and alkylated, and then digested by trypsin. Tryptic peptides (T1 from human tumour necrosis factor receptor 2 of etanercept, T15 and T27 from human IgG1 of the protein) were employed for detection and confirmation of the analyte. The limit of detection was 5 ng/mL, and the limit of confirmation 10 ng/mL. This method is specific for confirmation of etanercept, as assessed using the results from BLAST and SEQUEST searches. The results from SEQUEST searches also revealed an unexpected unique specificity of product ion spectrum of IgG1 T27 with only a single product ion for identification of etanercept. It is the first report for such a finding, to the authors' knowledge. The method was successful in analyses of the plasma samples collected post administration of etanercept to horses. Etanercept was detected up to 11 days post administration. This method will be helpful for confirmation of etanercept or other protein-based drugs consisting of human IgG1, in equine drug testing. Copyright © 2016 John Wiley & Sons, Ltd.

Plasma interleukin-6 concentration in Standardbred racehorses determined by means of a novel validated ELISA.

OBJECTIVE: To evaluate plasma interleukin 6 (IL-6) concentration in Standardbred racehorses by means of a novel ELISA following validation of the assay for use with equine plasma samples. SAMPLE: Plasma samples obtained from 25 Thoroughbreds for use in assay validation and from 319 Standardbred racehorses at rest 2 to 2.5 hours prior to warm-up and racing. PROCEDURES: A sandwich ELISA was developed with equine anti-IL-6 polyclonal antibody and the biotin-streptavidin chemical interaction to enhance sensitivity. The assay was validated for specificity, sensitivity, precision, and accuracy by use of both recombinant and endogenous proteins. RESULTS: For the assay, cross-reactivity with other human and equine cytokines was very low or absent. Serial dilution of plasma samples resulted in proportional decreases in reactivity, indicating high specificity of the method. Partial replacement of detection antibody with capture antibody or pretreatment of samples with capture antibody caused assay signals to significantly decrease by 55%. The inter- and intra-assay precisions were ≤ 13.6% and ≤ 9.3%, respectively; inter- and intra-assay accuracies were within ranges of ± 14.1% and ± 8.6%, respectively, at concentrations from 78 to 5,000 pg/mL, and the sensitivity was 18 pg/mL. Plasma IL-6 concentration varied widely among the 319 Standardbreds at rest (range, 0 to 193,630 pg/mL; mean, 6,153 pg/mL; median, 376 pg/mL). CONCLUSIONS AND CLINICAL RELEVANCE: This ELISA method proved suitable for quantification of IL-6 concentration in equine plasma samples. Plasma IL-6 concentration was high (> 10,000 pg/mL) in 9.1% of the Standardbred racehorses, which warrants further investigation.

Effect of long-term oral administration of a low dosage of clenbuterol on body fat percentage in working and nonworking adult horses.

OBJECTIVE: To determine the anabolic and lipolytic effects of a low dosage of clenbuterol administered orally in working and nonworking equids. ANIMALS: 8 nonworking horses and 47 polo ponies in active training. PROCEDURES: Each polo pony continued training and received either clenbuterol (0.8 µg/kg) or an equal volume of corn syrup (placebo) orally twice daily for 21 days, and then was evaluated for another 21-day period. Nonworking horses received clenbuterol or placebo at the same dosage for 21 days in a crossover trial (2 treatments/horse). For working and nonworking horses, percentage body fat (PBF) was estimated before treatment and then 2 and 3 times/wk, respectively. Body weight was measured at intervals. RESULTS: Full data sets were not available for 8 working horses. For working horses, a significant treatment effect of clenbuterol was detected by day 3 and continued through the last day of treatment; at day 21, the mean change in PBF from baseline following clenbuterol or placebo treatment was -0.80% (representing a 12% decrease in PBF) and -0.32%, respectively. By day 32 through 42 (without treatment), PBF change did not differ between groups. When treated with clenbuterol, the nonworking horses had a similar mean change in PBF from baseline from day 6 onward, which peaked at -0.75% on day 18 (an 8% decrease in PBF). Time and treatment had no significant effect on body weight in either experiment. CONCLUSIONS AND CLINICAL RELEVANCE: Among the study equids, long-term low-dose clenbuterol administration resulted in significant decreases in body fat with no loss in body weight.

Inhibitory effect of triamcinolone acetonide on synthesis of inflammatory mediators in the equine.

