Determination of Lactose in Lactose-Free Milk Products by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection

Importance of the Topic

The accurate determination of residual lactose in lactose‐free dairy products is critical for consumer safety, regulatory compliance, and quality control. Traditional enzymatic and spectroscopic methods can be time‐consuming or lack sufficient sensitivity for low‐level lactose detection. A robust analytical approach that can reliably quantify lactose and lactulose at trace levels supports product labeling, process validation, and analytical laboratories in the dairy industry.

Objectives and Study Overview

This study aimed to develop and validate a sensitive, accurate, and reproducible high‐performance anion‐exchange chromatography method with pulsed amperometric detection (HPAE‐PAD) for quantifying lactose and lactulose in various dairy matrices, including commercial lactose‐free milk and cheeses. The method’s performance was assessed in terms of linearity, detection limits, reproducibility, and sample recoveries.

Methodology and Instrumentation

The analytical workflow combined:

• Sample preparation using Carrez precipitation and OnGuard IIA cartridge cleanup.
• Chromatographic separation on a Thermo Scientific Dionex CarboPac PA20 analytical column (3 × 150 mm) with guard column (3 × 30 mm).
• Pulsed amperometric detection with disposable Au on PTFE electrodes under a standard carbohydrate waveform.
• Eluent gradients of deionized water, 200 mM NaOH, and sodium acetate mixtures, all maintained under inert gas to minimize carbonate contamination.

Instrument platform: Thermo Scientific Dionex ICS‐3000/5000+ with Chromeleon CDS software.

Main Results and Discussion

Linearity and Sensitivity:

• Lactose: linear range 0.25–100 mg/L, r² = 0.9966, MDL = 0.12 mg/L.
• Lactulose: linear range 0.5–100 mg/L, r² = 0.9942, MDL = 0.23 mg/L.

Reproducibility:

• Intra‐day retention time RSD ≤ 0.16% and peak area RSD ≤ 5.1% across key carbohydrates.
• Column‐to‐column reproducibility confirmed on three different column lots.

Sample Analysis:

• Whole milk (1:10 dilution) contained high lactose levels; lactose‐free milk exhibited trace lactose (0.6 mg/L).
• Lactose‐free Gouda and Havarti cheeses showed no detectable lactose, whereas cottage cheese and nonfat milk contained 2.17 and 0.6 mg/mL lactose, respectively.
• Recoveries of spiked lactose and lactulose ranged from 86% to 102% across matrices.

Benefits and Practical Applications

This HPAE‐PAD method offers:

• Direct detection without derivatization, reducing preparation time and potential errors.
• Sensitivity sufficient for trace lactose quantification in “lactose‐free” products.
• High reproducibility and ease of electrode replacement with disposable gold sensors.
• Applicability across diverse dairy matrices for quality assurance, regulatory compliance, and process monitoring.

Future Trends and Potential Applications

Advancements may include:

• Integration with automated sample preparation and inline cleanup for higher throughput.
• Expansion to other milk‐derived oligosaccharides and sugar acids.
• Coupling with mass spectrometry for structural confirmation in complex matrices.
• Use in real‐time process analytics during dairy product manufacturing.

Conclusion

The developed HPAE‐PAD method on a CarboPac PA20 column reliably quantifies lactose and lactulose at low mg/L levels in various dairy products. It combines high sensitivity, accuracy, reproducibility, and streamlined sample preparation, making it suitable for routine quality control and compliance testing of lactose‐free foods.

References

1. Marconi E, Messia CM, Amine A, Mascone D, Vernazza F, Stocchi F, Palleschi G. Heat-Treated Milk Differentiation by a Sensitive Lactulose Assay. Food Chem 2004;447–450.
2. Luzzana M, Agnellini D, Cremonesi P, Caramenti G, DeVita S. Milk Lactose and Lactulose Determination by the Differential pH Techniques. J Dairy Sci 2003;409–416.
3. Guan R, Liu D, Ye X, Yang K. Use of Fluorometry for Determination of Skim Milk Powder Adulteration in Fresh Milk. Int J Food Sci Technol 2005;1101–1106.
4. Monica RG, Mar V, Rosina LF. Effect of Homogenization on Protein Distribution and Proteolysis during Storage of Indirectly Heated UHT Milk. J Dairy Res 2002;589–599.
5. Moscone D, Bernado RA, Marconi E, Amine A, Palleschi G. Rapid Determination of Lactulose in Milk by Microdialysis and Biosensors. Anal Chim Acta 1999;325–329.
6. Lynch JM, Barbano DM. Determination of Lactose Content of Fluid Milk by Spectroscopic Enzymatic Analysis Using Weight Additions and Path Length Adjustment: Collaborative Study. J AOAC Int 2007;196–216.
7. Thermo Fisher Scientific. Technical Note 71: Proper Preparation, Storage, and Use of Eluents for HPAE-PAD. 2009.
8. Thermo Fisher Scientific. Material Safety Data Sheet: Potassium Hexacyanoferrate(III). 2016.