Development of in-house ELISA for detection of chloramphenicol in bovine milk with subsequent confirmatory analysis by LC-MS/MS.

This study was undertaken to develop and validate direct competitive ELISA for the determination of chloramphenicol residues in bovine milk. Antisera and an enzyme-tracer for chloramphenicol were prepared and used to develop an ELISA with inhibition concentrations, IC and IC, of 0.09 and 0.44 ng mL, respectively. Milk samples were spiked with standards equivalent to 0, 0.2, 0.3, 0.5, 1.0 & 1.5 ng mL and extracted in methanol. The mean recoveries were found to be 73-100% with coefficient of variance 7-11%. The decision limit (CCα) and detection capability (CCβ) were calculated as 0.10 and 0.12 ng mL, respectively. The results were found comparable with the commercial ELISA, having recoveries of 87 to 100%, CCα 0.09 ng mL and CCβ 0.12 ng mL. As per Commission Decision 2002/657/EC, in-house ELISA was further validated by using LC-MS/MS. Mass spectral acquisition was done by using electrospray ionization in the negative ion mode applying single reaction monitoring of the diagnostic transition reaction for CAP (m/z 152, 194 and 257). The calibration curve showed good linearity in concentrations from 0.025 to 1.6 ng mL with correction coefficient 0.9902. The mean recoveries were found to be 88 to 100%. The CCα was calculated as 0.057 ng mL and CCβ 0.10 ng mL. Since CCα and CCβ are less than half of the MRPL (0.15 ng mL), the test was found suitable for screening and quantification of CAP residues in bovine milk samples. Results of surveillance studies indicated that out of 31 analyzed milk samples, 12.9% samples were found with CAP residues but only 3.2% samples were declared positive with maximum concentration 0.31 ng mL, slightly above the MRPL.