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

Rationale Treatment of racehorses with bicarbonate solutions to manage acidosis and muscle cramps prior to competition is banned in Pennsylvania (PA). Use of excess bicarbonate in horses causes diarrhea, requiring treatment with an antibiotic such as metronidazole (MTNZ). At present no method exists for detecting MTNZ in equine plasma. Thus, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the detection, quantification and confirmation of MTNZ was developed. Methods The analyte was recovered from plasma by liquid-liquid extraction using methyl tert-butyl ether and separated on an ACE® C18 column with its guard column. The mobile phase comprised a mixture of 5 mM ammonium formate (pH 3.5) and acetonitrile (60:40; v/v). Mass analysis was performed on an LTQ XL linear ion trap mass spectrometer in positive electrospray ionization mode while accurate mass determination was also performed in positive electrospray ionization mode using high-resolution accurate mass spectrometry (HRAMS). Results The limit of detection (LOD), limit of confirmation (LOC) and lower limit of quantification (LLOD) were 1, 2 and 50 ng/mL, respectively. The analyte in plasma was stable at -20 and -70°C for 28 days, as well as for 24 h at 20°C in the autosampler. The percentage coefficients of variation (% CV) for the intra-day and inter-day precision for the LLOQ were 5.1:3.68 and 13.21:9.95, respectively, while the intra-day accuracy was from 98.71 to 101.57% and that of the inter-day was from 88.64 to 96.6%. The matrix effect was between 9 and 24%. The precursor → product ion transition m/z 172 → 128, a retention time of 2.92 min and the accurate mass of the [M+H]+ ion of the analyte (m/z 172.0173) were used as criteria for confirmation of the presence of MTNZ in equine plasma. Conclusions The method is highly sensitive and selective for the detection, identification and confirmation of MTNZ in equine plasma. Thus, illegal use of MTNZ in racehorses can be routinely monitored within the US State of Pennsylvania. The method is fast, sensitive, reproducible, and reliable.