A ROBUST LIQUID-CHROMATOGRAPHIC METHOD FOR MEASUREMENT OF MEDIUM COMPONENTS DURING PENICILLIN FERMENTATIONS

A reversed-phase gradient liquid chromatographic (LC) method was developed for measuring medium components formed during a penicillin V fed-batch fermentation. The method is based on a high concentration of sodium sulphate in the mobile phase and requires no chemical derivations of the fermentation sample. It is used for the quantitative analysis of p-hydroxyphenoxyacetic acid, isopenicillin N, 6-aminopenicillanic acid (6-APA), o-hydroxy-phenoxyacetic acid, phenoxyacetic acid, two isomers of penicilloic V acid, p-hydroxypenicillin V and penicillin V. The linear working range of the method is from 0.00125 to 0.125 g/l except for penicillin V which has a linear response from 0.005 to 0.500 g/l. For analyte concentrations below 0.010 g/l the response is influenced by the analyte concentration of the previously analyzed sample. The kinetics for the epimerisation of the two penicilloic V acid isomers was quantified. Decreasing values of the total peak area for the two isomers was observed during the epimerisation and to account for this a weight factor psi was introduced. Different methods for preparing standards of penicilloic V acid are discussed. The performance of the LC method was demonstrated during monitoring of 250 h of a fed-batch fermentation carried out on a complex medium. A peak for an as yet unidentified fermentation product was discovered and monitored during the entire fed-batch fermentation. Finally, the importance of a proper sample storage is discussed.