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目的:建立高效液相色谱法测定氨基葡萄糖(Glc N)样品中乙酰氨基葡萄糖(Glc NAc)。方法:采用氨基柱(250 mm×4.6 mm,5μm),以乙腈-磷酸盐缓冲液(65∶35,p H 7.5)为流动相,流速1.0 m L·min-1,柱温30℃,检测波长195 nm,进样体积20μL。结果:方法专属性强,Glc NAc与Glc N及各杂质分离良好;Glc NAc在1.94~14.70μg·m L-1浓度范围内线性良好(r=0.999 9),准确度高(回收率99.0%~104.2%,n=3),溶液在24 h内稳定(RSD为0.69%),精密度(RSD为1.5%~2.2%)和耐用性(RSD为1.6%~8.1%)均良好,灵敏度高(定量限0.98μg·m L-1)。经过测定,盐酸氨基葡萄糖粗品中Glc NAc含量为0.09%~0.18%,盐酸氨基葡萄糖成品和硫酸氨基葡萄糖氯化钾成品中未检出Glc NAc(含量<0.0049%)。结论:本法经方法学验证,能够快速、准确地测定出Glc N中Glc NAc的含量。
Objective: To establish a HPLC method for the determination of GlcNAc in Glc N samples. METHODS: Ammonia column (250 mm × 4.6 mm, 5 μm) was used with acetonitrile-phosphate buffer (65:35, p H 7.5) as the mobile phase at a flow rate of 1.0 mL · min-1 and the column temperature was set at 30 ℃. Wavelength 195 nm, injection volume 20μL. Results: GlcNAc was separated from Glc N and its impurities with good specificity. GlcNAc showed a good linearity (r = 0.999 9) within the range of 1.94 to 14.70 μg · m L-1 with a high accuracy (recovery of 99.0% ~ 104.2%, n = 3). The solution was stable within 24 h with a RSD of 0.69%. The precision (RSD 1.5% -2.2%) and durability (RSD 1.6% -8.1%) were good and the sensitivity was high (Limit of quantitation 0.98μg · m L-1). After determination, GlcNAc content of crude glucosamine hydrochloride was 0.09% ~ 0.18%, GlcNAc (content <0.0049%) was not detected in finished products of glucosamine hydrochloride and glucosamine potassium sulfate. Conclusion: This method is validated by methodology, which can determine the content of GlcNAc in Glc N quickly and accurately.