Direct quantification of lipopeptide biosurfactants in biological samples via HPLC and UPLC-MS requires sample modification with an organic solvent

Abstract

The rapid and accurate quantification of biosurfactants in biological samples is challenging. In contrast to the orcinol method for rhamnolipids, no simple biochemical method is available for the rapid quantification of lipopeptides. Various liquid chromatography (LC) methods are promising tools for relatively fast and exact quantification of lipopeptides. Here, we report strategies for the quantification of the lipopeptides pseudofactin and surfactin in bacterial cultures using different high- (HPLC) and ultra-performance liquid chromatography (UPLC) systems. We tested three strategies for sample pretreatment prior to LC analysis. In direct analysis (DA), bacterial cultures were injected directly and analyzed via LC. As a modification, we diluted the samples with methanol and detected an increase in lipopeptide recovery in the presence of methanol. Therefore, we suggest this simple modification as a tool for increasing the accuracy of LC methods. We also tested freeze-drying followed by solvent extraction (FDSE) as an alternative for the analysis of “heavy” samples. In FDSE, the bacterial cultures were freeze-dried, and the resulting powder was extracted with different solvents. Then, the organic extracts were analyzed via LC. Here, we determined the influence of the extracting solvent on lipopeptide recovery. HPLC methods allowed us to quantify pseudofactin and surfactin with run times of 15 and 20 min per sample, respectively, whereas UPLC quantification was as fast as 4 and 5.5 min per sample, respectively. Our methods provide highly accurate measurements and high recovery levels for lipopeptides. At the same time, UPLC-MS provides the possibility to identify lipopeptides and their structural isoforms.

High-throughput optimization of medium components and culture conditions for the efficient production of a lipopeptide pseudofactin by Pseudomonas fluorescens BD5

“…During the initial screening we tested 11 different media (Table 6 and “ Culture media ” section). Some of these media were previously proposed and tested by other authors for the production of BS [ 23 , 32 – 35 ] or the media were modified by us. Results are summarized in Table 1 .…”

“…PF3 and PF4 were identified in SPE extracts of cell-free supernatants from P. fluorescens BD5 cultures (Fig. 5 ), and they were analyzed using UPLC-MS/MS, as described before [ 32 ]. The 1053 and 1067 [H + ] m/z adducts were assigned to the newly identified peaks.…”

“…We measured PF analogues concentrations during cultivation of P. fluorescens BD5 in KB-Opt-Leu medium (Table 6 ), using previously established HPLC methods [ 32 ]. Under the described conditions, strain BD5 is capable of producing 8.8 ± 0.6 mg/L of PF3 and 20.1 ± 0.7 mg/L of PF4 in a total PF C of 1082.9 ± 24.5 mg/L.…”

“…PF concentration (PF C ) was measured using HPLC in cell-free culture supernatants, as described previously [ 32 ]. Two HPLC systems were used: system 1 consisted of a Beckman Gold 126 Pump and a Knauer Variable Wavelength Monitor equipped with a Macherey–Nagel C18 Isis column (50 mm × 4.6 mm, 1.8 μm) under the control of LP-Chrom software (Lipopharm, Poland).…”

The influence of Bacillus subtilis 87Y isolated from Eisenia fetida on the growth of pathogenic and probiotic microorganisms

“…The cultures were inoculated to A 600 = 0.1 and incubated for 24 h at 37°C with agitation (180 rpm). These cultures were centrifuged (13,500 rpm, 10 min) and the supernatants were used for ultra high-performance liquid chromatography (UHPLC, Aquity ARC Waters) analysis, equipped with a CORTEX C18 column (4.6 × 50 mm; 2.7 μm), according to Biniarz and Łukaszewicz [24].…”