N-Acetylglucosamine Assay and Glucosamine Limit Test Following the United States Pharmacopoeia Method

Chemical structure of N-acetylglucosamine

Introduction

N-Acetylglucosamine is an acetylated derivative of glucosamine and is found naturally in the outer shell of crustaceans. It is a popular nutritional supplement to reduce arthritic pain and stiffness.

A simple, precise, and sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) isocratic method is demonstrated for the assay of N-acetylglucosamine and glucosamine limit test following the USP monograph for dietary supplements.

In this method, two Chromolith^® Performance NH₂ columns, 100 x 4.6 mm and 50 x 4.6 mm were coupled with a zero dead volume connector for a total length of 150 mm. The method was evaluated according to USP general chapter <621>.

Experimental

Results

Figure 1.Blank chromatogram.

Assay– N-Acetylglucosamine

System Suitability– Standard Solution

Chromatogram for system suitability solution of N-Acetylglucosamine Standard (1 mg/mL) injection

Figure 2.N-Acetylglucosamine standard (1 mg/mL) injection.

System Suitability– System Suitability Solution

Chromatogram for system suitability solution of 1 mg/ml N-acetylglucosamine and 0.6 mg/ml glucosamine hydrochloride.

Figure 3.Chromatogram System Suitability solution (1 mg/mL N-acetylglucosamine and 0.6 mg/mL glucosamine hydrochloride).

Chromatogram for an N-acetylglucosamine sample solution (1 mg/ml)

Figure 4.Chromatogram N-acetylglucosamine sample solution (1 mg/mL).

Glucosamine content in N-Acetylglucosamine

Figure 5.Chromatogram for a 50 mg/mL N-acetylglucosamine sample solution.

Figure 6.Chromatogram of glucosamine standard solution 0.6 mg/mL.

Calibration curve obtained for glucosamine in its assay

Figure 7.Calibration function glucosamine.

Calibration curve obtained for N-acetylglucosamine in its assay

Figure 8.Calibration function N-acetylglucosamine.

Conclusion

As per the allowed changes in USP General chapter <621>, two Chromolith^® NH₂ (Amino propyl) columns coupled by a zero dead volume connector were used to determine N-acetylglucosamine according to USP. The method could satisfy the SST criteria on tailing factor, relative standard deviation, signal-to-noise ratio as well as resolution between N-acetylglucosamine and glucosamine for a standard solution.

For N-acetylglucosamine a LOD of 20.3 µg/mL and a LOQ of 61.5 µg/ml with linearity up to 1200 μg/mL were determined. For Glucosamine a LOD of 26.9 µg/mL and a LOQ of 81.7 µg/mL with linearity up to 720 μg/mL were determined. The injection volume and flow rate were scaled accordingly and the column back pressure was 20 bar. This is comparatively lesser than with a 3 µm particulate column (~100 bar) as per the current USP monograph method.

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References

1.

2022. N-Acetylglucosamine. Dietary Supplement Monographs, N-Acetylglucosamine. . https://doi.org/10.31003/uspnf_m5949_04_01