目的: 建立一种同时测定熥敷颈腰痛酊中大豆苷元、芒柄花黄素、芫花素、槲皮素、芦丁、芹菜素、山柰酚、木犀草素、葛根素含量的超高效液相色谱串联质谱法(UPLC-MS/MS)。方法: 采用Waters ACQUITY UPLC HSS T3色谱柱(100 mm×2.1 mm,1.8 μm),以0.1%甲酸水溶液为流动相A,甲醇为流动相B,梯度洗脱,流速为0.3 mL·min-1,柱温为45 ℃,进样量为2.0 μL;电喷雾(ESI)离子源,正、负离子模式多反应监测采集。结果: 芹菜素、芫花素、大豆苷元在0.2~1 000 ng·mL-1范围内线性良好,葛根素、芦丁、木犀草素、山柰酚、芒柄花黄素在0.2~2 000 ng·mL-1范围内线性良好,槲皮素在1.0~5 000 ng·mL-1范围内线性良好;分离度较好,精密度、重复性、重现性、稳定性试验RSD均小于5%(n=6);平均加样回收率在82.7%~111.8%,RSD为2.0%~5.4%(n=6)。6批熥敷颈腰痛酊中大豆苷元、芒柄花黄素、芫花素、槲皮素、芦丁、芹菜素、山柰酚、木犀草素、葛根素的含量范围分别为8.46~17.97 μg·mL-1、0.62~1.67 μg·mL-1、0.03~0.08 μg·mL-1、1.34~2.22 μg·mL-1、0.70~1.14 μg·mL-1、0.48~0.99 μg·mL-1、0.20~0.90 μg·mL-1、0.10~0.16 μg·mL-1、167.95~227.75 μg·mL-1。结论: 本方法专属性强,线性范围宽,准确高效,可同时对熥敷颈腰痛酊中9个有效成分进行含量检测,为该方建立有效的质量控制方法。
Objective: To establish an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for the simultaneous determination of daidzein, formononetin, genkwanin, quercetin, rutin, apigenin, kaempferol, luteolin and puerarin in Tongfu Jingyaotong tincture. Methods: The Waters ACQUITY UPLC HSS T3 Column (100 mm×2.1 mm,1.8 μm) was used with methanol and 0.1% formic acid (gradient elution) as the mobile phase at a flow rate of 0.3 mL·min-1. Column temperature was 45 ℃, and injection volume was 2.0 μL. Electrospray ion source was adopted with positive and negative ion modes and multi-reaction monitoring and acquisition. Results: Apigenin, genkwanin and daidzein had good linearity in the range of 0.2-1 000 ng·mL-1, puerarin, rutin, luteolin, kaempferol and formononetin in the range of 0.2-2 000 ng·mL-1, and quercetin in the range of 1.0-5 000 ng·mL-1. The resolution was good, and the RSDs of precision, repeatability, reproducibility and stability test were all below 5% (n=6). The average recoveries ranged from 82.7% to 111.8% with RSDs (n=6) of 2.0%-5.4%. The contents of daidzein, formononetin, genkwanin, quercetin, rutin, apigenin, kaempferol, luteolin and puerarin in 6 batches of Tongfu Jingyaotong tincture were 8.46-17.97 μg·mL-1, 0.62-1.67 μg·mL-1, 0.03-0.08 μg·mL-1, 1.34-2.22 μg·mL-1, 0.70-1.14 μg·mL-1, 0.48-0.99 μg·mL-1, 0.20-0.90 μg·mL-1, 0.10-0.16 μg·mL-1 and 167.95-227.75 μg·mL-1, respectively. Conclusion: This method has strong specificity, wide linear range, accuracy and efficiency, and can detect the contents of 9 active ingredients in Tongfu Jingyaotong tincture at the same time, which offers an effective quality control method for Tongfu Jingyaotong tincture.
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