人血浆中痕量灯盏乙素SPE-HPLC/MS/MS的建立及药动学研究

Determination of Trace Scutellarin by a SPE-HPLC/MS/MS Assay and Its Pharmcokinetics in Human Plasma

目的 建立人血浆中微量灯盏乙素的SPE-HPLC／MS／MS分析方法，分析临床用药每人60mg灯盏花素时，主要有效成分灯盏乙素的人体药动学过程和特征，评价受试制剂和参比制剂的生物等效性和相对生物利用度。方法 含有内标黄芩苷的血浆经固相萃取预处理后，HPLC-MS／MS分离、分析。色谱系统：ODS柱（4.6mm×250mm，5μm），梯度洗脱，流速1．0mL·min^-1．柱温35℃。质谱检测方式：选择反应检测（Selective Reaction Monitoring）。20名健康志愿者经随机交叉口服60mg受试制剂或参比制剂后，测定血药浓度的变化，计算药动学参数。结果 血浆中内源性杂质不干扰微量灯盏乙素分析。测定线性范围0.2～20．0μg·L^-1（r=0．9995），最低定量限0．2μg·L^-1，日内、日间精密度（RSD〈13％），灯盏乙素的提取回收率≥80％。受试制剂及参比制剂的生物半衰期分别为（2．27±0．58）和（2．25±0．44）h，达峰时间分别为（5，9±0．8）和（5．6±1.6）h，峰质量浓度分别为（12．02±2，23）和（11.68±2，67）μg·L^-1。受试制剂的相对生物利用度为（104．2±13，0）％。结论 建立的方法专属、准确、灵敏、简便，测定了健康受试者口服60mg小剂量灯盏花素制剂后的灯盏乙素血药浓度，估算灯盏乙素人体药动学参数。统计学结果表明，新制剂灯盏花素滴丸和市售灯盏花素片生物等效。

OBJECTIVE To establish a novel HPLC-MS/MS method for the determination of trace scutellarin and to investigate its pharmacokinetics and bioequivalence of scutellarin in human body. METHODS After adding baicalin （internal standard, IS）, the plasma sampies were prepared with Waters SPE cartridges and determined by HPLC-MS/MS. The separation was carried out on a ODS column （4.6mm×250mm,5μm） at 35℃ ,with gradient elution, at a flow rate of 1.0 mL·min^-1. HPLC-MS/MS was performed in the selected reaction monitoring （SRM） mode using target ions at m/z 463.0 - 287.0 for scutellarin and m/z 447.0 - 271.0 for baicalin（IS）. A randomized crossover design was performed in 20 healthy volunteers. In the two study periods, a single dose of 60 mg was administered to each volunteers. RESULTS The linear calibration curve was obtained in the concentration range of 0.2 - 20μg·L^-1 （ r = 0. 999 5）. The LOQ of scutellarin in plasma was 0.2μg·L^-1. The average recovery was more than 80%. The within- and between-day variations （RSD） were less than 13%. The ρmax of scutellarin in blood for the test and reference formulations were （12.02 ± 2.33） and （11.68 ± 2.67）μg·L^-1 at （5.9 ± 0.8） and （5.6 ± 1.6） h, respectively. The t 1/2 were （2.27 ± 0.58） and （2.25 ± 0.44 ） h, respectively. The relative bioavailability of the test formulation was（ 104.2 ± 13.0）%. CONCLUSION The method is proved to be special, sensitive, accurate and convenient. The pharmacokinetic profile of scutellarin in human after a single dose of 60 mg breviscapine ids discribed. The two formulations are bioequivalent.