Validation of UHPLC-MS/MS Method and Measurement Uncertainty Evaluation for Lactose Quantification in Lactose-Free and Regular UHT Milk

A fast, reliable, and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for lactose determination in commercial UHT milk samples (whole and skimmed regular milk, and lactose-free milk) was developed and validated. The sample preparation procedure involved a simple dilution of milk samples in acetonitrile/water (70:30, v/v) mixture followed by vortexing. Significant matrix effects were obtained in all milk matrices by hypothesis testing. The standard addition method and validation results were satisfactory for lactose quantification. The method was linear (r > 0.999) and approved by Grubbs and Cochran tests, within the dynamic working range (50-800 mu g L-1). The lower limits of quantification (LLOQ) ranged between 50 and 55 mu g L-1 for the milks studied. Recoveries ranged from 91 to 98%, and repeatability (RSD <= 4.1%) and intermediate precision (RSD <= 9.6%) were appropriate for all the spike levels. The measurement uncertainty (ranged 9.0 to 14%) was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM). LLOQ, accuracy, and precision data confirm the applicability of the proposed method for residual lactose quantification. Finally, the validated method was applied in regular UHT milk samples (whole and skimmed) and lactose-free UHT milk; the latter follows the limit established by Brazilian legislation.