Analytical validation of an advanced U-HPLC–MS/MS method for lactose detection in food supplements and pharmaceuticals

Lactose intolerance is common, so accurate detection of lactose in supplements and pharmaceuticals is critical. We validated an ultra-high performance liquid chromatography–tandem mass spectrometry (U-HPLC–MS/MS) method with high sensitivity and specificity for trace lactose in products labeled “lactose-free.” The workflow mitigates matrix effects through solid-phase extraction and filtration and explicitly accounts for α/β mutarotation to ensure correct identification and quantification. Validation per ISO and ICH Q2 confirmed robustness and performance superior to enzymatic/colorimetric assays for quality control and regulatory compliance. The method achieved excellent linearity ( R 2 > 0.995) over 0.05–10 ppm, recoveries of 95–105%, precision with RSD < 2%, LOQ 0.05 ppm, and matrix effect < 15%. These results enable reliable verification of “lactose-free” claims across diverse matrices, reducing false positives, enhancing reproducibility, and improving labeling transparency.