蛤蚧定喘丸中十种非法染色物的检测

Determination of ten illegal dyes in Gejie Dingchuan Pills

目的建立高效液相色谱法(HPLC)检测蛤蚧定喘丸中非法添加色素金胺O、柠檬黄、胭脂红、日落黄、亮黄、酸性红、甲基橙、金橙Ⅱ、赤藓红、碱性橙的方法,并用LC-MS/MS确证。方法样品用50%乙醇溶液提取,HPLC测定,流动相为乙腈-0.01 mol/L乙酸铵溶液(含0.5%冰乙酸),梯度洗脱,检测波长420 nm和500 nm。用LC-MS/MS进行确证,流动相为乙腈-0.01 mol/L乙酸铵溶液,梯度洗脱,离子源:ESI,扫描模式:正离子,在动态MRM模式下,以保留时间和质荷比对分离的组分给予定性。结果十种染色物线性相关系数均>0.999,定量限在0.3~2.8μg/g,回收率为96.3%~101.1%,RSD为0.6%~1.5%。123批次样品33批次检出金胺O。结论液质联用法定性准确、可靠,液相色谱含量测定法简便、快速、准确,两者相结合可快速、准确、灵敏地检测出蛤蚧定喘丸中十种染色物。

Objective To establish a quantification method of HPLC for determining illegally added auramine O, lemon carmine, sunset yellow, brilliant yellow, acid red, methyl orange, orange II, etythrosine and alkaline orange in yellow, Gejie Dingchuan Pills, and validate with LC-MS/MS method. Methods The sample was extracted by 50% ethanol, and determined by HPLC with a gradient elution of acetonitrile-0.01 mol/L ammonium acetate （contain 0.5% glacial acetic acid） and the detection wavelength was set at 420 nm and 500 nm. The identification was performed by LC-MS/MS, using a gradient elution of acetonitrile-0.01 mol/L ammonium acetate. The analytes were detected by an electro spray ionization tandem mass spectrometry with dynamic MRM. Qualitation of the auramine O based on the retention time and mass charge ratio. Results The calibration curve of ten dyes showed good linearity and correlation coefficients were all above 0.999, the limits of quantitation for ten dyes in samples were between 0.3-2.8 μg/g, and the average recoveries ranged from 96.3% to 101.1% with RSD of 0.6%-1.5%. Auramine O could be detected in 33 of 123 samples. Conclu- sion LC-MS/MS method is accurate and reliable for the qualitative identification and the HPLC method is simple, rapid and accurate for the content determination. The combination of HPLC and LC-MS/MS can detect ten dyes in Gejie Dingchuan Pills rapidly, accurately and sensitively.