Submitted to: Analytical and Bioanalytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/4/2011
Publication Date: 2/1/2012
Publication URL: handle.nal.usda.gov/10113/54055
Citation: Shelver, W.L., Varel, V.H. 2012. Development of a UHPLC-MS/MS method for the measurement of chlortetracycline degradation in swine manure. Analytical and Bioanalytical Chemistry. 402:1931-1939. Interpretive Summary: Chlortetracycline is an antibiotic frequently utilized in raising swine both as a medicine and as a growth promoter. Because chlortetracycline is excreted unchanged in the feces, relatively high amounts are found in hog wastes. Since this material is frequently used as fertilizer it is important to understand how concentrations of chlortetracycline are affected by processing of hog wastes to prevent environmental contamination. This study developed a method of measuring chlortetracycline and some of its degradation products in hog wastes enabling the tracking of the antibiotic. The method could measure several decomposition products simultaneously with chlortetracycline without interference from similar antibiotics. The study further showed higher temperatures markedly accelerated the decomposition of chlortetracycline as well as the formation of isochlortetracycline (a product with little or no antibiotic activity).
Technical Abstract: An ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed capable of simultaneously measuring chlortetracycline (CTC), epi-chlortetracycline (epi-CTC) and isochlortetracycline (ICTC), as well as other structurally related tetracyclines in swine manure. A simple sample preparation was used consisting of extraction, dilution, centrifugation, and ultrafiltration. The concentrations of analyte were calculated using d6-tetracycline as an internal standard in the matrix matched standard curve. A solvent gradient resolved the compounds in 3.5 minutes with an additional 1.5 minutes of re-equilibration allowing the analyses of a large number of samples in a short period of time. MS/MS was used as the detection method giving analyte confirmation in addition to a large dynamic range and high sensitivity. The UHPLC-MS/MS method successfully resolved multiple degradation products of CTC from the complex manure matrix. The method detection limits (MDL) ranged from 1.9 pg/uL for CTC to 7.3 pg/uL for ICTC and the calibration curve was linear from 1 to 10,000 pg/uL. The method was tested by measuring CTC and its degradation products as a function of time in incurred swine manure that had been incubated at three different temperatures (22, 38, and 55 C). CTC concentration at 22 C decreased 44% after 25 days; greater percentage decreases were observed when the manure was stored at elevated temperatures (96% and 98% for 38 and 55 C, respectively). The concentration of the microbiologically inactivate isomer, isochlortetracycline (ICTC), increased over the incubation period. At 22 C, ICTC continued to increase through 25 days of incubation; at 38 C, ICTC concentration plateaued on day 14; while at 55 C ICTC concentration plateaued on day 7, with concentration increases of 198%, 374%, and 282% for 22 C, 38 C, and 55 C respectively.