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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.

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.

Simultaneous separation and determination of 16 testosterone and nandrolone esters in equine plasma using ultra high performance liquid chromatography–tandem mass spectrometry for doping control

The potential for using testosterone and nandrolone esters in racehorses to boost the biological concentrations of these steroids and enhance athletic performance is very compelling and should be seriously considered in formulating regulatory policies for doping control. In order to regulate the use of these esters in racehorses, a sensitive and validated method is needed. In this paper, we report such a method for simultaneous separation, screening, quantification and confirmation of 16 testosterone and nandrolone esters in equine plasma by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Analytes were extracted from equine plasma by liquid-liquid extraction using a mixture of methyl tert-butyl ether and ethyl acetate (50:50, v/v) and separated on a sub-2 micron C(18) column. Detection of analytes was achieved on a triple-quadrupole mass spectrometer by positive electrospray ionization mode with selected reaction monitoring (SRM). Mobile phase comprised 2 mM ammonium formate and methanol. Deuterium-labeled testosterone enanthate and testosterone undecanoate were used as dual-internal standards for quantification. Limits of detection (LOD) and quantification (LOQ) were 25-100 pg/mL and 100-200 pg/mL, respectively. The linear dynamic range of quantification was 100-10,000 pg/mL. For confirmation of the presence of these analytes in equine plasma, matching of the retention time with mass spectrometric ion ratios from MS/MS product ions was used. The limit of confirmation (LOC) was 100-500 pg/mL. The method is sensitive, robust, selective and reliably reproducible.

Simultaneous separation and confirmation of amphetamine and related drugs in equine plasma by non‐aqueous capillary‐electrophoresis‐tandem mass spectrometry

A non-aqueous capillary electrophoresis-mass spectrometry (NACE-MS) method was developed for simultaneous separation and identification of 12 amphetamine and related compounds in equine plasma. Analytes were recovered from plasma by liquid-liquid extraction using methyl tertiary butyl ether (MTBE). A bare fused-silica capillary was used for separation of the analytes. Addition of sheath liquid to the capillary effluent allowed the detection of the analytes by positive electrospray ionization mass spectrometry using full scan data acquisition. The limit of detection (LOD) for the target analytes was 10-200 ng/mL and that of confirmation (LOC) was 50-1000 ng/mL in equine plasma. Capillary electrophoresis (CE) and mass spectrometry (MS) parameters were optimized for full CE separation and MS detection of the analytes. Separation buffer comprised 25 mM ammonium formate in acetonitrile/methanol (20: 80, v/v) plus 1 M formic acid. Sheath liquid was isopropanol-water-formic acid (50:50:0.5, v/v/v). Samples were hydrodynamically injected and separated at 25 kV. Analytes were electrokinetically separated and mass spectrometrically identified and confirmed. This simple, fast, inexpensive and reproducible method was successfully applied to post race equine plasma and research samples in screening for amphetamine and related drugs.

A controlled and sustained local gentamicin delivery system for inner ear applications.

Objective: Intratympanic gentamicin injection (ITGI) has gained acceptance worldwide for the treatment of Ménières disease. Reports assessing the efficacy of ITGI suffer from high variability between patients. This variability may be due to ITGI, which does not permit a sustained diffusion of gentamicin across the round window membrane. The present study investigates the effectiveness of a sustained local hydrogel system on the delivery of gentamicin into the inner ear for the treatment of Ménières disease. Methods: A matrix of hydrogel loaded with/without gentamicin was explored in vivo. Gentamicin was applied to the ear of mice either through ITGI or in the hydrogel system. Pharmacokinetics, hearing, and balance function were examined to study how the hydrogel system affected the gentamicin delivery and inner ear functions. Results: The 2 gentamicin delivery methods yielded different kinetics curves. The hydrogel system achieved sustained release during a 7-day period, with a flat plateau phase from Day 1 to Day 3 and slow descent in the subsequent days. The ITGI curve dramatically declined after the peak concentration at Day 1 and was almost eliminated by Day 3. The hydrogel system yielded noticeable balance dysfunction with no significant hearing changes. In contrast, ITGI exhibited no significant influences on the inner ear functions after applying the same dose of 40 kg of gentamicin. Conclusion: The hydrogel system established in this research allows for more sustained and consistent and efficient drug release than traditional ITGI for the transport of gentamicin into the inner ear, offering a new and exciting treatment of Ménières disease.

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.