A New HPLC Analysis of Residual Penicillins in Edible Animal Tissues by Pre-column Fluorescence Derivatization

Abstract

An analytical method was developed for the simultaneous determination of penicillins (benzylpenicillin, oxacillin, cloxacillin, dicloxacillin and nafcillin) remaining in edible tissues (muscle and kidney) of animals (bovine and swine) by pre-column fluorescence derivatization. For labeling a carboxyl group in penicillins, seven kinds of fluorescence derivatization reagents were tested: 3-bromamethyl-6, 7-dimethoxy-1-methyl-1, 2-dihydroquinoxaline-2-one (Br-DMEQ), 4-bromomethyl-7-methoxycoumarin (Br-Mmc), 5, 6-dimethoxy-2-(4-hydrazinocarbonylphenyl) benzothiazole (BHBT), 1-bromoacetylpyrene (BAP), 6, 7-dimethoxy-1-methyl-2(H)-quinoallnone-3-propinohydrazide(DMEQ-hydrazide), 2- (2, 3-naphthalimino)ethyl trifluoromethanesulfonate (NE-OTf) and 9-anthryldiazomethane (ADAM). BAP showed the best score on reactivity and was selected and used for the following examinations. Optimum conditions for HPLC analysis were determined with reference to the stability of each reagent, reactive temperature and time. Edible animal tissues were extracted with water and deproteinized with sulfuric acid and sodium tangstate, followed by concentration with C18 (solid-phase extraction) cartridge column. The acetonitrile eluate from the column was derivatized with BAP and 18-crown-6 reagents at 40°C for 30 min. The derivatized sample was analyzed by HPLC with reversed-phase mode. The mean recovery range was 73-96% for benzylpenicillin, 73-90% for oxacillin, 64-83% for cloxacillin, 62-71% for nafcillin and 61-67% for dicloxacillin in tissues. The detection limits for benzylpenicillin and other penicillins were about 2 and 5 ng/g in tissues, respectively. In the suspected case of penicillin residues, benzylpenicillin was determined (0.045-29.0μg/g) in bovine muscle and kidney by this method. The simple and sensitive method was expected for routine analysis of penicillins in edible animal tissues.