葛根芩连片中掺入染色剂的检测方法研究

目的:建立葛根芩连片中可能掺入的染色剂含量测定方法,并采用LC-MS/MS方法进行专属性确证。方法:采用HPLC法测定染色剂含量,使用Kromasil C₁₈(4.6 mm×250 mm,5μm)色谱柱,以乙腈-0.05 mol·L^-1乙酸铵溶液(含0.5%冰乙酸)为流动相,梯度洗脱,流速1.0 mL·min^-1,检测波长为420 nm和500 nm,柱温35℃。用LC-MS/MS进行确证,以乙腈-0.01 mol·L^-1乙酸铵溶液为流动相,梯度洗脱;离子源为电喷雾电离源(ESI),扫描模式为多反应监测(MRM)。结果:建立了HPLC法检测葛根芩连片中可能含有的10种染色剂含量的方法。方法学验证结果表明,柠檬黄、胭脂红、日落黄、亮黄、酸性红73、甲基橙、金橙Ⅱ、金胺O、赤藓红、碱性橙Ⅱ的线性范围分别为1.4986~49.9550、0.6727~22.4235、1.4978~49.9250、0.2235~7.4516、0.4470~14.9000、0.3980~13.2678、0.4970~16.5667、0.1906~6.3532、0.6953~23.1764和0.1193~3.9776μg;加样回收率(n=6)分别为97.2%(RSD=0.86%)、99.1%(RSD=0.48%)、98.6%(RSD=0.86%)、101.0%(RSD=1.2%)、98.2%(RSD=0.89%)、98.3%(RSD=1.0%)、97.5%(RSD=0.63%)、96.3%(RSD=0.40%)、97.8%(RSD=1.0%)和96.3%(RSD=0.96%)。在对11个厂家123批样品的检验中,有5个厂家33批样品检出金胺O,检出率为26.8%,含量在0.013~83.51μg·g^-1之间,其中1批样品同时检出金胺O和碱性橙Ⅱ。用LC-MS/MS进行专属验证,33批出现HPLC阳性峰样品均检出相应的金胺O和碱性橙Ⅱ。结论:本方法准确、便捷,具有良好的精密度、重现性和稳定性,线性关系良好,可用于葛根芩连片的质量控制。

Objective: To establish a method for the determination of dyeing agent content in Gegen Qinlian tablets, and exclusively prove it by LC-MS/MS method. Methods: The dyeing agents was determined by HPLC using a Kromasil C₁₈(4.6 mm×250 mm, 5 μm) chromatographic column. A gradient elution program of acetonitrile-0.05 mol·L^-1 ammonium acetate(including 0.5% glacial acetic acid) was adopted at the flow rate of 1.0 mL·min^-1. The detection wavelengths were 420 nm and 500 nm, and the column temperature was maintained at 35℃. The corroboration was performed by LC-MS/MS, using a gradient elution of acetonitrile-0.01 mol·L^-1 ammonium acetate. The analyzes were detected by an electro spray ionization tandem mass spectrometry with dynamic MRM. Results: HPLC analytical method was established for analysis of ten adulteration dyes in Gegen Qinilian tablets. The results of verification of methodology showed that the linear range of citron yellow, carminum, sunset yellow fcf, brilliant yellow, acid red 73, helianthin, orange Ⅱ, auramine O, erythrosine and basic orange Ⅱ were 1.498 6-49.955 0, 0.672 7-22.423 5, 1.497 8-49.925 0, 0.223 5-7.451 6, 0.447 0-14.900 0, 0.398 0-13.267 8, 0.497 0-16.566 7, 0.190 6-6.353 2, 0.695 3-23.176 4 and 0.119 3-3.977 6 μg, respective and the sample recovery were 97.2%(RSD=0.86%), 99.1%(RSD=0.48%), 98.6%(RSD=0.86%), 101.0% (RSD=1.2%), 98.2%(RSD=0.89%), 98.3%(RSD=1.0%), 97.5%(RSD=0.63%), 96.3%(RSD=0.40%), 97.8%(RSD=1.0%) and 96.3%(RSD=0.96%), respectively. The results of 123 batches of samples of 11 factories showed that the content of auramine O was detected in the range of 0.013-83.51 μg·g^-1 in 33 batches from samples of 5 factories and the dedection rate was 26.8%. Auramine O and basic orange Ⅱ were detected in 1 batch of sample. The results were verified by LC-MS/MS method. 33 batches of HPLC peak samples were detected positive corresponding auramine O and basic orange Ⅱ. Conclusion: The established method is accurate and convenient with good precision, reproducibility and stability which has good linear relationship, and it can be used for the quality control of Gegen Qinlian tablets.