Development and Validation of Liquid Chromatography-Tandem Mass Spectrometry Methods for the Quantification of Cefquinome, Ceftiofur, and Desfuroylceftiofuracetamide in Porcine Feces with Emphasis on Analyte Stability

Abstract

Cefquinome and ceftiofur are β-lactam antibiotics used for the treatment of bacterial infections in swine. Although these antimicrobials are administered intramuscularly, the exposure of the gut microbiota to these cephalosporins is not well described. This exposure can contribute to the emergence and spread of antimicrobials in the environment and to the possible spread of antimicrobial resistance genes. To assess the impact of drug administration on the intestinal excretion of these antimicrobials it is essential to measure the amounts of native compound and metabolites in feces. Two (ultra)-high-performance liquid chromatography-tandem mass spectrometry ((U)HPLC–MS/MS) methods were developed and validated, one for the determination of cefquinome and ceftiofur and the other for the determination of ceftiofur residues, measured as desfuroylceftiofuracetamide, in porcine feces. The matrix-based calibration curve was linear from 5 ng g−1 to 1000 ng g−1 for cefquinome (correlation coefficient (r) = 0.9990 ± 0.0007; goodness of fit (gof) = 3.70 ± 1.43) and ceftiofur (r = 0.9979 ± 0.0009; gof = 5.51 ± 1.14) and quadratic from 30 ng g−1 to 2000 ng g−1 for desfuroylceftiofuracetamide (r = 0.9960 ± 0.0020; gof = 7.31 ± 1.76). The within-day and between-day precision and accuracy fell within the specified ranges. Since β-lactam antibiotics are known to be unstable in feces, additional experiments were conducted to adjust the sampling protocol in order to minimize the impact of the matrix constituents on the stability of the analytes. Immediately after sampling, 500 µL of an 8 µg mL−1 tazobactam solution in water was added to 0.5 g feces, to reduce the degradation in matrix.

Intestinal Exposure to Ceftiofur and Cefquinome after Intramuscular Treatment and the Impact of Ceftiofur on the Pig Fecal Microbiome and Resistome

“…Feces were sampled in a sterile plastic cup after non-invasive rectal stimulation before the administration of ceftiofur and at 4, 6.5, 10, 23, 28, 31, 33.5, 46.5, 51, 54 and 57 h after the start of the treatment. It has been previously observed that the fecal microbiota can rapidly degrade the β-lactam ring of ceftiofur, its related metabolites and cefquinome [37,74]. Therefore, immediately after sampling, 500 µL of a tazobactam solution (8 mg•L −1 ) in water was added to an aliquot of 0.5 g sampled feces.…”

“…The fecal concentration of ceftiofur, desfuroylceftiofuracetamide and cefquinome was determined using (U)HPLC-MS/MS methods, as previously described [37]. The LOQ was 5 ng•g −1 for ceftiofur, 30 ng•g −1 for desfuroylceftiofuracetamide and 5 ng•g −1 for cefquinome.…”

“…After administration, ceftiofur is metabolized rapidly to its active residue, desfuroylceftiofur. Since desfuroylceftiofur is known to be unstable, an optimized sampling protocol was followed, and during analysis, the combinations of ceftiofur and its related residues (e.g., desfuroylceftiofur) were derivatized and measured as desfuroylceftiofuracetamide [37]. The second objective of this study was to explore the impact of ceftiofur treatment on the fecal microbiome and its resistome.…”

The Simultaneous Detection of Multiple Antibiotics in Milk and Pork Based on an Antibody Chip Biosensor

“…Rapid and efficient detection of drug residues in animal-derived food remains a key issue. Several residue detection methods have been reported, such as chromatographic methods [ 7 , 8 ], microbiological methods [ 9 , 10 , 11 ], and enzyme-linked immunosorbent assays (ELISA) [ 12 , 13 , 14 ]. Microbiological methods in residue screening on high throughput showed good performance but lack specificity and sensitivity.…”