Determination of benzylpenicillin in milk at the pg ml-1 level by reversed-phase liquid chromatography in combination with digital subtraction chromatography technique

A method for the determination of benzylpenicillin in milk at very low levels by a pre-column derivatisation LC-method is described. Nine millilitres of milk were precipitated and extracted. The sample was concentrated 40 times and the recovery of benzylpenicillin was about 90%. The derivatisation of benzylpenicillin was made by the beta-lactam ring specific formation of the mercuric mercaptide of the penicillenic acid in the presence of imidazole. A technique called digital subtraction chromatography (DSCh), which uses the built-in background compensation programme of the chromatographic data processor, was applied to the chromatographic step of the analysis. The blank sample required for this technique was obtained by the beta-lactam ring specific hydrolysis of benzylpenicillin by penicillinase. By this technique it was possible to reduce interfering background peaks on the chromatograms and to increase the specificity of the method. Standard curves were made in the range 0.2-1000 ng benzylpenicillin ml-1 milk. The relative standard deviation (RSD) at 1 ng ml-1 milk, corresponding to an injection of 3.9 ng benzylpenicillin-sodium converted to the mercuric mercaptide of the penicillenic acid, was +/- 7.8% and RSD at 10 ng ml-1 milk was +/- 1.6%.