Cornelius E. Uboh

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.

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.

Ultra-performance liquid chromatography/tandem mass spectrometry in high-throughput detection, quantification and confirmation of anabolic steroids in equine plasma

An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method for fast-throughput analysis of eight anabolic and androgenic steroids (AAS) in equine plasma is reported. Analytes were recovered by liquid-liquid extraction using methyl tert-butyl ether, separated on a 1.9 microm C(18) reversed-phase column, and analyzed in positive electrospray ionization mode on a triple quadrupole mass spectrometer with selected reaction monitoring (SRM) and full product ion scans. Two SRM ion transitions were monitored for each AAS during screening to obtain highly selective screening results. Full product ion spectra of excellent quality for AAS, at 100 pg/0.5 mL in plasma, devoid of interfering spectra from impurities in plasma, were obtained. To our knowledge, this is the first report on the acquisition of full product ion spectra at such a low analyte concentration and plasma volume using a triple quadrupole instrument. In addition to product ion intensity ratios obtained from three SRM scans for identifying AAS in equine plasma, full product ion spectra were used as supporting evidence for confirmation. For quantification, deuterium-labeled testosterone and stanozolol were used as internal standards (ISs). The limits of detection, quantification and confirmation were 6.25-12.5 pg/0.5 mL, 25 pg/0.5 mL and 50-100 pg/0.5 mL, respectively. There was no significant matrix effect on the analysis of all eight AAS. Intra-day precision and accuracy were 2-15% and 91-107%, respectively. Inter-day precision and accuracy were 1-21% and 94-110%, respectively. Total analysis time was 5 min. To date, the method has been successfully used in the analysis of >12,000 samples for AAS in plasma samples from racehorses competing in the State of Pennsylvania. The method is fast, selective, reproducible, and reliable.

Pharmacokinetic profile and pharmacodynamic effects of romifidine hydrochloride in the horse

Romifidine HCl (romifidine) is an α(2)-agonist commonly used in horses. This study was undertaken to investigate the pharmacokinetics (PK) of romifidine following intravenous (i.v.) administration and describe the relationship between PK parameters and simultaneously recorded pharmacodynamic (PD) parameters. Romifidine (80 μg/kg) was administered by i.v. infusion over 2 min to six adult Thoroughbred horses, and plasma samples were collected and analyzed using liquid chromatography-mass spectrometry. Limit of quantification was <0.1 ng/mL. PD parameters and arterial blood gases were measured for 300 min following romifidine administration. Statistical PD data analysis included mixed-effect modeling. After i.v. administration of romifidine, the plasma concentration-vs.-time curve was best described by a two-compartmental model. Terminal elimination half-life (t(1/2β) ) was 138.2 (104.6-171.0) min and volumes for central (V(c)) and peripheral (V(2)) compartments were 1.89 (0.93-2.39) and 2.57 (1.71-4.19) L/kg, respectively. Maximum plasma concentration (C(max)) was 51.9 ± 13.1 ng/mL measured at 4 min following commencement of drug administration. Systemic clearance (Cl) was 32.4 (25.5-38.4) mL · min/kg. Romifidine caused a significant reduction in heart rate and cardiac index and an increase in mean arterial pressure (P < 0.05). Sedation score and head height values were significantly different from the baseline values for 120 min (P < 0.05). The decline in cardiovascular and sedative effects correlated with the decline in plasma romifidine concentration (P < 0.05). In conclusion, a highly sensitive analytical technique for the detection of romifidine in equine plasma allowed detailed description of its PK profile. The drug produces long-lasting sedation in horses that corresponds with the long terminal elimination half-life of the drug.

Confirmatory Analysis of Continuous Erythropoietin Receptor Activator and Erythropoietin Analogues in Equine Plasma by LC−MS for Doping Control

Continuous erythropoietin receptor activator (CERA) is the third generation of recombinant human erythropoietin (rhEPO) medication that retains the effect of promoting red blood cell production but has longer duration of action in the body. CERA, rhEPO, and darbepoetin alpha (DPO) can be misused to enhance performance in both human and equine athletes. To deter such misuse, a very selective and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has now been developed for identification of CERA, rhEPO, and DPO in equine plasma. The method employs a new signature tryptic peptide, T8 ((54)MEVGQQAVEVWQGLALLSEAVLR(76), common to the three proteins), and improved immunoaffinity extraction. The analytes were extracted by anti-rhEPO antibodies from plasma samples that were pretreated with polyethylene glycol (PEG) 6000. The extracted analytes were digested by trypsin and analyzed by LC-MS/MS. The limit of identification was 0.5 ng/mL for CERA, 0.2 ng/mL for rhEPO, and 0.1 ng/mL for DPO in equine plasma; the limit of detection was 0.3 ng/mL for CERA, 0.1 ng/mL for rhEPO, and 0.05 ng/mL for DPO. Specificity of the method was assessed via BLAST and SEQUEST protein database searches, and the T8 is extremely specific at both peptide and product ion levels for the identification of CERA, rhEPO, and DPO. This method was successful in identifying CERA and DPO in plasma samples collected from research horses post the drug administrations. It provides a useful tool in the fight against blood doping with CERA, rhEPO, and DPO in racehorses. Additionally, the following two technical approaches adopted in this study may also be helpful in protein identifications and biomarker discoveries in a broad scope: precipitating plasma proteins with PEG 6000 to improve immunoaffinity extraction efficiency of the target proteins and making a large and more lipophilic peptide detectable at low concentrations by increasing its solubility in the sample solvent.

