High-performance thin-layer chromatography (HPTLC) for the simultaneous quantification of the cyclic lipopeptides Surfactin, Iturin A and Fengycin in culture samples of Bacillus species

•A HPTLC method for the quantification of Surfactin, Iturin A and Fengycin is presented.•A sample pretreatment protocol was established that enables further quantification of samples.•The introduced HPTLC method was validated as being highly accurate and precise.•The method is applicable to various Bacillus strains.

A high-performance thin-layer chromatography method has been established for the identification and simultaneous quantification of the cyclic lipopeptides Surfactin, Iturin A and Fengycin in Bacillus culture samples. B. subtilis DSM 10T, B. amyloliquefaciens DSM 7T and B. methylotrophicus DSM 23117 were used as model strains. Culture samples indicated that a sample pretreatment is necessary in order to run HPTLC analyses. A threefold extraction of the cell-free broth with the solvent chloroform/methanol (2:1, v/v) gave best results, when all three lipopeptides were included in the analysis. For the mobile phase, a two-step development was considered most suitable. The first development is conducted with chloroform/methanol/water (65:25:4, v/v/v) over a migration distance of 60 mm and the second development using butanol/ethanol/0.1% acetic acid (1:4:1, v/v/v) over a migration distance of 60 mm, as well. The method was validated according to Validation of Analytical Procedures: Methodology (FDA Guidance) with respect to the parameters linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy and recovery rate. A linear range with R2 > 0.99 was obtained for all samples from 30 ng/zone up to 600 ng/zone. The results indicated that quantification of Surfactin has to be performed after the first development (hRF = 44), while Fengycin is quantified after the second development (hRF = 36, hRF range = 20-40). For Iturin A, the results demonstrated that quantification is in favor after the first (hRF = 19) development, but also possible after the second (hRF = 59) development. LOD and LOQ for Surfactin and Iturin A after the first development, and Fengycin after the second development were determined to be 16 ng/zone and 47 ng/zone, 13 ng/zone and 39 ng/zone, and 27 ng/zone and 82 ng/zone, respectively. Results further revealed the highly accurate and precise character of the developed method with a good inter- and intraday reproducibility. For the precision and accuracy, expressed as % recovery and relative standard deviation, respectively, the determined values did not exceed ±15% as specified by the FDA Guidance. The recovery assay conducted for samples obtained from two strains with the solvent chloroform/methanol (2:1, v/v), which was determined to be most suitable if all three lipopeptides are of interest, gave recoveries of 96.5% and 99.6%, 68.6% and 71.6%, and 102.5% and 95.2% for Surfactin, Iturin A and Fengycin, respectively. Overall, a suitable and reliable method for the simultaneous quantification of the lipopeptides Surfactin, Iturin A and Fengycin in biological samples using HPTLC was successfully developed and validated.