HPLC-APCI-MS分析半边旗中二萜类化合物

ANALYSIS OF THE DITERPENOIDS IN THE EXTRACT OF PTERIS SEMIPINNATA L BY HPLC-APCI-MS

目的 建立一种准确可靠的分析半边旗中二萜类化合物的方法。方法 用高效液相色谱 (HPLC) 大气压化学电离源 (APCI) 质谱 (MS)联用的方式 ,以选择离子监测 (SIM)方式进行测定。主要的二萜类化合物 5F和 4F的准分子离子为 [M -H]- 1 ,发生源内分子离子碰撞活化解离 (CID)现象时 ,出现 [M -H]- 1 和 [M -H2 O -H]- 1 离子峰。采用 [M -H]- 1 为选择监测离子 ,以色谱保留时间和选择监测离子流峰面积定量 ,优选质谱条件和色谱条件。分析色谱柱为DiamonsilODS ,4 6mm× 15 0mm ,流动相为 3 0 %CH3CN 70 % 2mmol·L- 1 NH4 Ac,流速 1 0mL·min- 1 ,进样 5 μL。以测定半边旗中二萜类化合物 5F为例 ,对此方法进行了应用。结果 半边旗中二萜类化合物 5F为 1 18mg·g- 1 ,方法线性范围 0 0 5～ 2 5 μg,相关系数均优于 0 999,检出限达 0 4ng(进样 5 μL)。 5F的加标回收率为 97 8% (n =3 ) ,5F保留时间为 4 3min。结论 本法灵敏、准确、可靠 。

AIM To establish an accurate and reliable method for quantitative analysis of the diterpenoids in Pteris semipinnata L. METHODS A quadruple mass spectrometer coupled with atmospheric pressure chemical ionization interface was employed as a detector for HPLC. As to MS detector, selective ion monitoring (SIM) scan mode was used. For ent 11α hydroxy 15 oxo kaur 16 en 19 olic acid (5F) and ent 11α hydroxy 15 oxo kaur 16( R ) methyl 19 olic acid (4F), the majority of the diterpenoids in Pteris semipinnata L, the [M-H] -1 ion were observed, and the [M-H 2O-H] -1 ion could be observed from the collision induced dissociation spectua. [M-H] -1 was selected as the SIM ion in quantification, the mobile phase and the MS conditions were optimized. The mobile phase of HPLC was 30% CH 3CN 70% 2 mmol·L -1 NH 4Ac, analytical column was Diamonsil ODS (4 6 mm×150 mm), flow rate 1 0 mL·min -1 , inject volume 5 μL. The area of ion flow peak were used for quantitative determination. As an example of its application, this method was used to determine the content of 5F as an antitumor diterpenoid in Pteris semipinnata L. RESULTS The content of 5F accounted 1 18 mg·g -1 in Pteris semipinnata L sample. For 5F, RT is about 4 3 min, the standard curve showed good linearity over the range of 0 05～2 5 μg, γ=0 9998 ( n =5); the recovery was 97 8% ( n =5); the limit of detection was 0 4 ng (inject 5 μL ). CONCLUSION This method is highly sensitive,accurate and fast, which can be applied to study the antitumor drug of diterpenoids in Pteris semipinnata L and to establish the raw herb standard.