Detection and confirmation of 60 anabolic and androgenic steroids in equine plasma by liquid chromatography-tandem mass spectrometry with instant library searching

In 2008, Pennsylvania (PA) became the first State in the USA to ban and enforce the ban on the use of anabolic and androgenic steroids (AAS) in equine athletes by using plasma for analysis. To enforce the ban, a rapid and high-throughput method for analysis of 60 AAS in equine plasma was developed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analytes were recovered from plasma by liquid-liquid extraction (LLE) using methyl tert-butyl ether, separated on a reversed-phase C₁₈ column and analyzed by electrospray ionization mass spectrometry. Multiple-reaction monitoring (MRM) scan was employed for screening. When the MRM signal of an analyte exceeded 1000 counts per second (cps), information-dependent acquisition (IDA) triggered generation of an enhanced product ion (EPI) scan of the analyte. A library for the analytes was simultaneously established using the EPI spectrum. Unambiguous identification of any of the 60 AAS in a test sample was based on both the presence of MRM response within the correct retention time (t(R)) window and a qualitative match between EPI spectrum of the test sample and that of the reference drug standard stored in the library. Total analysis time was 7 min. The limit of detection (LOD) and limit of confirmation (LOC) for most of the analytes were 0.01-2 ng/mL and 0.1-10 ng/mL, respectively. Recovery of the analytes from plasma by LLE was 74-138%. The method was successfully verified and is routinely used in the screening of post-race equine plasma samples for the presence of these 60 AAS. The method is rapid, sensitive, reproducible, and reliable.

Pharmacokinetics of intra-articular, intravenous, and intramuscular administration of triamcinolone acetonide and its effect on endogenous plasma hydrocortisone and cortisone concentrations in horses

OBJECTIVE To compare pharmacokinetics of triamcinolone acetonide (TA) following i.v., intra-articular (i.a.), and i.m. administration and determine its effect on plasma concentrations of hydrocortisone and cortisone. ANIMALS 6 Thoroughbreds. PROCEDURES TA (0.04 mg/kg) was administered i.v., i.m., or i.a., and plasma TA, hydrocortisone, and cortisone concentrations were determined. RESULTS I.v. administration of TA was fitted to a 2-compartment model. Median distribution half-life was 0.50 hours (range, 0.24 to 0.67 hours); elimination half-life was 6.1 hours (range, 5.0 to 6.4 hours). Transfer half-life of TA from joint to plasma was 5.2 hours (range, 0.49 to 73 hours); elimination half-life was 23.8 hours (range, 18.9 to 32.2 hours). Maximum plasma concentration following i.a. administration was 2.0 ng/mL (range, 0.94 to 2.5 ng/mL), and was attained at 10 hours (range, 8 to 12 hours). Maximum plasma concentration following i.m. administration was 0.34 ng/mL (range, 0.20 to 0.48 ng/mL) and was attained at 13.0 hours (range, 12 to 16 hours); concentration was still quantifiable at 360 hours. Hydrocortisone plasma concentrations were significantly different from baseline within 0.75, 2, and 1 hours after i.v., i.a., and i.m. administration, respectively, and remained significantly different from baseline at 96 and 264 hours for i.v. and i.a. administration. Following i.m. administration of TA, plasma concentrations of hydrocortisone did not recover to baseline concentrations by 360 hours. CONCLUSIONS AND CLINICAL RELEVANCE Pharmacokinetics of TA and related changes in hydrocortisone were described following i.v., i.a., and i.m. administration. A single administration of TA has profound effects on secretion of endogenous hydrocortisone.

Pharmacokinetics of fentanyl administered transdermally and intravenously in sheep

OBJECTIVE To investigate the pharmacokinetics of fentanyl administered transdermally and IV in sheep. ANIMALS 21 adult female sheep. PROCEDURES Fentanyl was administered IV to 6 healthy sheep. Transdermal fentanyl patches (TFPs) were applied to 15 sheep 12 hours prior to general anesthesia and surgery. Seria blood samples were collected for 18 hours after IV injection and 84 hours after TFP application. Fentanyl concentrations were quantified via liquid chromatography-mass spectrometry, and pharmacokinetic values were estimated. RESULTS All sheep completed the study without complications. Following a dose of 2.5 g/kg administered IV, the half-life was 3.08 hours (range, 2.20 to 3.36 hours), volume of distribution at steady state was 8.86 L/kg (range, 5.55 to 15.04 L/kg), and systemic clearance was 3.62 L/kg/h (range, 2.51 to 5.39 L/kg/h). The TFPs were applied at a mean dose of 2.05 g/kg/h. Time to maximum plasma concentration and maximal concentration were 12 hours (range, 4 to 24 hours) and 1.30 ng/mL (range, 0.62 to 2.73 ng/mL), respectively. Fentanyl concentrations were maintained at >0.5 ng/mL for 40 hours after TFP application. CONCLUSIONS AND CLINICAL RELEVANCE IV administration of fentanyl resulted in a short half-life. Application of a TFP resulted in stable blood fentanyl concentrations in sheep.