Stable Isotope Labeling by Carbon-13 in Bacteria Culture for the Analysis of Residual Avermectin Using Stable Isotope Dilution Liquid Chromatography Tandem Mass Spectrometry

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This study describes the development of a new stable isotope labeling method by carbon-13 in bacteria culture (SILCB) for the analysis of residual antibiotics via liquid chromatography with tandem mass spectrometry (LC-MS/MS). Stable isotope dilution (SID) LC-MS/MS with QuEChERS was employed to determine avermectin, particularly avermectin B1a (AV-a) and B1b (AV-b), based on completely ¹³C-labeled internal standards (¹³C-ISs) obtained from the SILCB. Our SILCB was developed from an optimal inorganic medium using ¹³C₆-glucose for Streptomyces avermitilis (14893 strain). A rough extract containing ¹³C-ISs was purified via high-speed countercurrent chromatography with a volatile two-phase solvent system composed of n-hexane/ethyl acetate/methanol/0.5% formic acid in water (7/3/5/5/, V/V). The purified ¹³C-ISs were evaluated to confirm the presence of completely ¹³C-labeled ions with m/z 938 > 326 and m/z 923 > 309 for AV-a and AV-b, respectively. The QuEChERS approach with the ¹³C-ISs procedure achieved acceptable recovery rates in beef meat samples of 99.5 – 100.0% (RSD < 2.0%, n = 6). For the analysis of residual antibiotics in foodstuffs by SID-LC-MS/MS and QuEChERS, the SILCB represents a significant improvement over previous methods suffering from cumbersome sample preparation and matrix effects.