Prolonged administration of oral phenylbutazone and firocoxib in horses has no impact on selected cytokine and growth factor concentrations in platelet-rich plasma and autologous protein solution.

OBJECTIVE: To determine the effects of prolonged administration of the oral NSAIDs phenylbutazone and firocoxib on concentrations of cytokines and growth factors in platelet-rich plasma (PRP) and autologous protein solution (APS). ANIMALS: 6 adult University owned horses. METHODS: Horses were randomized to receive phenylbutazone (1 g, orally, q 12 h) or firocoxib (57 mg, orally, q 24 h) for 6 days. Blood was obtained and processed for APS (Pro-Stride) and PRP (Restigen) before the administration of NSAIDs and at 7 days (1 day following cessation of NSAIDs). Horses underwent a two-week washout period, during which blood was obtained at 14 days and 21 days. The protocol was repeated with a crossover design. PRP and APS were analyzed for concentrations of platelets, leukocytes, and several cytokines (IL-1ß, IL-10, IL-6, IL-8, and tumor necrosis factor-α) and growth factors (PDGF, FGF-2, and TGF-ß1) using immunoassays. Plasma was evaluated for drug concentrations. RESULTS: No significant differences existed in concentrations of growth factors and cytokines before or after prolonged administration of NSAIDs. There were significant differences in concentrations of leukocytes and platelets in PRP compared to APS, with higher concentrations of leukocytes at the day 7 time point (T) in APS (phenylbutazone) and in concentrations of platelets in APS at T0 (firocoxib) and in APS at T7 (phenylbutazone). CLINICAL RELEVANCE: Veterinarians can recommend the administration of these oral NSAIDs prior to obtaining blood for PRP and APS provided a single-day washout period is instituted.

Simultaneous quantification and confirmation of oxycodone and its metabolites in equine urine using ultra-high performance liquid chromatography-tandem mass spectrometry.

Oxycodone, an opioid commonly used to treat pain in humans, has the potential to be abused in racehorses to enhance their performance. To understand the pharmacokinetics of oxycodone and its metabolites in horses, as well as to detect the illegal use of oxycodone in racehorses, a method for quantification and confirmation of oxycodone and its metabolites is needed. In this study, we developed and validated an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method that can simultaneously quantify and confirm oxycodone and eight metabolites in equine urine. Samples were subjected to enzymatic hydrolysis and then liquid-liquid extraction using ethyl acetate. The analyte separation was achieved on a Hypersil Gold C18 sub-2 µm column and analytes were detected on a triple quadrupole mass spectrometer. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 25-50 pg/mL and 100 pg/mL, respectively. Excellent linearity of the calibration curves was observed over a range of 100-10000 pg/mL for all nine analytes. Retention time, signal-to-noise ratio, and product ion ratios were utilized as confirmation criteria, with the limits of confirmation (LOC) ranging from 100 to 250 pg/mL. The data from a pilot pharmacokinetic (PK) study suggested that oxycodone metabolites have longer detection periods in equine urine compared to oxycodone itself; thus, the detection of metabolites in equine urine extends the ability to detect oxycodone exposure in racehorses.

Single-dose nonsteroidal anti-inflammatory drugs in horses have no impact on concentrations of cytokines or growth factors in autologous protein solution and platelet-rich plasma.

OBJECTIVE: To determine the effects of a single dose of the NSAIDs phenylbutazone, firocoxib, flunixin meglumine, and ketoprofen on concentrations of growth factors and cytokines in autologous protein solution (APS) and platelet-rich plasma (PRP). ANIMALS: 6 adult university-owned horses. METHODS: For the first phase, 6 horses were randomized to receive ketoprofen (1,000 mg) or flunixin meglumine (500 mg) IV. Blood was obtained and processed for APS (Pro-Stride) and PRP (Restigen) before and 6 hours after administration of NSAIDs. Horses underwent a 2-week washout period, after which the protocol was repeated using a crossover design. For the second phase, following at least a 2-week washout period, the study protocol was repeated with phenylbutazone (1 g) or firocoxib (57 mg) administered orally. Plasma was collected 6 hours after administration for evaluation of drug concentrations, and APS and PRP were analyzed for concentrations of drug, platelets, leukocytes, and several growth factors and cytokines (PDGF, fibroblast growth factor, TGF-ß1, IL-1ß, IL-10, IL-6, IL-8, and tumor necrosis factor-α) before and 6 hours after administration of NSAIDs using immunoassays. RESULTS: There were no significant differences in concentrations of cytokines or growth factors before or after administration of any NSAID. There were significant differences in concentrations of leukocytes and platelets based on both product and time. NSAID concentrations in plasma were not significantly different from concentrations in APS and PRP. CLINICAL RELEVANCE: These results help guide clinicians on the appropriate use of these NSAIDs in conjunction with the processing of APS and PRP, which is unlikely to significantly alter the final product after single-dose administration.

