HPLC法测定二甲双胍格列本脲片(Ⅱ)中格列本脲的有关物质

目的：建立HPLC法测定二甲双胍格列本脲片（Ⅱ）中格列本脲的有关物质。方法：色谱柱为C₈柱（250 mm×4.6 mm，5 μm），流动相A为pH 3.5的磷酸二氢铵溶液（取磷酸二氢铵1.725 g，加水300 mL溶解，用磷酸调节pH至3.5±0.05），流动相B为乙腈，梯度洗脱，流速为1.0 mL·min^-1，柱温为40℃，检测波长为230 nm。结果：格列本脲与已知杂质及强制破坏产生的降解产物均分离良好；格列本脲杂质Ⅰ、格列本脲杂质Ⅱ、格列本脲杂质B、格列本脲杂质C、格列本脲杂质D、格列本脲杂质E和格列本脲的定量下限分别为0.037、0.016、0.021、0.074、0.049、0.073和0.049 μg·mL^-1，检测下限分别为0.011、0.004 7、0.006 2、0.022、0.015、0.022和0.015 μg·mL^-1；格列本脲杂质Ⅰ、Ⅱ、B、C、D、E和格列本脲的质量浓度分别在0.037 43~2.245 5 μg·mL^-1（r=0.999 9）、0.015 62~0.780 8 μg·mL^-1（r=1.000）、0.020 76~0.778 5 μg·mL^-1（r=1.000）、0.073 69~0.736 9 μg·mL^-1（r=0.999 9）、0.049 30~0.739 5 μg·mL^-1（r=0.999 9）、0.073 28~0.732 8 μg·mL^-1（r=0.999 9）和0.051 49~0.386 2 μg·mL^-1（r=0.999 7）范围内与峰面积呈良好的线性关系；杂质Ⅰ、Ⅱ、B、C、D、E低、中、高3种浓度的平均回收率（n=9）分别为106.1%、102.6%、101.0%、100.0%、101.3%和99.0%。3批样品有关物质测定结果显示，各已知杂质的含量均低于0.2%，其他最大单个杂质的含量均低于0.1%，除杂质Ⅰ外，杂质总量均低于0.5%。结论：本方法可以用于二甲双胍格列本脲片（Ⅱ）中格列本脲的有关物质测定。

Objective: To establish an HPLC method for the determination of related substances of glyburide in glyburide and metformin hydrochloride tablets (II).Methods: C₈ (250 mm×4.6 mm, 5 μm) column was adopted in this study, and the mobile phase A was monobasic ammonium phosphate buffer solution (dissolve 1.725 g of monobasic ammonium phosphate in 300 mL of water, and adjust to pH 3.5±0.05 with phosphoric acid);and the mobile phase B was acetonitrile with gradient elution at the flow rate of 1.0 mL·min^-1.The column temperature was 40℃, and the detection wavelength was 230 nm.Results: Glyburide was well separated from the known impurities and the forced degradation products.The limits of quantitation were 0.037, 0.016, 0.021, 0.074, 0.049, 0.073, 0.049 μg·mL^-1 for glyburide impurityⅠ glyburide impurity, Ⅱ glyburide impurity, B glyburide impurity, C glyburide impurity, D glyburide impurity, E and glyburide, respectively.The limits of detection were 0.011, 0.004 7, 0.006 2, 0.022, 0.015, 0.022 and 0.015 μg·mL^-1.There was a good linearity separately over the ranges 0.037 43-2.245 5 μg·mL^-1 (r=0.999 9) of glyburide related impurityⅠ, 0.015 62-0.780 8 μg·mL^-1 (r=1.000) of glyburide related impurityⅡ, 0.020 76-0.778 5 μg·mL^-1 (r=1.000) of glyburide related impurity B, 0.073 69-0.736 9 μg·mL^-1 (r=0.999 9) of glyburide related impurity C, 0.049 30-0.739 5 μg·mL^-1 (r=0.999 9) of glyburide related impurity D, 0.073 28-0.732 8 μg·mL^-1 (r=0.999 9) of glyburide related impurity E, 0.051 49-0.386 2 μg·mL^-1 (r=0.999 7) of glyburide.The average recovery rates (n=9) were 106.1%, 102.6%, 101.0%, 100.0%, 101.3% and 99.0% at the concentration level of low, medium and high concentrations, respectively.The results of related substances in the three batches of samples showed that the content of each known impurities was less than 0.2%, and the content of the other single impurities was less than 0.1%.Except for impurityⅠ, the content of totol impurities was less than 0.5%.Conclusion: The method is suitable for the determination of related substances of glyburide in glyburide and metformin hydrochloride tablets (II).

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