Journal of Agronomy, Technology and Engineering Management J Agron Technol Eng Manag 2022, 5(1), 697-703. www.fimek.edu.rs/jatem Article The Validation of a Method for the Determination of Chloramphenicol in Milk Sanja Novković 1 , Vojislava Bursić 2 , Tijana Stojanović 2 , Bojana Špirović Trifunović 3 , Nikola Puvača 4 , Aleksandra Petrović 2 , Dušan Marinković 2 , and Gorica Vuković 1,3,* 1 Field test doo, Beograd, Vinogradska 150b, Serbia. 2 University of Novi Sad, Faculty of Agriculture, Trg Dositeja Obradovića 8, 21000 Novi Sad, Serbia. 3 University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11080 Zemun, Serbia. 4 Department of Engineering Management in Biotechnology, Faculty of Economics and Engineering Management in Novi Sad, University Business Academy in Novi Sad, Cvećarska 2, 21000 Novi Sad, Serbia. * Correspondence: goricavukovic@yahoo.com Received: 26 November 2021; Accepted: 08 February 2022 Abstract: The liquid chromatography tandem mass spectrometry (LC-MS/MS) method with the liquid-liquid extraction and SPE (Waters OASIS HLB column) cleaning process was validated for the determination of chloramphenicol (CAP) residues in milk. The optimisation of the mass spectrometry parameters was performed by injecting the CAP standard solutions into the electrospray ion (ESI) source. The mass spectrometer was operated in the negative ESI mode. The validation data fulfilled the requirements established in the Regulations Decisions 2020/657/EC. During the validation process the chloramphenicol-D5 as an internal standard was used. The obtained results indicated the good linearity (R 2 > 0.99) within the range of 0.1–2.0 μg/kg. The mean recovery for spiking levels was 96.5 ±10.59%. The limit of quantification (LOQ) was 0.1 μg /kg. The repeatability was 10.6%. The method fulfilled all the 2020/657/EC guidelines and thus can be extended for the routine analysis of CAP residues in milk. Keywords: validation; chloramphenicol; LC-MS/MS; milk. 1. Introduction The milk and dairy products are considered to be the essential components of a balanced human diet as they have tremendous nutritional values regarding a good proportion of proteins, fats and important minerals [1]. According to Puvača et al. [2], significant health benefits of milk and its products are related to the proteins, especially their nutritive value and biological properties. In the veterinary practice the usage of antibiotics is extensive - from the treating of diseases and microbial infections to the dietary supplements [3]. Chloramphenicol (CAP), or the 2,2-dichloro-N-[(1R,2R)-1,3-dihydroxy-1-(4-nitrophenyl)propan-2-yl]acetamide, is a broad-spectrum antibiotic, isolated from Streptomyces venezuelae, which belongs to the amphenicol drug family and has been used through the history as a cure for various infections in the veterinary medicine. Its chemical structure comprises both the lipophilic and hydrophilic groups, as well as the substituents (Figure 1). Since the polar and nonpolar groups are present, it is soluble in organic solvents (acetone, ethanol, ethyl acetate and methanol) and slightly soluble in water [4].