Factors affecting untargeted detection of doping agents in biological samples.

Doping control is essential for sports, and untargeted detection of doping agents (UDDA) is the holy grail for anti-doping strategies. The present study examined major factors impacting UDDA with metabolomic data processing, including the use of blank samples, signal-to-noise ratio thresholds, and the minimum chromatographic peak intensity. Contrary to data processing in metabolomics studies, both blank sample use (either blank solvent or plasma) and marking of background compounds were found to be unnecessary for UDDA in biological samples, the first such report to the authors' knowledge. The minimum peak intensity required to detect chromatographic peaks affected the limit of detection (LOD) and data processing time for untargeted detection of 57 drugs spiked into equine plasma. The ratio of the mean (ROM) of the extracted ion chromatographic peak area of a compound in the sample group (SG) to that in the control group (CG) impacted its LOD, and a small ROM value such as 2 is recommended for UDDA. Mathematical modeling of the required signal-to-noise ratio (S/N) for UDDA provided insights into the effect of the number of samples in the SG, the number of positive samples, and the ROM on the required S/N, highlighting the power of mathematics in addressing issues in analytical chemistry. The UDDA method was validated by its successful identification of untargeted doping agents in real-world post-competition equine plasma samples. This advancement in UDDA methodology will be a useful addition to the arsenal of approaches used to combat doping in sports.

Use of Liquid Chromatography--Tandem Mass Spectrometry to Quantify and Confirm the Fentanyl Metabolite N-[1-(2-Phenethy-4-Piperidinyl)] Maloanilinic Acid in Equine Urine for Doping Control.

Fentanyl, a powerful synthetic mu opioid receptor agonist, is banned in equine sports by the Association of Racing Commissioners International and the Fédération Équestre Internationale. The presence of fentanyl in equine blood has been confirmed during routine post-race screening for doping substances in the authors' laboratory. While fentanyl can be detected and confirmed in blood, it is rapidly metabolized, and screening for the metabolite N-[1-(2-phenethy-4-piperidinyl)] maloanilinic acid (PMA) in equine urine is expected to allow for a longer detection time. In this study, a quantitative and confirmatory liquid chromatography--tandem mass spectrometry (LC-MS-MS) method was developed for PMA analysis in equine urine. PMA was extracted by solid phase extraction, separated on a C18 column and detected using a triple quadrupole mass spectrometer. The mass spectrometer was operated in positive-ion mode, and multiple reaction monitoring was used to monitor product ions m/z 188, m/z 281 and m/z 323. The method was validated for extraction recovery, matrix effect, specificity, sensitivity, precision and accuracy, carryover and processed sample stability according to the guidelines of the US Food and Drug Administration for bioanalysis. The limits of detection and quantification were 5 and 10 pg/mL, respectively. Linearity was obtained over the concentration range of 10-10,000 pg/mL. To confirm PMA in equine urine, LC retention time, diagnostic product ions (m/z 188, m/z 281 and m/z 323) and product ion ratio were used as the criteria. The lowest concentration for confirmatory analysis was validated at 50 pg/mL. The method was applied to measure the PMA concentrations in equine urine following intravenous administration of fentanyl to a research horse and has confirmed the presence of PMA in post-race urine samples. This method is a valuable addition to the arsenal of equine doping control methods to combat illegal doping and protect racehorse health.

Automated identification of unknown doping agents in confiscation samples by flow-injection mass spectrometry and mass spectral library searches.

Rapid and accurate identification of unknown compounds within suspicious samples confiscated for sports doping control and law enforcement drug testing is critical, but such analyses are often conducted manually and can be time-consuming. Here, we report a methodology for automated identification of unknown substances in confiscation samples by rapid automatic flow-injection analysis on a liquid chromatography coupled to high-resolution mass spectrometry system and identifying unknown compounds with Compound Discoverer software. The developed methodology was validated by comparing the automated identification results with those obtained from manual syringe-infusion experiments and manual tandem mass spectral library searches. The automated methodology resulted in far higher throughput and remarkably shorter turnaround time for analysis when compared with manual procedures and, in most cases, yielded more compounds. As this is the first such report to the authors' knowledge, this methodology may potentially transform analysis of confiscated samples in sports doping control and law enforcement drug testing.

