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目的:建立高效液相色谱方法,用于非法染色的关黄柏饮片中金胺O的检测。方法:采用TLC、HPLC-DAD法对样品中的金胺O进行快速筛查:用70%乙醇为提取溶剂,以硅胶G为吸附剂,二氯甲烷-乙酸乙酯-甲醇-氨水(4∶5∶1∶1)的下层溶液为展开剂,进行TLC筛查;采用ZORBAX SB-C_(18)色谱柱(4.6 mm×250 mm,5μm),柱温30℃,流动相为乙腈-0.025 mol·L~(-1)磷酸二氢钾溶液(含0.2%三乙胺,并用磷酸调节pH为3.0)(35∶65),流速1 mL·min~(-1),检测波长432 nm,进行定性鉴别。进一步利用HPLC-MS/MS法对阳性样品进行验证,采用Eclipse Plus C_(18)(4.6 mm×100 mm,3.5μm)色谱柱,以乙腈-0.05 mol·L~(-1)醋酸铵水溶液(冰醋酸调pH 4.5)(32∶68)为流动相,流速0.3 mL·min~(-1),柱温30℃,采用电喷雾离子源正离子、子离子扫描模式,母离子m/z 268.0,子离子扫描范围m/z 20~300,干燥气流速11 L·min~(-1),裂解电压90.0 V,碰撞能量25 eV;同时,建立HPLC测定阳性样品中的金胺O定量方法,色谱条件同HPLC-DAD筛查方法。结果:采用TLC、HPLC法对30批市售品进行快速筛查,发现6批样品检出金胺O,并用HPLC-MS/MS对可疑样品进行确认,三者测定结果一致;HPLC法定量测定,金胺O进样量在0.021 8~1.52μg范围内线性关系良好,阳性检出样品中金胺O含量为39.9~324.2μg·g~(-1)。结论:TLC、HPLC-DAD与HPLC-MS/MS的方法,三者相结合,可快速、准确、灵敏地检测出关黄柏中金胺O。
Objective: To establish a high performance liquid chromatography (HPLC) method for the detection of Auramine O in Guanzhong Pork Pieces, which is illegally stained. Methods: Rapid screening of Auramine O in samples was carried out by TLC and HPLC-DAD methods: 70% ethanol as extraction solvent, silica gel G as adsorbent, dichloromethane-ethyl acetate-methanol-ammonia water 5: 1: 1) as the developing solvent and TLC screening. The mobile phase was ZOLLBAX SB-C18 column (4.6 mm × 250 mm, 5 μm) with a mobile phase of acetonitrile-0.025 mol L -1 potassium dihydrogen phosphate solution containing 0.2% triethylamine and adjusted to pH 3.0 with phosphoric acid (35:65) at a flow rate of 1 mL · min -1 at a detection wavelength of 432 nm Qualitative identification. The positive samples were further identified by HPLC-MS / MS. The elution was performed on an Eclipse Plus C_ (18) (4.6 mm × 100 mm, 3.5 μm) column with acetonitrile-0.05 mol·L -1 ammonium acetate aqueous solution The pH was adjusted to 4.5 with acetic acid (32:68) as the mobile phase at a flow rate of 0.3 mL · min ~ (-1) and the column temperature was set at 30 ℃. The precursor ion m / z 268.0 , Ion scan range m / z 20 ~ 300, dry gas flow rate 11 L · min -1, cleavage voltage 90.0 V and collision energy 25 eV. At the same time, Chromatographic conditions with HPLC-DAD screening method. Results: Rapid screening of 30 batches of commercial products by TLC and HPLC showed that Auramine O was detected in 6 batches of samples and suspicious samples were confirmed by HPLC-MS / MS. The injection rate of Auamine O was linear in the range of 0.021 8 ~ 1.52μg. The content of Auamine O in the positive samples was 39.9 ~ 324.2μg · g ~ (-1). Conclusion: The combination of TLC, HPLC-DAD and HPLC-MS / MS can be used to detect cinchona O rapidly, accurately and sensitively.