Studies on the formation and forming mechanism of the related substance E in potassium clavulanate production by HPLC-MS/MS

Abstract

The objective of this study was to investigate the formation and forming mechanism of the related substance E in potassium clavulanate production. The impurity with retention time of 11.1 min in potassium clavulanate final product was confirmed as the related substance E by high performance liquid chromatography with tandem mass spectrometric detection (HPLC-MS/MS).The related substance E analysis during the production of clavulanic acid showed that this impurity could be formed during both the fermentation and purification processes, especially in the later fermentation stage, filtration concentration and back-extraction procedure. Clavulanic acid was the precursor of the related substance E. Studies on its forming mechanism showed that the related substance E was formed by the combination of the imino group of one molecule of clavulanic acid with the carboxyl group of another molecule of clavulanic acid with the opening of β-lactam ring. Results of a multi-factor orthogonal test confirmed that the concentration of clavulanic acid was the dominant factor to accelerate the reaction, while the temperature was another contributing factor. The pH 5.0-6.5 had little impact on the generation of the related substance E.Uniterms: Potassium clavulanate/production. Clavulanic acid/formation of the Related substance E. Related substance E/formation. Related substance E/forming mechanism High performance liquid chromatography/biotechnological process. O objetivo deste estudo foi investigar a formação da substância E e o respectivo mecanismo na produção de clavulanato de potássio. Confirmou-se a impureza com tempo de retenção de 11,1 min no produto final, clavulanato de potássio, como substância E, por meio de cromatografia líquida de alta eficiência, em conjunto com detecção por espectrometria de massas (CLAE-MS-MS). A análise da substância relacionada E durante a produção do ácido clavulânico mostrou que essa impureza pode ser formada tanto durante a fermentação quanto durante os processos de purificação, especialmente no estágio final de fermentação, filtração, concentração e procedimento de extração. O ácido clavulãnico foi o precursor da substância E. Estudos no mecanismo de sua formação mostraram que a substância E formou-se pela combinação do grupo imina da molécula do ácido clavulânico com o grupo carboxílico de outra molécula de ácido clavulânico, com a abertura do anel β-lactâmico. Resultados do teste ortogonal multifatorial confirmaram que a concentração do ácido clavulânico foi o fator dominante para acelerar a reação, enquanto a temperatura foi outro fator que contribuiu. O pH de 5,0 a 6,5 teve pouco impacto na geração da substância E. Unitermos: Clavulanato de potássio/produção. Ácido clavulânico/formação da substância E relacionada. Substância E relacionada/mecanismo de formação Cromatografia líquida de alta eficiência/processo biotecnológico.

Degradation Kinetics of Clavulanic Acid in Fermentation Broths at Low Temperatures

“…Additionally, this molecule has different potential candidate nodes (C atoms bonded to N and/or O) for suffering nucleophilic attacks from the substances present in the medium, e.g., CA itself, amino acids, and other metabolites with electronegative groups. In this regard, Zhong et al [30] proposed a mechanism of formation of the degradation product known as substance E; this mechanism involves several steps, including the irreversible reaction of an active intermediate with an additional molecule of CA leading to the reported decomposition product.…”

Impacts of horizontal gene transfer on the compact genome of the clavulanic acid-producing Streptomyces strain F613-1

“…Streptomyces clavuligerus F613-1 is an industrial producer that can produce up to 4.87 g/l of clavulanic acid in 125-m 3 fermentation tanks (Zhong et al 2014). Similar to S. clavuligerus ATCC 27064, F613-1 showed productive sporulation on BSCA plates after 7-8 days (Table 1).…”

“…To produce clavulanic acid, S. clavuligerus was grown on liquid seed medium (2% soybean meal, 2% glycerol, 1.2% cornstarch, 0.5% yeast extract, 0.08% potassium phosphate, pH 8.0) and then inoculated into a fermentation tank containing 3.0% soybean meal, 2.5% soy isolate, 3.0% maltdextrin, 1.0% glycerol trioleate, 0.25% potassium phosphate, 0.1% magnesium chloride, 0.02% sodium chloride, and 0.05% foamaster, pH 7.0; supplemented 1.5% glycerol was added during the fermentation process. The concentration of clavulanic acid was detected as described (Zhong et al 2014), with a Thermo Scientific Hypersil ODS-3 (C18) 5 µm column.…”

Complete Genome Sequence of Streptomyces clavuligerus F613-1, an Industrial Producer of Clavulanic Acid

“…A complete genome sequence of an important industrial producer, S. clavuligerus F613-1 ( 7 ), was reported in this study, which showed good clavulanic acid production and poor ability to produce 5S clavam compounds.…”