A rapid and simple method for the determination of organic acids in proteolytic enzymes by capillary electrophoresis with indirect ultraviolet detection

The use of organic acids (e.g. acetic acid and gluconic acid) as additives during protease production is regarded as one of the simplest alternatives to increase enzyme stability and activity in many industrial processes. However, no methods have been described for the determination of organic acids in proteases and their contents have not been established yet. In this work, a novel, rapid and simple method for the determination of organic acids in proteolytic enzymes by capillary electrophoresis (CE) with indirect ultraviolet (UV) detection has been developed. Under the optimized conditions, the method was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ) and infra-day and inter-day repeatability. Later, a sample pretreatment based on a hydroalcoholic microextraction was carefully optimized to obtain good recovery and repeatability and determine acetic and gluconic acids in a commercial protease sample. The complete procedure was validated using the standard-addition calibration method, finding matrix effects on the studied compounds. Finally, acetic acid and gluconic acid were quantified at 80 mg/Kg (0.0080% (m/m)) and 69 mg/Kg (0.0069% (m/m)) in the protease sample, respectively.