Validated High-Performance Liquid Chromatographic (HPLC) Method for the Simultaneous Quantification of 2,3-Butanediol, Glycerol, Acetoin, Ethanol, and Phosphate in Microbial Cultivations

The aim of this study was to validate an analytical method for the simultaneous quantification of 2,3-butanediol, glycerol, acetoin, ethanol and phosphate (PO43-) by high-performance liquid chromatography (HPLC) coupled with a refractive index detector. The validation was performed according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) - Guideline Q2 (R1). The parameters of linearity, limits of detection and quantification, precision and accuracy were evaluated. The accuracy was evaluated using standards solutions and a microbial cultivation sample. The linearity (r ≥ 0.99, n = 3) of the method was defined in the range from 0.18 to 2.52 g L−1 for phosphate (PO43-); 0.5 to 10.0 g L−1 for glycerol, acetoin and ethanol; 0.375 to 7.5 g L−1 for meso-2,3-butanediol; and 0.125 to 2.5 g L−1 for (S,S)- or (R,R)-2,3-butanediol. The limits of detection and quantification were below the concentration range used in the method. The average recovery rate, intra-day and inter-day precisions for all compounds were 98.71, 0.09 and 0.50%, respectively. The results for the accuracy ranged between 97.97 and 101.18% for all compounds in the standard solution. The method was demonstrated to be precise, specific, and reproducible for the quantification of all compounds evaluated, ensuring the reliability. Further, this method was successfully applied to samples from bioprocesses with low matrix effects and an average recovery of 99.55%.