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.

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.

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.

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.