Simultaneous quantification of lipopeptide isoforms by UPLC-MS in the fermentation broth from Bacillus subtilis CNPMS22

摘要

The rapid and accurate quantification of lipopeptide families in biological samples are challenging. We present the development and validation of a method for simultaneous quantification of three families of lipopeptides (iturins, fengycins, and surfactins) and their isoforms, as well as the homologous series. The method was optimized in UPLC-MS for a column temperature at 65 degrees C, injection volume of 5L, and sample temperature of 10 degrees C. The SIM mode was used for detection and quantification of lipopeptides exhibiting ions [M+H](+) and [M+2H](2+). Since the maximum mass detection threshold of the equipment is 1250Da and the fengycins have ions between 1435 and 1505Da, the ions [M+2H](2+) were chosen for fengycin identification. The monitored ions were as follows: m/z 1043.5, 1057.5, 1071.5, 718.3, 725.4, 739.4, 732.4, 746.4, 753.4, 1008.6, 1022.6, and 1036.6. The compounds were separated by reverse-phase chromatography using a C18 analytical column in a total time of 19min. Standard curves were linear with r(w) 0.99 for all analytes. Intra- and inter-day precision for samples (50, 250, and 750gL(-1)) were within recommended limits. The proposed analytical method was capable of simultaneously quantifying 12 isoforms and homologous series of lipopeptide families in biological samples, thus making it an important industrial tool in the evaluation of lipopeptide production processes.