牛黄消炎片中孔雀石绿的检测方法研究

目的： 建立牛黄消炎片中孔雀石绿的检测方法。 方法： 采用HPLC法鉴别和定量，使用Phenomenon Gemini C₁₈色谱柱（250 mm×4.6 mm，5 μm），以乙腈-0.05 mol·L^-1醋酸铵溶液（冰醋酸调pH 4.5）（40：60）为流动相，流速1 mL·min^-1，检测波长618 nm；采用HPLC-MS/MS法验证，以乙腈-5 mmol·L^-1醋酸铵溶液（冰醋酸调pH 4.5）（40：60）为流动相，电喷雾离子化源，正离子检测方式，对孔雀石绿的准分子离子峰m/z 329 ⁺进行选择离子监测及二级全扫描质谱分析。 结果： HPLC鉴别方法分离良好，检出限在0.03 μg·mL^-1；阳性样品的选择离子监测图及二级全扫描质谱图与孔雀石绿对照品一致；含量测定方法在0.33～7.85 μg·mL^-1线性关系良好，平均回收率为99.3%。 结论： 本方法专属性强，结果准确，可用于牛黄消炎片中孔雀石绿的定性、定量检测。

Objective: To develop a specific method for the detection of malachite green in Niuhuang Xiaoyan tablets. Methods: An HPLC method was adopted for identification and determination using a Phenomenon Gemini C₁₈ column(250 mm×4.6 mm,5 μm)with a mixture of acetonitrile and 0.05 mol·L^-1 ammonium acetate solution(adjusted to pH 4.5 with glacial acetic acid)(40:60)as the mobile phase at a flow rate of 1 mL·min^-1.The detection wavelength was set at 618 nm.Meanwhile,the positive results were confirmed by HPLC-MS/MS with ESI⁺ ion source.The mixture of acetonitrile and 5 mmol·L^-1 ammonium acetate solution(adjusted to pH 4.5 with glacial acetic acid)(40:60)was used as the mobile phase.The ion at m/z 329 was selected for monitoring and MS/MS analysis. Results: Good separation was obtained in HPLC identification.The detection limit was 0.03 μg·mL^-1.The selected ion monitoring chromatogram and full scan MS² spectra of positive samples were consistent with the reference substance of malachite green.For malachite green,the calibration curve was linear in the range of 0.33-7.85 μg·mL^-1 and the average recovery was 99.3%. Conclusion: The proposed method is exclusive,accurate for qualitative detection and quantitative determination of malachite green in Niuhuang Xiaoyan tablets.