Fast and sensitive determination of lactose in lactose-free products using HPAE-PAD

Significance of the Topic

Consumer demand for lactose free products has grown sharply as awareness of lactose intolerance increases. Residual lactose in enzymatically treated dairy and baked goods can cause symptoms in sensitive individuals, so accurate measurement of trace lactose levels is essential for product labeling, quality control and regulatory compliance.

Objectives and Study Overview

This study aimed to develop and validate a high-performance anion-exchange chromatography method with pulsed amperometric detection (HPAE-PAD) for rapid, sensitive separation and quantification of lactose and related isomers in lactose free dairy and bakery products. Eleven commercial samples labeled as lactose free were analyzed for residual lactose, allolactose, lactulose and epilactose.

Methodology and Instrumentation

Samples were prepared by aqueous extraction, protein and fat precipitation with Carrez reagents, and cleanup using OnGuard IIA cartridges. Chromatographic separation was achieved on a Dionex CarboPac PA210-4μm column with a guard, using a gradient of potassium hydroxide eluent generated by a Dionex EGC 500 KOH cartridge. Detection employed a disposable gold working electrode with pulsed amperometry on a Thermo Scientific Dionex ICS-5000+ HPIC system controlled by Chromeleon CDS software. Standards were prepared from 1000 mg/L stocks of lactose, allolactose, lactulose and epilactose.

Key Results and Discussion

The method resolved lactose from isomeric sugars in under 9 minutes, with full cycle time of 30 minutes. Calibration curves were linear or quadratic with coefficients of determination above 0.9998 over 0.25 to 20 mg/L. Limits of detection ranged from 0.009 to 0.017 mg/L and limits of quantification from 0.029 to 0.056 mg/L. Recoveries in fortified dairy and cookie matrices were 80–110% for lactose and lactulose; allolactose recoveries in aged cheese and sandwich cookie were not calculated due to co-elution of minor interferents.

Benefits and Practical Applications of the Method

• The direct detection approach of HPAE-PAD avoids derivatization, reducing preparation time and error.
• Sensitivity allows quantification of lactose well below common regulatory thresholds for lactose free labeling.
• Fast separation and robust cartridge-based eluent generation support high sample throughput for QA/QC laboratories.

Future Trends and Potential Applications

Integration of automated sample preparation and inline cleanup could further increase throughput. Expanding the method to other dairy derivatives, plant-based milks and low sugar formulations will meet evolving market needs. Advances in electrode materials and waveform optimization may improve detection of even lower analyte levels.

Conclusion

A validated HPAE-PAD method using the CarboPac PA210-4μm column provides a rapid, sensitive and accurate tool for measuring residual lactose and related sugars in lactose free products. The approach supports reliable labeling and quality control of dairy and bakery items marketed to lactose intolerant consumers.

References

1. Commission Directive 2006/141/EC of 22 December 2006 on infant formula and follow-on formula, Official Journal of the European Union, 2006.
2. EFSA NDA Panel, Scientific opinion on lactose thresholds in lactose intolerance and galactosaemia, EFSA Journal, 2010;8(9):1777.
3. US DHHS FDA, Grade A Pasteurized Milk Ordinance, 2009 revision.
4. Langer AJ et al, Nonpasteurized dairy products, disease outbreaks and state laws, Emerg Infect Dis, 2012;18(3):385–391.
5. Seki N, Saito H, Lactose as a source for lactulose and other functional lactose derivatives, Int Dairy J, 2012;22:110–115.
6. Fransen CTM et al, Novel lactose isomers by β-galactosidase, Carbohydr Res, 1998;314:101–114.
7. Adachi S, Formation of lactulose and tagatose from lactose in heated milk, Nature, 1958;181:840–841.
8. Kuschel B et al, Analysis of prebiotics in milk: lactose, lactulose and epilactose, Biospektrum, 2017;23:833.
9. van Riel J, Olieman C, Anion-exchange chromatographic determination of saccharides in dairy, Carbohydr Res, 1991;215(1):39–46.
10. Monti L et al, Determination of sugars in hard cheese by HPAEC-PAD, Food Chem, 2017;220:18–24.
11. Thermo Fisher Scientific, Application Note 248: Determination of lactose in lactose-free milk products by HPAEC-PAD, 2017.
12. van Scheppingen WB et al, Sensitive determination of lactose in low-lactose dairy products with HPAEC-PAD, J Chromatogr B, 2017;1060:395–399.
13. Thermo Fisher Scientific, Technical Note 71: Eluent preparation for HPAE-PAD, 2017.
14. Thermo Fisher Scientific, Application Note 1158: HPAE-PAD determination of carbohydrates in honey, 2017.