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.