Quantitative Analysis of Carbohydrates by LC/MS

Significance of the Topic

Accurate quantitation of carbohydrates is fundamental in food quality control, nutritional analysis and industrial process monitoring. Traditional HPLC methods often rely on refractive index or post-column derivatization, which can limit sensitivity and throughput. Coupling liquid chromatography with mass spectrometry and post-column solvent addition offers improved ionization efficiency for underivatized saccharides, enhancing detection stability and expanding dynamic range.

Study Objectives and Overview

This application note presents a methodology for quantitative analysis of seven key monosaccharides and disaccharides (ribose, xylose, rhamnose, fructose, glucose, sucrose and maltose) using LC/APCI-MS with post-column methanol-chloroform addition. The goal is to demonstrate the approach’s linearity, precision and applicability to real food matrices such as mirin (sweet sake) and soft drinks.

Methodology and Instrumentation

A hydrophilic interaction column (Shodex Asahipak NH2P-50, 150 × 2.0 mm, 5 μm) was employed with an acetonitrile/water (3:1) mobile phase at 0.2 mL/min. After separation, a post-column pump delivered methanol–chloroform (4:1) at 0.075 mL/min to promote formation of chloride adducts ([M+Cl]–). Ionization was achieved in negative APCI mode on a Shimadzu LCMS-2020, operating under the following conditions:

• Probe voltage –3.5 kV, temperature 400 °C
• Nebulizing gas 4.0 L/min, drying gas 5.0 L/min
• DL temperature 250 °C, block heater 200 °C
• SIM monitoring at m/z 184.8, 198.8, 214.8 and 376.9

Main Results and Discussion

Calibration curves for each analyte exhibited excellent linearity (R ≥ 0.9996) over ranges from 0.1 to 100 mg/L. Precision, expressed as RSD for the lowest calibration level, ranged from 0.5% (glucose) to 10.2% (sucrose). Representative SIM chromatograms confirmed baseline separation of all seven carbohydrates. Analysis of food samples (10-fold dilution, ultrafiltration, 1000-fold final dilution, 1 µL injection) yielded the following concentrations:

• Mirin: 360 mg/mL glucose, 41.7 mg/mL maltose
• Soft drink: 8.6 mg/mL fructose, 12.1 mg/mL glucose, 32.4 mg/mL sucrose

Benefits and Practical Applications

The described LC/APCI-MS method delivers:

• High sensitivity for underivatized saccharides via chloride adduct formation
• Wide linear range and robust precision
• Minimal sample preparation and elimination of derivatization steps
• Compatibility with complex food matrices

Used Instrumentation

• Shimadzu LCMS-2020 with APCI in negative mode
• Shodex Asahipak NH2P-50 column (150 × 2.0 mm, 5 µm)
• Post-column solvent addition pump for methanol–chloroform (4:1)

Future Trends and Opportunities

Advancements may include integration with high-resolution MS for structural confirmation, on-line sample cleanup for fully automated workflows, and expansion to comprehensive carbohydrate profiling in metabolomics. Adoption of novel ionization techniques and micro- or nano-flow formats could further boost sensitivity and reduce solvent consumption.

Conclusion

This study validates a straightforward, highly sensitive LC/APCI-MS approach for quantitative carbohydrate analysis without chemical derivatization. The method’s strong linearity, precision and adaptability to real samples make it a valuable tool for food testing, nutritional research and quality assurance.