Quantification of Lactulose and Epilactose in the Presence of Lactose in Milk using a dual HPLC analysis
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The valuable lactose derivatives lactulose and epilactose can be derived from lactose either by the Lobry de Bruyn-Alberda van Ekenstein transformation during heat treatments or by enzymatic conversion using cellobiose 2-epimerases (EC 18.104.22.168). The chromatographic determination of lactose, lactulose, and epilactose in milk is challenging, due to the variable ratio of the three saccharides and their similar retention properties. In this work, a dual high-performance liquid chromatography (HPLC) analysis for the quantification of lactose, lactulose, and epilactose in milk samples was developed and validated. The samples originated from an enzymatic lactose conversion using the cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus. Application of this enzyme led to the formation of high lactulose concentrations (28.0 g/L) in milk. The dual HPLC analysis utilized a combination of two chromatographic separation techniques, configured in two parallel systems. After precolumn derivatization, the samples were analyzed as follows: Method 1 determined the concentration of lactose and epilactose using a C18 column with an ion-pair reagent as eluent, coupled with a UV detector. Method 2 determined the concentration of lactulose using a trimodal stationary phase (hydrophilic interaction, anion- and cation-exchange properties) with acetonitrile/ammonium formiate buffer as eluent, coupled with an evaporative light scattering detector. Both methods were validated in terms of linearity, precision and recovery. The revealing detection limits in the milk samples were 3.32 mg/L for lactose, 4.73 mg/L for epilactose and 139 mg/L for lactulose. The dual HPLC analysis presented allows accurate lactose, lactulose, and epilactose separation in complex food matrices such as milk.
KeywordsPrecolumn derivatization Ion-pair HPLC HILIC/IEX HPLC Lactulose Epilactose Milk
Many thanks go to Michael Merz and Prof. Jörg Hinrichs (Institute of Food Science and Biotechnology) for helpful discussions.
Compliance with Ethical Standards
Conflict of interest
Eva Rentschler declares that she has no conflict of interest. Beatrice Kuschel declares that she has no conflict of interest. Manuel Krewinkel declares that he has no conflict of interest. Wolfgang Claaßen declares that he has no conflict of interest. Claudia Glück declares that she has no conflict of interest. Bo Jiang declares that he has no conflict of interest. Wanmeng Mu declares that he has no conflict of interest. Timo Stressler declares that he has no conflict of interest. Lutz Fischer declares that he has no conflict of interest. This article does not contain any studies with human or animal subjects.
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