Determination of β-lactams and their biosynthetic intermediates in fermentation media by pre-column derivatisation followed by fluorescence detection
Article
Shah, A. and Adlard, M. 1988. Determination of β-lactams and their biosynthetic intermediates in fermentation media by pre-column derivatisation followed by fluorescence detection. Journal of Chromatography B: Biomedical Sciences and Applications. 424, pp. 325-336. https://doi.org/10.1016/S0378-4347(00)81109-3
Type | Article |
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Title | Determination of β-lactams and their biosynthetic intermediates in fermentation media by pre-column derivatisation followed by fluorescence detection |
Authors | Shah, A. and Adlard, M. |
Abstract | 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. |
Publisher | Elsevier |
Journal | Journal of Chromatography B: Biomedical Sciences and Applications |
ISSN | 1570-0232 |
Publication dates | |
31 Jan 1988 | |
Online | 12 Nov 2001 |
Publication process dates | |
Deposited | 01 Nov 2017 |
Submitted | 18 May 1987 |
Accepted | 07 Sep 1988 |
Output status | Published |
Digital Object Identifier (DOI) | https://doi.org/10.1016/S0378-4347(00)81109-3 |
Language | English |
https://repository.mdx.ac.uk/item/87417
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