Glucocorticoids (corticosteroids) are widely used anti-inflammatory agents in veterinary medical practice. These drugs are considered doping agents because they mask pain and thus, increase injury potential in equine athletes. They exhibit anti-inflammatory property by binding to glucocorticoids receptor (GR) to control the transcription of pro- and anti-inflammatory cytokines and enzymes involved in the synthesis of bioactive eicosanoids. To evaluate the role of triamcinolone acetonide (TA) on concentrations of bioactive eicosanoids in equine plasma, TA (0.04 mg/kg) was intravenously administered to horses. Before (0 h) and after TA administration, equine whole blood (EWB) samples were collected and challenged with either methanol (vehicle), calcium ionophore A-23187 (CI) or lipopolysaccharide (LPS) to stimulate ex-vivo synthesis of eicosanoids. Plasma concentrations of eicosanoids were quantified using LC-MS/MRM. Results showed that thromboxane B2 (TXB2) was not affected by TA administration when EWB was stimulated with CI. However, after LPS treatment, TXB2, PGE2, PGF2α and 15-(s)-HETE decreased during 2-8 h post-TA administration but recovered to concentrations which were not significantly different from those of pre-TA administration (0 h), after 24 h. When EWB was treated with CI, LTB4 was suppressed post-TA administration compared to 0 h. When EWB collected after TA administration was stimulated with LPS, LTB4 was not significantly different from those of 0 h. Administration of a therapeutic dose of TA (0.04 mg/kg, iv) in the horse suppressed biosynthesis of bioactive eicosanoids indicating the anti-inflammatory role of TA in the horse.

Identification of sample donor by 24-plex short tandem repeat in a post-race equine plasma containing dexamethasone.

BACKGROUND: Animal sport such as horseracing is tainted with drug abuse as are human sports. Treatment of racehorses on race day with therapeutic medications in most cases is banned, and thus, it is essential to monitor the illicit use of drugs in the racing horse to maintain integrity of racing, ensure fair competition and protect the health, safety and welfare of the horse, jockeys and drivers. In the event of a dispute over the identity of the sample donor, if the regulator can provide evidence that the DNA genotype profile of the post-race sample matched that of the alleged donor, then the potential drug violation case might be easily resolved without legal challenges. CASE DESCRIPTION: We present a case study of a racehorse sample that tested positive for dexamethasone in a post-race plasma sample in Pennsylvania (PA) but the result was challenged by the trainer of the horse. Dexamethasone is a synthetic glucocorticoid widely used in the management of musculoskeletal problems in horses but its presence in the horse during competition is banned by the PA Racing Commissions. The presence of dexamethasone in the post-competition plasma sample was confirmed using liquid chromatography-tandem mass spectrometry. However, this finding was challenged by the trainer of the horse alleging that the post-race sample was not collected from his/her horse and thus petitioned the Commission to be absolved of any wrong-doing. To resolve the dispute, a DNA test was ordered by the PA Racing Commission to identify the correct donor of the dexamethasone positive sample. For this purpose, a 24-plex short tandem repeat analysis to detect 21 equine markers and three human markers was employed. The results indicated that all the samples tested had identical DNA profiles and thus, it was concluded that the samples were collected from the same horse and that the probability of drawing a false conclusion was approximately zero (1.5 × 10(-15)). CONCLUSIONS: The plasma sample confirmed for the presence of dexamethasone was collected from the alleged horse.

Interleukin-1ß inhibits synthesis of 5-lipooxygenase in lipopolysaccharide-stimulated equine whole blood.

Interleukin-1ß (IL-1ß) is a pro-inflammatory cytokine. It induces the synthesis of prostaglandin E2 (PGE2) catalyzed by cyclooxygenase (COX) and microsomal prostaglandin E synthase (m-PGES). Besides its pro-inflammatory properties, PGE2 also exhibits anti-inflammatory properties by inhibiting synthesis of 5-lipooxygenase (5-LO) products which are in themselves, pro-inflammatory mediators. Thus, inhibition of 5-LO products is beneficial in regulating immune-responses and pro-inflammatory processes. To investigate the hypothesis that IL-1ß is responsible for the increase in the synthesis of PGE2 and in the reduction of 5-LO products, equine whole blood (EWB) was treated with lipopolysaccharide (LPS). In vitro treatment of EWB with LPS resulted in increased expression of IL-1ß while expression of 5-LO was suppressed. Quantification of eicosanoids using liquid-chromatography-mass spectrometry/multiple reaction monitoring (LC-MS/MRM) showed increased concentrations of prostaglandins and decreased 5-LO products in LPS-treated EWB. Pretreatment of EWB with IL-1ß followed by calcium ionophore A23187 (CI) reduced synthesis of 5-LO products. However, pretreatment of EWB with COX-2 inhibitor (NS-398) or m-PGES-1 inhibitor (CAY 10526) and IL-1ß followed with CI resulted in a significant (p<0.0001) increase in 5-LO products. Pretreatment of EWB with phospholipase C inhibitor (U73122) followed with LPS reduced PGE2 production but increased 5-LO products. The result of this study indicated that increased PGE2 production led to reduction in 5-LO products in LPS-treated EWB via IL-1ß. However, other pathways, cytokines and mediators may be involved in inhibiting 5-LO products but the present study did not include those other potential pathways. Inhibition of 5-LO products by PGE2 in EWB may regulate the initiation and pathogenesis of inflammatory responses in the horse.

