Evaluation of HPLC and MEKC methods for the analysis of lipopeptide antibiotic iturin A produced by Bacillus amyloliquefaciens

Abstract

Two methods, high-performance liquid chromatography (HPLC) and micellar elec- trokinetic chromatography (MEKC), for analysis of multicomponent lipopeptide antibiotic iturin A are described. After conducting a series of optimizations, the HPLC separation of iturin A2, A3, A4, A5, A6, A7, and A8 isomers were achieved by a NPS RP-C18 column with an isocratic elution of acetonitrile/10 mM ammonium acetate (40:60, v/v), and flow rate of 1 mL/min. On the other hand, the MEKC separation achieved at 25 °C using an applied voltage of 20 kV, and back- ground electrolyte consisting of 20 mM boric acid buffer (pH 8.72) with 50 mM sodium dodecyl sulfate (SDS) and 10% (v/v) acetonitrile. The calibration curve was linear to 20-200 µg/mL and 50-500 µg/mL for HPLC and MEKC with R 2 = 0.9954 and R 2 = 0.9967, respectively. The detec- tion limits of iturin A from the strain B128 cultivation medium were in the 2.5-7.0 µg/mL and 5.0-13.0 µg/mL ranges, and the reproducibility of within-day assay was 98.1 and 98.7%, for HPLC and MEKC, respectively (a UV detector was applied in the both cases). Both methods were selective, robust, and specific, allowing reliable quantification of iturin A, and could be useful for clinical and biomedical investigations of related antibiotics.