Determination of β-lactams and their biosynthetic intermediates in fermentation media by pre-column derivatisation followed by fluorescence detection

This paper describes a novel and sensitive pro-column derivatisation method for the detection and quantitation of β-lactams and their biosynthetic precursors at trace levels in fermentation media. Filtered broths from fermentations of strains of Penicillium chrysogenum and Cephalosporium acremonium, after deproteination and centrifugation, were incubated with 9-fluorenylmethylchloroformate for 5 min at 20°C in 0.2 M borate buffer at pH 7,7. Following two-fold pentane extraction of the reagent hydrolysis product, the aqueous layer was injected directly onto a C18 reversed-phase column, and products were detected spectrofluorimetrically with excitation and emission wave-lengths of 260 and 313 nm, respectively. Detection limits of 0.01 and 0.05 μg ml−1 were achieved for both 6-aminopenicillanic acid (6-APA) and isopenicillin N in borate buffer and filtered fermentation broths, respectively, using a 10-μl injection volume. A linear calibration for 6-APA in fermentation broth was obtained for a very wide concentration range (0.05-100 μg ml−1). Detection limits for solutions of cephalosporin C, deacetylcephalosporin C and deacetoxycephalosporin C in broth were all 0.25 μg ml−1. The detection limit for the β-lactam precursor δ-(L-aminoadipyl)-L-α-cysteinyl-D-valine (ACV) dimer in borate buffer was 0.5 μg ml−1. The cephalosporins and ACV dimer gave linear plots in the ranges 3–25 and 1–100 μg ml−1, respectively. Repeated analysis of 6-APA at a concentration of 10 μg ml−1 in filtered broth gave a mean peak area of 2.5 · 106 with a standard deviation of 2.6 · 105 using a 10-μl injection volume. Ampicillin spiked into deproteinated blood serum gave a linear calibration in the concentration range 2-100 μg ml−1.