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.

This study describes the development and validation of a liquid chromatography–tandem mass spectrometry method to detect the fentanyl metabolite, N-[1-(2-Phenethy-4-Piperidinyl)] Maloanilinic Acid (PMA), in horse urine as a way to improve doping control in equine sports. This method is particularly useful because fentanyl is rapidly metabolized and its detection in urine allows for a longer period of detection.

Method Developed for PMA Analysis

• This study focused on developing an effective method to analyze the presence of PMA, a fentanyl metabolite in horse urine. For this, the researchers used a technique known as liquid chromatography–tandem mass spectrometry (LC-MS-MS), which allows the accurate identification and quantification of substances in samples. This method is necessary because while fentanyl can be detected in blood, it quickly gets metabolized, limiting its detection period. However, checking for PMA in urine is advantageous as it allows for a longer detection time.

Procedure and Method Validation

• PMA was first extracted from urine samples using a process called solid phase extraction. This extraction was followed by its separation on a C18 column, and then finally detected by using a triple quadrupole mass spectrometer. This spectrometer was operated in positive-ion mode, monitoring product ions with specific mass-to-charge ratios.
• The developed method was then validated for several factors as per the guidelines of the US Food and Drug Administration for bioanalysis. These factors included extraction recovery, matrix effect, specificity, sensitivity, precision and accuracy, carryover, and processed sample stability. The limits of detection and quantification were ascertained to be 5 and 10 pg/mL, respectively with the linearity obtained over the concentration range of 10-10,000 pg/mL.
• For confirmatory analysis, the researchers used LC retention time, diagnostic product ions, and product ion ratio as the determining criteria. The lowest concentration for confirmatory analysis was validated at 50 pg/mL.

Application and Importance of the Method

• After being satisfactorily validated, the method was used to measure the PMA concentrations in horse urine post the intravenous administration of fentanyl to a research horse. The researchers found that the method was effective in confirming the presence of PMA in post-race urine samples.
• This research, therefore, represents a valuable addition to the toolbox of equine doping control methods. Its application can help combat illegal doping and subsequently contribute to protecting the health of racehorses.