Development and validation of a rapid RP-HPLC-UV method for lactose quantification in milk and its application in lactose hydrolysis monitoring.

The increasing prevalence of lactose intolerance necessitates reliable analytical methods for lactose detection and effective strategies for lactose reduction in dairy products. In this study, a reverse-phase HPLC-UV method was optimized and validated for the detection and quantification of lactose in milk. As lactose lacks a UV chromophore, samples were derivatized using para-aminobenzoic acid (PABA) and sodium cyanoborohydride, followed by ten-fold dilution with methanol: water (70:30) before injection onto an RP-C18 column. The method provided good chromatographic resolution, with retention times of 6.0, 6.9, and 9.1 min for lactose, glucose, and PABA, respectively. Excellent linearity was obtained (R2 = 0.9952), with limits of detection and quantification of 0.002 M and 0.008 M, respectively, and precision below 2%. Application of the method revealed that lactase treatment reduced lactose concentration to below 1 g/100 g within 4 h, achieving low-lactose milk, whereas dahi culture and Kluyveromyces lactis showed slower lactose hydrolysis. The method demonstrated applicability for monitoring lactose reduction during hydrolysis and for analysis of low-lactose milk within the validated analytical range.