HPLC Methods for Determination of Dihydroxyacetone and Glycerol in Fermentation Broth and Comparison with a Visible Spectrophotometric Method to Determine Dihydroxyacetone

Abstract

High-performance liquid chromatographic (HPLC) methods were respectively developed for the quantitative determination of dihydroxyacetone (DHA) and glycerol in the fermentation broth. Validation parameters such as linearity, precision, accuracy, and specificity, limit of detection (LOD), and limit of quantitation (LOQ) were determined. Both HPLC methods were carried out on a Lichrospher 5-NH2 column with a mobile phase constituted of acetonitrile and water (90:10, v/v). The linearity range for DHA was 2.00-12.00 mg/mL with a correlation coefficient (r) of 0.9994. The LOD and LOQ were 0.06 and 1.20 mg/mL, respectively. The linearity range for glycerol was 0.50-20.00 mg/mL with a correlation coefficient of 0.9998. The LOD and LOQ were 0.22 and 0.50 mg/mL, respectively. Also, the HPLC method to determine DHA was compared with an existing visible spectrophotometric method. Statistical analysis by F-test and t-test showed no significant difference at 95% confidence level between the two methods when applied to low DHA concentrations while a large deviation existed in the determinations of high DHA concentrations. The HPLC method was more accurate to determine high DHA concentrations.