Validation of a HILIC Method for the Analysis of Ergothioneine in Fermentation Broth

Abstract

A hydrophilic interaction liquid chromatography method has been established for the quantification of ergothioneine (EGT) in fermentation broth. Chromatographic separation was conducted on a Venusil hydrophilic interaction liquid chromatography (HILIC) column (250 × 4.6 mm, 5 μm) at an elution rate of 1.0 mL/min with an isocratic mobile phase consisting of acetonitrile/20 mmol/L ammonium acetate solution (85: 15, v/v) adjusted to pH 6.0 with acetic acid. Analytes were detected at 254 nm using a UV-VIS detector. The injection volume was 10 μL, and the column temperature was 40°C. The limits of detection and limits of quantification were 63 and 21 μg/L, respectively. Excellent linearity [correlation coefficient (R2) = 0.9999] was achieved for EGT quantification in the range of 5-400 mg/L. The relative standard deviations of repeatability, intermediate precision and stability were 1.47, 1.03 and 1.66%, respectively, and EGT recoveries were within 99.2-100.8%. The chromatographic peak corresponding to EGT in the HILIC spectrum was confirmed using ESI-MS. In general, the method developed here is simple, reliable, accurate, and stable and may be useful for routine analyses in EGT biosynthesis research.