Determination of lactose in lactose-free dairy products using HPAE coupled with PAD and MS dual detection

Importance of the topic

Lactose intolerance is widespread and drives consumer demand for lactose‐free dairy products. Accurate measurement of residual lactose is essential for labeling compliance, quality control, and consumer safety. Dual detection strategies combining high‐performance anion‐exchange chromatography (HPAE) with pulsed amperometric detection (PAD) and mass spectrometry (MS) deliver both quantification and confirmatory identification of lactose and its thermally or enzymatically derived isomers.

Objectives and study overview

This study aimed to develop and validate a rapid, reliable method to determine trace levels of lactose, allolactose, lactulose, and epilactose in commercially available lactose‐free dairy products. The method leverages HPAE separation paired with simultaneous PAD and single‐quadrupole MS detection to achieve low detection limits and high selectivity, meeting labeling requirements for lactose‐free claims (<0.01% to 0.1% lactose). Five sample types (skim milk, half & half, cultured smoothie, yogurt, sour cream) labeled as “99–100% lactose‐free” were analyzed to demonstrate method performance.

Methodology and instrumentation used

Ultrapure water extracts of dairy samples were clarified by Carrez precipitation and OnGuard IIA anion trap cleanup, then injected onto a Thermo Scientific Dionex ICS‐5000+ HPIC system. A Dionex CarboPac PA210‐4 μm column ran at 0.8 mL/min with a KOH gradient (19 mM to 100 mM back to 19 mM). The outlet stream was split (~45% to PAD, ~55% to MS). A Dionex ERD 500 electrolytic desalter converted KOH to water before the MS inlet. PAD employed a gold disposable electrode and Ag/AgCl reference with a TN21 waveform. MS detection used an ISQ EC single‐quadrupole with a heated electrospray ionization probe in negative mode, monitoring m/z 341.3 for all target analytes.

Main results and discussion

• Chromatographic Resolution: All four sugars were baseline‐separated within 15 min, including isomeric pairs (lactulose vs. epilactose).
• Calibration and Linearity: Standards from 0.05 mg/L to 5 mg/L fit quadratic calibration curves (r2 > 0.999) for both PAD and MS. MS detection showed 2–3× lower LODs (0.003–0.004 mg/L) and LOQs (0.009–0.016 mg/L) compared to PAD.
• Sample Analysis: Lactose in commercial lactose‐free samples ranged from 0.004 g/100 g to 0.163 g/100 g. Products labeled 100% lactose‐free typically contained <0.05 g/100 g lactose; one cultured smoothie (99% lactose‐free label) had ~0.16 g/100 g. Allolactose concentrations measured by PAD were ~2–3× higher than by MS, suggesting possible co‐elution or ion suppression.
• Recovery and Precision: Spike recoveries for lactose and isomers were 85–110% by both detectors. RSDs for retention time and quantitation were <1% and sample run‐to‐run precision was maintained over typical sequence lengths.

Benefits and practical applications of the method

• Dual detection confers both accurate quantification (PAD) and compound confirmation (MS).
• Low detection limits enable compliance with strict “lactose‐free” thresholds.
• Rapid analysis (<30 min/run) supports high‐throughput quality control.
• Minimal make‐up solvents or additional sample handling required due to optimized ESI interface.

Future trends and application possibilities

• Extension to other carbohydrate isomers and prebiotic oligosaccharides in complex foods.
• Integration with high‐resolution MS for structural elucidation of unknown sugar derivatives.
• Automation of sample prep using online cleanup and 96‐well formats for screening large product portfolios.
• Regulatory adoption for standard methods in dairy analytics and labeling enforcement.

Conclusion

The combined HPAE‐PAD/MS method provides a sensitive, selective, and efficient approach to quantify and confirm residual lactose and related isomers in lactose‐free dairy products. Its low detection limits, robust recoveries, and compatibility with high‐throughput workflows make it a valuable tool for industry and regulatory laboratories monitoring lactose content.

References

• U.S. FDA Grade A Pasteurized Milk Ordinance (2009 Revision).
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• EFSA NDA Panel. EFSA Journal 2010, 8(9), 1777.
• van Scheppingen, W.B. et al. J. Chromatogr. B 2017, 1060, 395–399.
• Thermo Scientific Application Note 72632, AN72632-EN.