Quantification of Mono and Disaccharides in Foods

Importance of the Topic

The accurate measurement of mono- and disaccharides in food products is critical for nutritional labeling, monitoring added sugars, and addressing public health concerns such as obesity and diabetes. Reliable quantification supports regulatory compliance and informs consumers and manufacturers about sugar intake.

Objectives and Study Overview

This application study aimed to improve a high-performance liquid chromatography–mass spectrometry (HPLC-MS) method for simultaneous separation and quantification of six common sugars—fructose, galactose, glucose, sucrose, lactose, and maltose—in diverse food and feed matrices. The revised protocol was tested across multiple sample types including infant formulas, milk powders, oatmeal, cocoa powder, and chicken feed.

Methodology and Instrumentation

The method coupled a hydrophilic interaction liquid chromatography (HILIC) column with single-quadrupole mass detection.

• Instrumentation
• ACQUITY Arc System for gradient delivery
• ACQUITY QDa Mass Detector in electrospray ionization mode
• XBridge BEH Amide XP, 2.5 µm, 3.0×150 mm column
• Empower 3 CDS software for data processing
• Chromatographic Conditions
• Run time: 25 minutes per injection
• Column temperature: 90 °C
• Mobile phase: acetonitrile:water:methanol (90:6:4) with diethylamine and guanidine hydrochloride additives
• Flow rate: 0.8 mL/min, injection volume: 1 µL
• Mass Spectrometry
• Positive ESI, capillary voltage 0.8 kV, probe temperature 600 °C
• Single Ion Recording channels monitored chloride adducts of each sugar
• Calibration used quadratic regression with 1/x weighting across seven concentration levels
• Sample Preparation
• Matrix removal via Carrez reagents to precipitate proteins and salts
• Extraction in ethanol:water and acetonitrile
• Internal standards: glucose-13C6 and lactose-13C6 at 20 mg/L

Main Results and Discussion

Baseline separation of all six sugars was achieved within 25 minutes, including challenging pairs such as galactose and glucose. High salt concentrations up to 500 mM did not affect peak shapes or resolution. Calibration curves exhibited excellent linearity (R² > 0.995). Recoveries in spiked infant formula matrices ranged from 90% to 125%, and repeatability studies demonstrated relative standard deviations below 5.8%. Application to real samples revealed expected sugar profiles: high lactose in milk powders and infant formulas, sucrose as the dominant sugar in oatmeal, soy-based formula, and chicken feed, and elevated galactose and glucose in lactose-free milk.

Benefits and Practical Applications

• Reliable separation of isomeric sugars without interferences
• Short analysis time compared to traditional HPAEC methods
• High sensitivity and selectivity afforded by MS detection
• Robust performance in complex and high-salt matrices

Future Trends and Opportunities

Advancements may include coupling high-resolution mass spectrometry for simultaneous profiling of larger oligosaccharides, integration with automation for high-throughput screening, and expansion to emerging sugar substitutes and novel sweeteners relevant to current food trends.

Conclusion

The presented HILIC-MS method delivers accurate, precise, and rapid quantification of common mono- and disaccharides in a variety of food matrices. Its robustness against matrix interferences and salts, combined with a streamlined 25-minute runtime, makes it suitable for routine quality control and nutritional labeling workflows.

Reference

1. FDA Food Labeling Regulation, 21 CFR Part 101 (2016)
2. Benvenuti et al., Waters Application Note 720005767EN (2016)
3. Chavez-Servin et al., J. Chromatogr. A, 1043 (2004) 211–215
4. Waters XBridge BEH Amide XP Care and Use Manual 720004162EN (2012)