VALIDATION OF AN ANALYTICAL METHOD FOR THE QUANTIFICATION OF 1-KESTOSE, GLUCOSE AND SUCROSE IN THE REACTION PRODUCTS OF A FRUCTOSYLTRANSFERASE

Abstract

The enzymatic synthesis of fructooligosaccharides (FOS) from sucrose by the action of fructosyltransferases produces a mixture of different carbohydrates. The concentration of each carbohydrate involved must be quantified to follow its variation as the kinetics of the reaction progresses. This requires a method such as high-performance liquid chromatography (HPLC) with refractive index detector (IR). The aim of this work was to validate the HPLC-IR analytical method for the quantification of total and individual carbohydrates in mixtures of 1-kestose, sucrose and glucose. For this study, the validation parameters recommended by the FDA (Food and Drug Administration), the ICH (International Council for Harmonisation) and the EMEA (European Medicines Agency) were followed. The method was able to quantify carbohydrates with adequate levels of precision, accuracy and linearity in ranges of 1.6-8.7, 4.3-22.0 and 7.0-41.0 mg/mL for glucose, 1-kestose and sucrose, respectively. The lowest limit of detection was for glucose (0.2 mg/mL) and the highest for sucrose (0.8 mg/mL), while the lowest and highest limit of quantification were also for glucose (0.6 mg/mL) and sucrose (1.8 mg/mL). The detection (0.7 mg/mL) and quantification (1.4 mg/mL) limits for 1-kestose were intermediate. The HPLC-IR method was used to calculate the concentration of the carbohydrates involved in the reaction kinetics of a recombinant fructosyltransferase (Sa1-SSTrec). This analytical technique can be implemented for the analysis of samples from the FOS production process at both laboratory and industrial scale, but also the technique is useful to detect 1-kestose in functional foods based in fruit juices.