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Development and validation of a rapid RP-HPLC-UV method for lactose quantification in milk and its application in
Snigdha Paul1, Deep Anmol Singh2, Ravinder Kaushik3
1School of Health Sciences and Technology, UPES, Dehradun, Uttarakhand, 248007, India; School of Health and Medical Sciences, Adamas University, Kolkata, West Bengal, India.
Abstract:
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.
