基于UPLC-MS/MS技术的更年宁水蜜丸中藏柴胡掺伪方法的研究

Adulteration of Bupleurum marginatum var.stenophyllum in Gengnianning water-honeyed pill by UPLC-MS/MS

目的 建立更年宁中藏柴胡掺伪检查方法,考察生产企业是否存在使用藏柴胡替代柴胡投料的情况。方法 采用超高效液相色谱 - 三重四极杆质谱联用技术,选择 Waters BEH C_(18)色谱柱(2.1 mm×50 mm,1.7 μm),流动相为乙腈(A)-0.1% 甲酸水溶液(B),梯度洗脱,流速0.3 mL·min^(-1),柱温 35℃,进样量 1 μL;采用电喷雾离子源(ESI),负离子模式,进行多反应监测(MRM),选择 m/z 943.5 → 635.4、m/z 943.5 → 797.5 和 m/z 943.5 → 781.5 为尼泊尔柴胡皂苷 K 的检测离子对。结果 尼泊尔柴胡皂苷 K 质量浓度在 93.3 ~ 3734 ng·m L^(-1)内与峰面积呈现良好的线性关系(r = 0.9998);精密度、重复性、稳定性(24 h)试验的 RSD 均小于2.0%;3 个浓度平均加样回收率为 96.4% ~ 99.6%(RSD 均< 3.0)。更年宁中藏柴胡掺伪比例在 3% ~ 100% 内与尼泊尔柴胡皂苷 K 质量浓度的线性关系良好(r = 0.9947)。以掺伪藏柴胡不得过 10% 为限度,对 44 批更年宁样品进行筛查,结果 5 批更年宁掺伪藏柴胡 22% ~ 36%,超出拟订限度。结论 该方法准确、可靠,可用于更年宁中藏柴胡掺伪检查及市场监管。

Objective To establish the detection method for the adulteration of Bupleurum marginatum var.stenophyllum in Gengnianning water-honeyed pill,and to find out whether Bupleurum marginatum var.stenophyllum was used to replace Bupleuri Radix in the manufacturing enterprises.Methods UPLC-MS/MS was performed on Waters BEH C_(18) column (2.1 mm×50 mm,1.7μm),with acetonitrile and 0.1%formic acid as the mobile phase in gradient elution at 0.3 mL·min~(-1).The column temperature was 35℃and the injection volume was 1μL.A triple quadrupole mass spectrometer was used for the detection in negative ion mode with an electrospray ion source.m/z 943.5→635.4,m/z 943.5→797.5 and m/z 943.5→781.5 were selected as the ion pairs and multiple reaction monitoring (MRM) mode was used for the determination of nepasaikosaponin K.Results Nepasaikosaponin K showed a good linearity at 93.3~3734 ng·m L~(-1) (r=0.9998).The RSDs of precision,repeatability and stability (24 h) were all lower than 2.0%.The average recoveries at three spiked levels were 96.4%~99.6%(RSD<3.0%).The adulteration ratio of Bupleurum marginatum var.stenophyllum in Gengnianning water-honeyed pill showed a good linearity to the content of nepasaikosaponin K within 3%~100%(r=0.9947).Totally 44 batches of Gengnianning water-honeyed pill were screened with the limit that the doping of Bupleurum marginatum var.stenophyllum should not exceeding 10%.The doping of Bupleurum marginatum var.stenophyllum in 5 batches of Gengnianning water-honeyed pill was 22%~36%,which exceeded the proposed limit.Conclusion The method is accurate and reliable,can be used to detect the adulteration of Bupleurum marginatum var.stenophyllum in Gengnianning water-honeyed pill.