Quantification of lactose using ion-pair RP-HPLC during enzymatic lactose hydrolysis of skim milk

► We developed a sensitive quantification method for lactose in original milk matrix using ion-pair RP-HPLC. ► For lactose a LOD of 2mg L−1 and a LOQ of 6mgL−1 was achieved using UV detection. ► With fluorescence detection a LOD for lactose of 0.13mgL−1 and a LOQ of 0.45mgL−1 was achieved. ► Using the commercial β-galactosidase Godo-YNL2 “lactose-free” milk was produced. The correct labelling of dairy foods as “lactose-free” requires a suitably sensitive and valid analytical method for the quantification of lactose in complex food matrices. Thus, an ion-pair RP-HPLC method for the simultaneous determination of lactose, glucose and galactose in original skim milk was investigated. The samples derived from an enzymatic lactose hydrolysis approach (0.5L) using the commercial β-galactosidase Godo-YNL2. After derivatisation with p-aminobenzoic acid and sodium cyanoborohydride, the samples were injected on a RP-C18 column. Tetrabutylammonium hydrogen sulphate was used as the ion-pair reagent in the eluent system. The sugars were quantified using photometric- (UV; 303nm) and fluorescence-detection (λex 313nm, λem 358nm). The overall run time was 27min. The limits of detection (LOD) were estimated at 2mgL−1 (UV detection) and at 0.13mgL−1 (fluorescence detection). The limits of quantification were 6mgL−1 (UV detection) and 0.45mgL−1 (fluorescence detection). Thus, this analytical method is suitable for sensitive lactose quantification in milk systems of less than 10mgL−1.