Study On The Distribution Of Main Components Of Kudiezi Injection In Rat Brain And Brain Tissue

Kudiezi injection has been playing an important role in treatment of cardiovascular and cerebrovascular diseases. However, it is not clear about the effective constituents. Since the consitituents absorbed in target organ impact the effect of Kudiezi injections. Therefore it’s necessary to develop a sensitive method for the quantification of trace components in biological sample by LC-MS. Firstly, based on LTQ-Orbitrap, a rapid method was established to screen and identify the consitituents in rat brain and heart after an intravenous administration of Kudiezi injection. Secondly, a sensitive and rapid method was developed to determine four flavones in Kudiezi injection by UPLC-ESI-MS/MS. Finally, the determined flavones were used to study pharmacokinetics of Kudiezi injection in rat plasma, brain and heart.The main research contents and results are as follows:1. Identification of consitituents in rat brain and heart after an intravenous administration o f Kudiezi injectionThe consitituents in rat brain and heart were identified after an intravenous administration of Kudiezi injection detected by full scan-dynamic exclusion and parent ion list method using HPLC-LTQ-Orbitrap. According to the high resolution information data, molecular formulae of compounds were predicted. As a result, five consitituents were screened and identified in normal rat brain, sixteen consitituents in MCAO model rat brain and twenty-five consitituents in nomal rat heart based on the fragmentation information characteristics of different compounds.2. Rapid determination of four flavones in Kudiezi injection by UPLC-ESI-MS/MSIn this study, a method was developed for the rapid determination of four flavones in Kudiezi injection by UPLC-ESI-MS/MS. The analysis was performed on a Waters ACQUITY BEH C18 column (2.1 mm×50 mm,1.7 μm) with the mixture of acetonitrile-water as the mobile phase at a flow rate of 0.4 mL·min-1, the column temperature was 40 ℃. Multiple reaction monitoring (MRM) scanning was employed for quantification in ESI negative ion mode. Complete separation was achieved within 3 min for the four compounds. The regression equations showed good linear relationships within their respective linear ranges. The established analysis method was confirmed, and the results satisfied with the requirement of related regulations.3. A quantitative method was developed and validated for the simultaneous determination of four flavones of Kudiezi injection in rat plasma, brain and heart.In this study, a simple, rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) was developed for the simultaneous determination of four flavones from Kudiezi injection in rat plasma, brain and heart. The analysis was performed on a Waters ACQUITY BEH C18 column (2.1 mm×50 mm,1.7 μm) with the mixture of acetonitrile-water as the mobile phase at a flow rate of 0.4 mL·min-1, the column temperature was 40 ℃. Multiple reaction monitoring (MRM) scanning was employed for quantification in ESI negative ion mode. The regression equations showed good linear relationships within their respective linear ranges. The established analysis method was confirmed, and the results satisfied with the requirement of related regulations.4. Pharmacokinetic study of Kudiezi injection in rat plasma, brain and heart.The UPLC-MS/MS method was successfully applied in a pharmacokinetic study of Kudiezi injection in rat plasma, brain and heart. Pharmacokinetic parameters were performed using Phoenix WinNonlin Ver.6.3. The results demonstrated that the four compounds were eliminated quickly in rat plasma and has a strong ability to combine with organization. The flavonoid glycosides metaclass are more likely to get into the heart and brain tissue than flavonoid glycosides. With plasma, brain and heart concentration-time profiles, the study achived the main pharmacokinetic parameters of Luteolin, Luteolin-7-O-β-D-glucoside, Apigenin, Apigenin-7-O-β-D-glucoside to reflect the pharmacokinetic process of Kudiezi injection in rat.