Determination of Glucosamine in Chondroitin Sulfate-Containing Dietary Supplements Using HPAE-PAD

Importance of the Topic

Glucosamine and chondroitin sulfate are key components in joint health supplements. Reliable quantification of glucosamine (GlcN) in formulations containing high-molecular-weight chondroitin sulfate (CS) is essential for quality control, label compliance, and stability assessment. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) offers the selectivity and robustness needed for such analyses.

Objectives and Study Overview

This study adapts an existing HPAE-PAD method to determine GlcN in CS-containing dietary supplements. It evaluates accuracy, precision, ruggedness, and the detection of degradation products across various commercial forms: tablets, capsules, liquids, and powders.

Methodology and Instrumentation

• Sample Preparation: Dissolution in ultra-pure water, centrifugation, and dilution to a target GlcN concentration (10 µM).
• Chromatographic Conditions: CarboPac PA20 column (3×150 mm), 20 mM KOH eluent (isocratic), 0.5 mL/min, 30 °C, 10 µL injection, 7.5 min run time.
• Detection: Pulsed amperometric detection using a disposable gold working electrode and Ag/AgCl reference electrode with a four-step potential waveform.
• Instrumentation: Dionex ICS-3000 RFIC-EG system, DP/SP pump, EGC II KOH cartridge, CR-ATC, DC module with ED detector, AS autosampler, Chromeleon software.
• Reagents and Standards: Gravimetric preparation of GlcN and related carbohydrate standards; deionized water (>18 MΩ·cm) degassed under helium.

Main Results and Discussion

• Linearity and Sensitivity: GlcN calibration was linear (r²>0.9999) from 0.30 to 340 µM; routine range 2.1–38 µM was employed.
• Precision: Over 1,030 injections of a challenging sample, retention-time RSD was <0.7% and peak-area, efficiency, and asymmetry remained stable over seven days.
• Accuracy: Recoveries of 98–109% in spiked water and matrix-matched samples confirmed method accuracy.
• Supplement Analysis: Measured GlcN levels were 98–119% of label claims. No GlcN was found in a GlcN-free formulation. Trace mono- and disaccharides were resolved and quantified at <5% of GlcN.
• Degradation Study: A product coeluting with mannosamine increased over time and with heat exposure, indicating potential for stability monitoring.

Benefits and Practical Applications

This HPAE-PAD approach provides rapid (≤7.5 min) and high‐throughput (>100 samples/day) measurement of GlcN in diverse supplement matrices. Automated eluent generation minimizes contamination and operator workload, supporting routine quality assurance and stability testing.

Future Trends and Applications

• Extension to other glycosaminoglycans and complex carbohydrates in supplements.
• Integration with automated sample handling and real-time degradation studies.
• Adaptation for regulatory compliance and addition to pharmacopoeial monographs.
• Coupling with mass spectrometry for structural confirmation.

Conclusion

The described HPAE-PAD method using a CarboPac PA20 column and electrolytic eluent generation is robust, selective, and efficient for glucosamine quantification in chondroitin sulfate–containing supplements. It demonstrates excellent accuracy, precision, and ruggedness, and can also detect glucosamine degradation products for stability monitoring.

Reference

• Dionex Corporation. Determination of Glucosamine in Dietary Supplements Using HPAE-PAD; Application Note 197, LPN 2001.
• Dionex Corporation. Eluent Preparation for High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection; Technical Note 71, LPN 1932-01, 2007.
• Dionex Corporation. Determination of Oxalate in Urine by Ion Chromatography; Application Note 36, LPN 034797, 1992.
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