Determination of natural penicillins in fermentation media by high-performance liquid chromatography using pre-column derivatisation with 1-hydroxybenzotriazole

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Abstract
The separation and determination of trace amounts of 6-aminopenicillanic acid (6-APA) and penicillins G, V, X, K and penicillin G penicilloic acid in fermentation broths has been achieved using pre-column derivatisation with 1-hydroxybenzotriazole. Fermentation broth samples containing penicillins were mixed with benzoic anhydride and left to stand at room temperature for 3 min. The latter step was essential for the stabilisation of derivatives of amino group-containing penicillins. The resulting mixtures were then treated with aqueous 1-hydroxybenzotriazole containing mercury(II) chloride and incubated at 60 °C for 45 min. Aliquots of the resulting solutions were injected on to a reversed-phase C-18 column and separated by gradient elution using a mobile phase consisting of phosphate buffer, sodium thiosulphate and acetonitrile. Following separation, the derivatised penicillins were detected spectrophotometrically at 328 nm. This method is both sensitive and selective and allowed the simultaneous detection of penicillins G, V and X at concentrations down to 1 ng ml–1 with retention times of 35 min or less. The detection limits for 6-APA, penicillin K and penicillin G penicilloic acid were 10, 100 and 100 ng ml–1, respectively. Ten injections of antibiotic-free broths which had been spiked with penicillins (1 µg ml–1) and subsequently derivatised gave peak area standard deviations of 8–10% for penicillins G, V, X, K and 6-APA. Ampicillin can be employed as an internal standard, and this together with all previously mentioned penicillins can be conveniently separated and detected using a gradient or isocratic HPLC procedure. The method described can also be used to determine simultaneously penicillin G penicilloic acid and penicillin G in the same solution.

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