Development, optimization and validation of an HPLC-ELSD method for the analysis of enzymatically generated lactulose and saccharide by-products

• Four columns were compared regarding their ability to separate several saccharides.
• Sample pretreatment for a high recovery of lactulose was established.
• A HPLC-ELSD method for the quantification of lactulose was optimized and validated.
• The process of the enzymatic generation of lactulose was analyzed.

The aim of this study was to develop an HPLC-ELSD method for the quantification of lactulose in complex sugar solutions. Lactulose is a well-known prebiotic and supports the alleviation of digestive disorders. The enzymatic generation of lactulose requires fructose as nucleophilic acceptor. By-products such as glucose and galactose are generated. Four amino-modified silica-columns were tested and compared. The most suitable column based on peak resolution was used to optimize the method. Furthermore, sample preparation was optimized for the recovery of analytes. During the validation step, the following parameters were determined (e.g. for lactulose): recovery (106 ± 7%), precision (98%), correctness (99%), limit of detection (3.9 mg/L), limit of quantification (13.4 mg/L) and linearity (0.993). The validated method was applied to samples from an enzymatic process for the production of lactulose at the laboratory scale. A final lactulose concentration of 6.7 ± 0.4 g/L was determined.