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.

Pharmacokinetics and pharmacodynamics of oral and intravenous metoprolol tartrate in clinically healthy horses.

Cardiac drugs with defined pharmacological parameters in horses are limited. The objective of this study was to characterize the pharmacokinetic properties and cardiovascular effects of intravenous and oral metoprolol tartrate (MET) in horses. In a 2-period randomized cross-over design, MET was administered IV (0.04 mg/kg) and PO (6 mg/kg) once to six healthy adult horses. Horses were monitored via continuous telemetry and non-invasive blood pressure (NIBP). Blood samples were serially collected for 72 h post-administration, and concentrations were determined by LC-MS/MS. Pharmacokinetics were modeled using a 3-compartment model and non-linear least squares regression. Median (range) MET concentration was 110 (40.1-197) ng/ml collected 1 min (0.0167 h) after a bolus IV administration. Maximum concentration (Cmax ) after PO administration was 2135 (1590-4170) ng/ml at 0.5 (0.25-0.5) hours. Oral bioavailability was 54% (17-100%). Median apparent volume of distribution was 0.39 (0.17-0.58) l/kg, clearance was 12.63 (11.41-18.94) ml/kg/min, and elimination half-life was 21.1 (7.46-34.36) minutes. No clinically relevant effects of IV or PO metoprolol were noted on cardiac rhythm or NIBP. Sweating was the most common side effect. The metoprolol doses used in this study achieve plasma concentrations reported to achieve ß-blockade in humans.

Novel Algorithms for Comprehensive Untargeted Detection of Doping Agents in Biological Samples.

To address the limitations of current targeted analytical methods that can only detect known doping agents, a novel methodology that permits untargeted drug detection (UDD) has been developed to help in the fight against doping in sports. Fifty-seven drugs were spiked into blank equine plasma and were treated as unknowns since their exact masses and chromatographic retention times were not utilized for detection. The spiked drugs were extracted from the plasma samples and were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). The acquired LC-HRMS raw data files were processed using metabolomic software for compound detection and identification. For UDD with the resultant data, a mathematical model was created, and two algorithms were generated to calculate the ratio of the mean (ROM) and outlier index (OLI). Using ROM and OLI, the majority of the 57 drugs were accurately detected by name (52 of 57) or chemical formula (1 of 57). The limit of detection for the drugs was from tens of picograms to nanograms per milliliter. Xenobiotics and endogenous substances relevant to doping control were also identified using this untargeted approach following their extraction from real-world race samples, thus validating the UDD methodology. To the authors' knowledge, this is the first completely UDD methodological approach and represents significant advance toward using artificial intelligence for the detection of both known and emerging doping agents in sports.

Use of high resolution/accurate mass full scan/data-dependent acquisition for targeted/non-targeted screening in equine doping control.

High-resolution mass spectrometry (HRMS) is a very powerful technology for equine doping control analysis. The more recently developed hybrid type of Orbitrap-based HRMS instrument allows for both targeted and non-targeted screening analyses in a single liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) run. In the present study, an LC-HRMS/MS method was developed and validated to detect prohibited substances in equine sports. The substances were recovered from equine plasma by liquid-liquid extraction (LLE) using methyl tert-butyl ether and were separated on a C18 reversed-phase column using mobile phases of 5 mM ammonium formate and acetonitrile. A 7.5 min LC gradient was employed to elute substances and results indicated that the LC method generated sharp and symmetric chromatographic peaks. An in-house equine doping compound database and a spectral library were built to increase method specificity for substances of interest. Five criteria, i.e. accurate mass, retention time, isotope pattern, selected HRMS/MS fragment ions (compound database) and HRMS/MS spectra (spectral library), were employed for targeted screening. We utilized these criteria to validate targeted detection of 451 substances within our in-house equine doping compound database. By using all five criteria in screening, the false screening positive rate is significantly reduced. A screening strategy and a Microsoft Excel macro were developed to facilitate interpretation and reporting of results. As the simultaneous acquisition of the full scan HRMS data provides the opportunity for retrospective non-targeted analysis, our findings highlight the use of this novel methodology as a simple, rapid, and reliably reproducible strategy to meet the challenge of identifying an increasing number of doping substances that could potentially impact the integrity of the horse racing community.

