Evaluating raffinose family oligosaccharides and their decomposition products by ion chromatography – a method development and advanced repeatability study

During the germination of legumes, raffinose family oligosaccharides (RFOs) are decomposed into mono- and disaccharides. As legumes are a traditional part of the human nutrition, storage carbohydrates are generally incorporated in the daily meals and can cause flatulence or even abdominal pain. However, their decomposition products can be metabolized without any difficulty. To date, no validated method is available to quantify the main decomposition products, glucose, and galactose simultaneously with the RFOs and the starch decomposition product maltose in a single measurement without derivatization. To provide a suitable method and fill the existing knowledge gap, a highly sensitive ion chromatography method was established. By using an optimized gradient, the separation of the RFOs, namely raffinose, stachyose, and verbascose, from their decomposition products, including glucose, galactose, fructose, and saccharose was accomplished. An advanced repeatability study proved that the method could be used for up to 4 days until the sample or eluent degradation made further measurement unfeasible. To improve the repeatability while avoiding to overload the chromatogram with several internal standards, a mathematical approach was presented to cope with different decomposition characteristics of the analytes and the internal standard. This study presents a modified and validated method allowing the measurement of all relevant carbohydrates in the germination of legumes. By using only a single method, a high number of samples can be processed. Therefore, a deeper and more detailed insight into the changes in the carbohydrate spectrum during the germination of legumes becomes feasible.