Sensitive hydrophilic interaction liquid chromatography/tandem mass spectrometry method for rapid detection, quantification and confirmation of cathinone-derived designer drugs for doping control in equine plasma.

RATIONALE: Cathinone derivatives are new amphetamine-like stimulants that can evade detection when presently available methods are used for doping control. To prevent misuse of these banned substances in racehorses, development of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method became the impetus for undertaking this study. METHODS: Analytes were recovered via liquid-liquid extraction using methyl tert-butyl ether. Analyte separation was achieved on a hydrophilic interaction column using liquid chromatography and mass analysis was performed on a QTRAP mass spectrometer in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM). Analyte identification was carried out by screening for a specified MRM transition. Quantification was conducted using an internal standard. Confirmation was performed by establishing a match in retention time and ion intensity ratios comparison. RESULTS: The method was linear over the range 0.2-50 ng/mL. The specificity was evaluated by analysis of six different batches of blank plasma and those spiked with each analyte (0.2 ng/mL). The recovery of analytes from plasma at three different concentrations was >70%. The limits of detection, quantification and confirmation were 0.02-0.05, 0.2-1.0 and 0.2-10 ng/mL, respectively. The matrix effect was insignificant. The intra-day and inter-day precision were 1.94-12.08 and 2.58-13.32%, respectively. CONCLUSIONS: The method is routinely employed in screening for the eleven analytes in post-competition samples collected from racehorses in Pennsylvania to enforce the ban on the use of these performance-enhancing agents in racehorses. The method is sensitive, fast, effective and reliably reproducible.

Isolation of RNA from equine peripheral blood cells: comparison of methods.

Gene expression studies in equine research involve the use of whole blood samples as a vital source of RNA. To determine the optimal method for RNA isolation from equine whole blood, we compared three RNA isolation strategies using different commercially available kits to evaluate the yield and quality of equine RNA. All 3 methods produced RNA with high quality. Though it did not produce the highest yield, combining the quality, yield and the need for the downstream application in our project, LeukoLOCK™ total RNA isolation system was the best RNA extraction method.

Detection, quantification, and identification of dermorphin in equine plasma and urine by LC-MS/MS for doping control.

Dermorphin is a unique opioid peptide that is 30-40 times more potent than morphine. It was misused and went undetected in horse racing until 2011 when intelligence obtained from a few North American race tracks suggested its use. To prevent such misuse, a reliable analytical method became necessary for detection and identification of dermorphin in post-race horse samples. This paper describes the first liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for such a purpose. Equine plasma and urine samples were pre-treated with ethylenediamine tetra-acetic acid and urea prior to solid-phase extraction (SPE) on Oasis MCX cartridges. Resulting eluates were dried under vacuum and analyzed by LC-MS/MS for dermorphin. The matrix effect, SPE efficiency, intra-day and inter-day accuracy and precision, and stability of the analyte were assessed. The limit of detection was 10 pg/mL in plasma and 20 pg/mL in urine, and the limit of confirmation was 20 pg/mL in plasma and 50 pg/mL in urine. Dermorphin in plasma is stable at ambient temperature, but its diastereomer is unstable. With isotopically labeled dermorphin as an internal standard, the quantification range was 20-10,000 pg/mL in plasma and 50-20,000 pg/mL in urine. The intra-day and inter-day accuracy was from 91 % to 100 % for the low, intermediate, and high concentrations. The intra-day and inter-day coefficients of variation were less than 12 %. The method differentiates dermorphin from its diastereomer. This method is very specific for identification of dermorphin in equine plasma and urine, as assessed by BLAST search and targeted SEQUEST search, and by MS/MS spectrum library search. The method has been successfully applied to analysis of samples collected following dermorphin administration to research horses and of official post-race samples.

Validated UHPLC-MS-MS method for rapid analysis of capsaicin and dihydrocapsaicin in equine plasma for doping control.

A method involving ultra high-performance liquid chromatography-tandem mass spectrometry was developed and validated for the analysis of capsaicin and dihydrocapsaicin in equine plasma. The analytes were recovered from plasma by liquid-liquid extraction using methyl tert-butyl ether and separated on a sub-2 micron column. The mobile phase was composed of 2 mM ammonium formate and methanol. A triple quadrupole mass spectrometer was used to detect the analytes in positive electrospray ionization mode with selected reaction monitoring. The limits of detection, quantification and confirmation for both analytes were 0.5, 1.0 and 2.5 pg/mL, respectively. The linear dynamic range of quantification was 1.0-1,000 pg/mL. During storage, both analytes in equine plasma were unstable at room temperature but stable at -20 and -70°C. The retention time and product ion ratios were employed as the criteria for confirmation of the presence of the analytes in plasma. The total analysis time was 2 min. The method is fast, selectively sensitive, reproducible, reliable and fully validated.