Method Development and Application: Solid Phase Extraction (SPE) Clean-up and Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC/MS/MS) Quantification of Pirlimycin in Dairy Cow Feces and Urine

Abstract Text: In the last two decades antibiotic excretion by livestock has received significant attention because of the contribution of excreted antibiotics to bacterial antibiotic resistance. Reliable and accurate quantification of antibiotics in feces and urine is critical to assess environmental loading of antibiotics by the livestock industry in the effort to maintain or improving the sustainability of animal agriculture. Pirlimycin, a lincosamide antibiotic, is one of the most commonly used antibiotics for the treatment of mastitis in dairy cows. There is no published data on pirlimycin loading to the environment via fecal and urinary excretion, probably due to inadequate methodology to quantify pirlimycin in fecal and urine matrices. Therefore, the objective of this study was to develop and validate an analytical method to qualify and quantify pirlimycin in dairy cow feces and urine. Samples were extracted with methanol+0.05 M phosphate buffer (70:30, v/v). Sample extracts were cleaned using solid phase extraction (SPE) via elution through hydrophilic-lipophilic-balanced (HLB) cartridges and filtering through 0.2 μm filters. Clarified extracts were analyzed for pirlimycin using ultra performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS). This method was sensitive with a limit of quantification (LOQ) of 1.47 ng/g wet feces and 0.90 ng/mL urine. The developed method recovered 80 to 108% of pirlimycin spiked in feces and recovery was 89 to 98% in urine. Repeatability and reproducibility of the method was estimated by intra- and inter-day variations [residual standard deviation (RSD) %], and ranged from 2.3 to 13% and 2.3 to 14% for feces and urine, respectively. With the application of this method to samples collected in the 10 hours following intramammary dosing, pirlimycin was detected at 61.2 to 71.8 ng/g and 153 to 254 ng/mL in feces and urine, respectively. This sensitive, accurate, and robust method can be used to quantify trace amounts of antibiotics in dairy cow feces and urine, and may help in the assessment of fate and environmental impact of antibiotics used on farms.

Keywords: pirlimycin, dairy cow feces and urine, ultra performance liquid chromatography-tandem mass spectrometry