Determination of Low Level Lactose in Dairy Products Using UHPLC-MS

Significance of the Topic

Accurate measurement of low-level lactose in dairy products is essential to support the growing demand for lactose-free and low-lactose foods, ensure compliance with regulatory thresholds, and protect consumers with lactose intolerance from adverse effects.

Objectives and Overview of the Study

This work describes a robust UHPLC-MS method for the determination of lactose at trace levels in diverse dairy matrices, using a Waters ACQUITY Arc System coupled with a QDa mass detector and a HILIC amide column. The method targets a limit of quantitation (LOQ) below common lactose-free thresholds (2.5 mg per 100 g), complete chromatographic separations, and a total run time of 25 minutes per injection.

Methodology and Instrumentation

Sample preparation involves extraction with ethanol–water, protein precipitation using Carrez reagents, and cleanup by centrifugation and membrane filtration. A 13C6-lactose internal standard is added to normalize ionization variability. Calibration standards range from 0.2 to 100 µg/mL with quadratic fitting and 1/x weighting.

Instrumentation Used

• ACQUITY Arc UHPLC System
• ACQUITY QDa Single Quadrupole Mass Detector
• XBridge BEH Amide XP Column, 3.0 × 150 mm, 2.5 µm
• Empower 3 Chromatography Data Software

Main Results and Discussion

The method achieved an LOQ of approximately 2.5 mg lactose per 100 g of final product (S/N=10) in a complex soy-based infant formula matrix. Co-elution studies of nine isomeric sugars showed only cellobiose co-elutes with lactose, but it is rarely present in dairy. Calibration curves yielded R2 values >0.995. Precision across various dairy samples was high (RSD <4.3%), and recoveries in spiking experiments ranged between 90% and 110%. Analysis of NIST SRM 1849a infant formula returned 109% of the certified lactose value.

Benefits and Practical Applications

• Sensitive quantitation of lactose at levels below international regulatory limits for lactose-free claims
• Minimal matrix interference due to HILIC separation and MS detection
• Robust performance across diverse dairy products including milk, yogurt, cheese, and butter
• Supports quality control and label claim verification in food manufacturing

Future Trends and Applications

Advancements may include integration with high-resolution mass spectrometry for broader sugar profiling, automation of sample preparation for high throughput, and adaptation to other carbohydrate analysis challenges in food and beverage industries.

Conclusion

The presented UHPLC-MS approach delivers a rapid, accurate, and reproducible workflow for low-level lactose determination in dairy foods, meeting stringent regulatory requirements and supporting the production of lactose-free and low-lactose products.

Reference

1. Deng Y., Misselwitz B., Dai N., Fox M. Lactose Intolerance in Adults: Biological Mechanism and Dietary Management. Nutrients 2015, 7(9), 8020–8035.
2. Scientific Opinion on lactose thresholds in lactose intolerance and galactosaemia, EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA). EFSA J. 2010, 8(9), 1777.
3. Chavez-Servin J. L., et al. Analysis of Mono- and Disaccharides in Milk Based Formulae by High Performance Liquid Chromatography with Refractive Index Detection. J. Chromatogr. A 2004, 1043(2), 211–215.
4. XBridge XP 2.5 µm Columns. Waters Care and Use Manual 720004162EN, March 2012.