College of Pharmacy, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 61186, Republic of Korea.
National Development Institute of Korean Medicine, 288 Udeuraendeu-gil, Anyang-myeon, Jangheung-gun, Jeollanam-do 59338, Republic of Korea.
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Sep 15;1126-1127:121743. doi: 10.1016/j.jchromb.2019.121743. Epub 2019 Aug 2.
The purpose of this study was to develop a method for simultaneous analysis of fourteen major active components in Gumiganghwal-tang tablet widely prescribed for cold related diseases using UPLC-ESI-MS/MS. Twelve of these 14 components were separated using 0.1% formic acid and acetonitrile as a mobile phase by gradient elution at a flow rate of 0.3 mL/min equipped with a KINETEX C column (2.1 × 50 mm, 1.7 μm). The remaining two components were separated using 10 mM aqueous ammonium formate containing 0.01% formic acid and acetonitrile as a mobile phase by gradient elution at a flow rate of 0.2 mL/min equipped with an Inertsil C-3 column (2.1 × 100 mm, 2.0 μm). Quantitation of this analysis was performed on a triple quadrupole mass spectrometer using electrospray ionization technique operating in multiple reaction monitoring mode. Full validation of the analysis method was carried out, including its linearity, selectivity, sensitivity, precision, accuracy, recovery, and stability. Chromatograms showed high resolution, sensitivity, and selectivity without interference by impurities. Calibration curves of all 14 components ranged from 0.5 to 1000 ng/mL, displaying excellent linearity (correlation coefficients >0.99). The relative standard deviations (RSD) of intra- and inter-day were <11.75%. Recoveries were within the range 95.41-103.24% (RSD value of 1.62-9.09%). These results demonstrate that the developed method is simple, rapid, reliable, specific, accurate, and sensitive for the quantification of bioactive components of Gumiganghwal-tang. The developed method was successfully applied to the analysis of Gumiganghwal-tang tablet. The developed UPLC-ESI-MS/MS method could be useful not only for quality control, but also for effectiveness and safety evaluation of Gumiganghwal-tang tablet.
本研究旨在开发一种使用 UPLC-ESI-MS/MS 同时分析广用于治疗感冒相关疾病的古秘方 Gumiganghwal-tang 片的 14 种主要活性成分的方法。这 14 种成分中的 12 种采用 0.1%甲酸和乙腈作为流动相，通过在 0.3 mL/min 的流速下进行梯度洗脱，使用 KINETEX C 柱（2.1×50mm，1.7μm）进行分离。另外两种成分采用 10 mM 水溶液中含有 0.01%甲酸和乙腈作为流动相，通过在 0.2 mL/min 的流速下进行梯度洗脱，使用 Inertsil C-3 柱（2.1×100mm，2.0μm）进行分离。该分析的定量采用电喷雾电离技术在三重四极杆质谱仪上进行，采用多反应监测模式。对分析方法进行了全面验证，包括线性、选择性、灵敏度、精密度、准确度、回收率和稳定性。色谱图显示出高分辨率、灵敏度和选择性，没有杂质干扰。所有 14 种成分的校准曲线范围为 0.5 至 1000ng/mL，呈现出极好的线性（相关系数>0.99）。日内和日间的相对标准偏差（RSD）<11.75%。回收率在 95.41-103.24%范围内（RSD 值为 1.62-9.09%）。这些结果表明，所开发的方法简单、快速、可靠、具有特异性、准确且灵敏，可用于 Gumiganghwal-tang 的生物活性成分定量分析。该方法已成功应用于 Gumiganghwal-tang 片的分析。所开发的 UPLC-ESI-MS/MS 方法不仅可用于质量控制，还可用于 Gumiganghwal-tang 片的有效性和安全性评价。
