基于HPLC-TOF-MS和HPLC-MS/MS技术的产复康颗粒主要成分定性定量及药动学研究

Qualitative, quantitative and pharmacokinetic analysis of major constituents in Chanfukang granules by HPLC-TOF-MS and HPLC-MS/MS

目的运用高效液相色谱-高分辨飞行时间质谱联用(HPLC-TOF-MS)技术和高效液相色谱串联三重四级杆质谱(HPLC-MS/MS)法,对产复康颗粒化学成分进行快速鉴定及定量分析,并进行初步药动学研究。方法 HPLC-TOF-MS法采用ZORBAX Eclipse Plus C18(100 mm×3.0 mm,1.8μm)色谱柱;流动相为乙腈(A)-0.1%甲酸水(B),梯度洗脱,体积流量0.2 m L/min;质谱采用正、负离子扫描,扫描范围m/z 100~2000。HPLC-MS/MS法采用Waters XBridge?BEH C18(150 mm×4.6 mm,2.5μm)色谱柱;流动相为乙腈(A)-0.1%甲酸水(B),梯度洗脱,体积流量0.4 m L/min;Scheduled MRM正负离子切换监测模式定量分析。ig给予SD大鼠产复康颗粒(0.5、5 g/kg),给药后于不同时间点颈静脉取血0.2 m L,分离血浆,HPLC-MS/MS法检测产复康颗粒入血成分及含量。结果 HPLC-TOF-MS法鉴定产复康颗粒中24种成分;建立的HPLC-MS/MS方法分离度良好,各方法学验证均符合要求,对13种主要成分进行定量分析,水苏碱、黄芪甲苷和益母草碱的含量较高,可能是其主要活性成分;药动学结果显示,仅在给药剂量5 g/kg时,血浆中可检测到水苏碱和益母草碱,水苏碱的消除较慢,益母草碱快速消除。结论该方法简便、准确,重复性好,可用于产复康颗粒中多种成分的鉴定和含量的测定,为药材的质量控制、药动学研究提供了参考。

Objective To establish qualitative and quantitative analysis methods for identification and determination of multi-constituent of Chanfukang granules（CFKG） using high performance liquid chromatography-time-of-flight mass spectrometry（HPLC-TOF-MS） and high performance liquid chromatography-triple quadrupole mass spectrometry（HPLC-MS/MS）, and a preliminary pharmacokinetic study was carried out. Methods An Agilent ZORBAX Eclipse Plus C18 column（100 mm × 3.0 mm, 1.8 μm） and a Waters XBridge？ BEH C18 column（150 mm × 4.6 mm, 2.5 μm） were used with 0.1% formic acid aqueous solution-acetonitrile as mobile phases by gradient elution. The compounds were detected by electrospray ion source in both positive and negative mode with multiple reaction monitoring（MRM） mode. SD rats were ig with CFKG（0.5, 5 g/kg）. Blood samples of 0.2 m L were taken from jugular vein at different time points and plasma components and contents were determined by HPLC-MS/MS. Results Twenty-four constituents were identified by HPLC-TOF-MS qualitative analysis and thirteen constituents were quantitatively detected by HPLC-MS/MS. The established HPLC-MS/MS method had a good separation, and all the legal verification met the requirements. The content of stachydrine, Leonurine, and astragaloside IV were high, which may be the main active ingredient. The pharmacokinetic results showed that, only when the dose was 5 mg/kg, stachydrine and Leonurine can detected. Elimination of stachydrine was slow and elimination of Leonurine was fast. Conclusion A rapid and efficient method for studying the chemical constituents was established, which could provide reference for the quality control of Chanfukang granules.