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.

Identification of ex vivo catabolites of peptides with doping potential in equine plasma by HILIC-HRMS.

Bioactive peptides pose a great threat to sports integrity. The detection of these peptides is essential for enforcing their prohibition in sports. Identifying the catabolites of these peptides that are formed ex vivo in plasma may improve their detection. In the present study, the stability of 27 bioactive peptides with protection at both termini in equine plasma was examined under different incubation conditions, using HILIC coupled to HRMS. Of the 27 peptides, 13 were stable after incubation at 37°C for 72 hr, but the remaining 14 were less stable. Ex vivo catabolites of these 14 peptides were detected using their theoretical masses generated in silico, their appearance was monitored over the time course of incubation, and their identity was verified by their product ion spectra. Catabolites identified for chemotactic peptide, DALDA, dmtDALDA, deltorphins I and II, Hyp6 -dermorphin, Lys7 -dermorphin, and dermorphin analog are novel. A d-amino acid residue at position 2 or 1 of a peptide or next to its C-terminus protected the relevant terminal from degradation by exopeptidases, but such a residue at position 3 did not. A pGlu residue or N-methylation at the N-terminus of a peptide did not protect its N-terminal. Ethylamide at the C-terminus of a peptide provided the C-terminal protection from attacks by carboxypeptidases. The C-terminal Lys amide in DALDA, dmtDALDA, and Lys7 -dermorphin was susceptible to cleavage by plasma enzymes, which is the first report, to the authors' knowledge. The results from the present study provide insights into the stability of peptides in plasma.

Effects of acepromazine and xylazine on subjective and objective assessments of forelimb lameness.

BACKGROUND: To facilitate lameness evaluation, sedatives such as xylazine and acepromazine are regularly used in the clinical setting, despite concerns that they may confound lameness assessment. OBJECTIVES: The objective of this study was to determine the effect of low doses of acepromazine and xylazine on subjective and objective lameness assessment. STUDY DESIGN: Randomised, blinded, crossover study. METHODS: Six horses with experimentally induced solar pain were evaluated over a 1-hour period after treatment with intravenous xylazine (0.1 or 0.2 mg/kg), intravenous acepromazine (0.02 or 0.04 mg/kg), intravenous saline (1 mL) or local analgesia (4 mL 2% mepivacaine administered subcutaneously). Lameness was assessed objectively with inertial sensors and subjectively on a scale from 0 to 5. Lameness assessments were compared with logistic regression analysis to account for the repeated measures and cross-over study design (P < .05). RESULTS: Xylazine (0.1 and 0.2 mg/kg) or acepromazine (0.02 and 0.04 mg/kg) did not result in significant differences in objective lameness assessment (vector sum) or average subjective lameness grade. Local analgesia was associated with a decrease in subjective lameness grade (OR 0.32 [0.11-0.92], P = .03). Objective measures of lameness (vector sum) were significantly decreased 45 minutes (vector sum 41.8, P = .04) and 60 minutes (vector sum 47.3, P = .03) following local analgesia administration compared with baseline (vector sum 121.4). MAIN LIMITATIONS: Extrapolation of the experimental model of moderate lameness used in this study to broad range of clinical lameness situations should be performed carefully. CONCLUSIONS: These results support the use of low doses of xylazine or acepromazine to facilitate forelimb lameness evaluation up to 1 hour in duration.

A comprehensive approach to detecting multitudinous bioactive peptides in equine plasma and urine using hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry.

