Simultaneous quantification and confirmation of oxycodone and its metabolites in equine urine using ultra-high performance liquid chromatography-tandem mass spectrometry.
Abstract: 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.
Copyright © 2024. Published by Elsevier B.V.
Publication Date: 2024-04-12 PubMed ID: 38615430DOI: 10.1016/j.jchromb.2024.124125Google Scholar: Lookup
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Summary
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This research article is about the development and validation of a method to simultaneously confirm and quantify oxycodone and its metabolites in horse urine, for preventing the misuse of this painkiller as a performance-enhancer in racehorses.
About the Research
- The article centers on the development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. The method is designed to simultaneously quantify and confirm the presence of oxycodone and eight of its metabolites in equine urine.
- This research was driven by the need to monitor and detect the potentially illegal use of oxycodone, a strong pain-relieving opioid, in racehorses for performance enhancement purposes.
Methodology
- The method involved the use of enzymatic hydrolysis and liquid-liquid extraction using ethyl acetate to process the equine urine samples.
- The analytes, substances analyzed in the lab, were separated on a Hypersil Gold C18 sub-2 µm column. They were then detected on a triple quadrupole mass spectrometer, an instrument that can identify and quantify compounds based on their mass-to-charge ratio.
Results
- The limit of detection (LOD) was determined to be between 25-50 pg/mL. The lower limit of quantification (LLOQ) was 100 pg/mL. This indicates the smallest amount of analyte that can be accurately identified and quantified, respectively.
- Excellent linearity of the calibration curves was observed over a range of 100-10000 pg/mL for all nine analytes, demonstrating good accuracy and precision of measurements across this range.
- Through retention time, signal-to-noise ratio, and product ion ratios, confirmation criteria were established, resulting in the limits of confirmation (LOC) ranging from 100 to 250 pg/mL.
- Finally, preliminary pharmacokinetic (PK) study results suggested that oxycodone metabolites have longer detection periods in equine urine than oxycodone itself, extending the ability to detect oxycodone exposure in racehorses.
Cite This Article
APA
You Y, Missanelli JR, Proctor RM, Haughan J, Robinson MA.
(2024).
Simultaneous quantification and confirmation of oxycodone and its metabolites in equine urine using ultra-high performance liquid chromatography-tandem mass spectrometry.
J Chromatogr B Analyt Technol Biomed Life Sci, 1238, 124125.
https://doi.org/10.1016/j.jchromb.2024.124125 Publication
Researcher Affiliations
- Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, 382 West Street Road, Kennett Square, PA 19348, USA; Pennsylvania Equine Toxicology and Research Laboratory, 220 East Rosedale Avenue, West Chester, PA 19382, USA. Electronic address: ywyou@upenn.edu.
- Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, 382 West Street Road, Kennett Square, PA 19348, USA; Pennsylvania Equine Toxicology and Research Laboratory, 220 East Rosedale Avenue, West Chester, PA 19382, USA.
- Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, 382 West Street Road, Kennett Square, PA 19348, USA; Pennsylvania Equine Toxicology and Research Laboratory, 220 East Rosedale Avenue, West Chester, PA 19382, USA.
- Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, 382 West Street Road, Kennett Square, PA 19348, USA.
- Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, 382 West Street Road, Kennett Square, PA 19348, USA; Pennsylvania Equine Toxicology and Research Laboratory, 220 East Rosedale Avenue, West Chester, PA 19382, USA.
Conflict of Interest Statement
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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