Analysis of metronidazole in equine plasma using liquid chromatography/tandem mass spectrometry and high-resolution accurate mass spectrometry.

This research article presents a new method using liquid chromatography and mass spectrometry to detect a specific antibiotic called metronidazole in horse’s blood. The purpose of this method is to monitor the illegal use of certain substances in racehorses in the state of Pennsylvania, USA.

Introduction

• The study focuses on the development of a new method to detect the antibiotic metronidazole (MTNZ) in horse’s blood. This is crucial because the illegal use of substances like bicarbonate solutions and antibiotics in racehorses to improve their performance is a concerning issue in Pennsylvania.
• Bicarbonate treatments are used to manage acidosis and muscle cramps in horses, but their misuse often results in diarrhea, necessitating the use of antibiotics like MTNZ. Because there were no existing methods to detect MTNZ in the horses’ blood, this study aimed to change that.

Methods

• The team used a method called liquid chromatography/tandem mass spectrometry (LC/MS/MS), along with high-resolution accurate mass spectrometry (HRAMS) for accurate mass determination.
• The analyte (the substance being measured – in this case, MTNZ) was extracted from horse plasma using methyl tert-butyl ether.
• Then they used an ACE® C18 column for separation and a combination of ammonium formate and acetonitrile for the mobile phase.
• The mass analysis was performed using an LTQ XL linear ion trap mass spectrometer in positive electrospray ionization mode.

Results

• The limit of detection (LOD), limit of confirmation (LOC) and lower limit of quantification (LLOD) of MTNZ were established as 1, 2 and 50 ng/mL respectively. This indicates that the developed method is highly sensitive.
• The MTNZ was found to be stable in plasma at -20 and -70°C for 28 days, and for 24 hours at 20°C in the autosampler. This validates that the method is reliable and provides consistent results.
• The performance of the method was further ensured by assessing the accuracy and precision within an acceptable range.
• The presence of MTNZ in horse plasma was confirmed using various parameters like a specific ion transition, a retention time and the accurate mass of the analyte.

Conclusions

• The research concludes that the developed method is highly efficient, reliable and selective for detecting MTNZ in horse blood. This will allow the authorities to regularly monitor the illegal use of MTNZ in racehorses.
• The methodology is quick, sensitive and reproducible which makes it an impactful tool to ensure fairness in horse racing competitions.