Bioactive peptides possess pharmacological effects and can be illicitly used in sports. To deter such misuse, an untargeted method using high resolution mass spectrometry (HRMS) has been developed for comprehensive detection of multitudinous exogenous peptides in equine plasma and urine. Forty-four peptides were extracted using mixed-mode solid-phase extraction (SPE) from plasma and urine, separated with a hydrophilic interaction liquid chromatography (HILIC) column, and detected on an HRMS instrument. Ammonium formate as a mobile phase additive had effects on HILIC retention and charge state distribution of the peptides. The acetonitrile percentage in the reconstitution solution affected the solubility of peptide neat standards and peptides in plasma and urine extracts differently. The stability of the peptides in plasma at ambient temperature was assessed. The limit of detection (LOD) was 10-50 pg/mL for most of the peptides in plasma, and ≤ 500 pg/mL for the remaining. LOD was 100-400 pg/mL for the majority of the analytes in urine, and ≤ 4000 pg/mL for the others. The method was used successfully to analyze incurred plasma and urine samples from research horses administered dermorphin. Even in the absence of reference standards, dermorphin metabolites (aFGYPS-NH2 , YaFG, and YaF) were identified. These results demonstrate that data generated with this method can be retrospectively reviewed for peptides that are unknown at the time of sample analysis without requiring re-analysis of the sample. This method provides a powerful novel tool for detection of numerous bioactive peptides and their metabolites in equine plasma and urine for doping control.

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.

Doping control analysis of four JWH-250 metabolites in equine urine by liquid chromatography-tandem mass spectrometry.

JWH-250 is a synthetic cannabinoid. Its use is prohibited in equine sport according to the Association of Racing Commissioners International (ARCI) and the Fédération Équestre Internationale (FEI). A doping control method to confirm the presence of four JWH-250 metabolites (JWH-250 4-OH-pentyl, JWH-250 5-OH-pentyl, JWH-250 5-OH-indole, and JWH-250 N-pentanoic acid) in equine urine was developed and validated. Urine samples were treated with acetonitrile and evaporated to concentrate the analytes prior to the analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The chromatographic separation was carried out using a Phenomenex Lux® 3 µm AMP column (150 x 3.0 mm). A triple quadrupole mass spectrometer was used for detection of the analytes in positive mode electrospray ionization using multiple reaction monitoring (MRM). The limits of detection, quantification, and confirmation for these metabolites were 25, 50, and 50 pg/mL, respectively. The linear dynamic range of quantification was 50-10000 pg/mL. Enzymatic hydrolysis indicated that JWH-250 4-OH-pentyl, JWH-250 5-OH-pentyl, and JWH-250 5-OH indole are highly conjugated whereas JWH-250 N-pentanoic acid is not conjugated. Relative retention time and product ion intensity ratios were employed as the criteria to confirm the presence of these metabolites in equine urine. The method was successfully applied to post-race urine samples collected from horses suspected of being exposed to JWH-250. All four JWH-250 metabolites were confirmed in these samples, demonstrating the method applicability for equine doping control analysis.

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.

Detection and confirmation of α-cobratoxin in equine plasma by solid-phase extraction and liquid chromatography coupled to mass spectrometry.

α-Cobratoxin (CTX) is a large peptide (71 amino acids) with strong analgesic effect and may be misused in sports such as horse racing. To prevent such misuse, a sensitive method is required for detection and confirmation of the toxin in equine samples. CTX was extracted from equine plasma using an optimized mixed-mode solid-phase extraction (SPE) procedure. Extracted CTX was reduced with dithiothreitol and alkylated with iodoacetamide, and then was digested by trypsin at 56°C for 30min. The digest was analysed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and tryptic peptides T2 (3CFITPDITSK12) and T4 (24TWCDAFCSIR33) were monitored for detection and confirmation of CTX. The limit of detection (LOD) was 0.05ng/mL for CTX in plasma, and the limit of confirmation (LOC) 0.2ng/mL. Unlike small peptides consisting of the 20 canonical amino acids, CTX was stable in equine plasma at ambient temperature for at least 24h. The developed analytical method was successfully applied to analysis of incurred plasma samples; CTX was detected in plasma collected 15min through 36h post subcutaneous administration of CTX (2.0mg dose) to a research horse, and confirmed 30min through 24h. Additionally, an approach named "reliable targeted SEQUEST search" has been proposed for assessing the specificity of T2 at product ion spectrum level for confirmation of CTX. T2 is uniquely specific for CTX, as evaluated with this approach and BLAST search. Furthermore, the effect of dimethyl sulfoxide (DMSO) as a mobile phase additive on electrospray (ESI) response of T2 and T4, background noise level and signal to noise ratio (S/N) was examined; DMSO increased signal intensity of T2 and T4 by a factor of less than 2. It is the first report that DMSO raised background noise level and did not improve S/N for the peptides, to the authors' knowledge. The developed analytical method may be applicable for analysis of CTX in plasma from other species such as greyhound dogs or even human beings.

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.

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.