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, IC20 and IC50, of 0.09 and 0.44 ng mL-1, respectively. Milk samples were spiked with standards equivalent to 0, 0.2, 0.3, 0.5, 1.0 & 1.5 ng mL-1 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-1, respectively. The results were found comparable with the commercial ELISA, having recoveries of 87 to 100%, CCα 0.09 ng mL-1 and CCβ 0.12 ng mL-1. 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-1 with correction coefficient 0.9902. The mean recoveries were found to be 88 to 100%. The CCα was calculated as 0.057 ng mL-1 and CCβ 0.10 ng mL-1. Since CCα and CCβ are less than half of the MRPL (0.15 ng mL-1), 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-1, slightly above the MRPL.
Keywords: Chloramphenicol; bovine milk; confirmation; in-house ELISA; mass spectrometry.