Pharmacokinetic Study Of Active Phenolics In Traditional Chinese Medicine By High Performance Liquid Chromatography With Chemiluminescence Detection

Chemiluminescence (CL) is defined as the emission of electromagnetic radiation produced by chemical reactions which yield one of the reaction products in an electronic excited state producing light on falling to the ground state. Because the emission intensity is a function of the concentration of the chemical species involved in the CL reaction, measurement of emission intensities can be used for analytical purposes. HPLC coupled to chemiluminescence (HPLC–CL) detection has become in the last years quite a useful detecting tool due to its high selectivity, high sensitivity, simplicity, and low cost. A wide variety of HPLC–CL methods have been developed for the clinical, pharmaceutical, environmental and food analysis. Active phenolics with a wide range of biological activity are main ingredient of most Traditional Chinese Medicine. In recent years, increasing attention have been attracted to active phenolics, however, the current research is mainly focused on the pharmacological effects of the phenolic compounds, while the pharmacokinetic study was relatively rare .In this study, novel and sensitive methods based on high-performance liquid chromatography coupled with chemiluminescence detection were developed for the quanittation of active phenolics of traditional Chinese medicine in biological specimens. And conbined with microdialysis sampling, the disadvantages of conventional sampling method was overcomed and dynamic monitoring of free phenolic compounds was achieved. The research work was illustrated in detailed as fellows:Part I. Determination of kaempferol in rat plasma by high-performance liquid chromatography with chemiluminescence detection and application to a pharmacokinetic studyA simple and sensitive method based on high-performance liquid chromatography (HPLC) coupled with chemiluminescence detection was developed for the quantitation of kaempferol (KA) in rat plasma. Isorhamnetin (IS) was used as an internal standard. Plasma samples were prepared only by acidification with 20% phosphoric acid and protein precipitation using methanol. Good separations of kaempferol and internal standard were achieved with an isocratic elution using a mobile phase consisting of methanol and aqueous 0.4% phosphoric acid (47: 53, v/v) within 25 min. The detection limit for kaempferol was 1.0×10-9 g·mL-1. The mean accuracy was within 80.0%– 100.2%, and the intra-day and inter-day precision had RSD (%) < 5.0. The sample preparation method was able to produce high recovery (≥80.0%). The proposed method with high sensitivity, wide linear range and simple pretreatment has been successfully applied to a pharmacokinetic study in SD rats after oral administration of 2500 mg·kg-1 BW or 1250 mg·kg-1 BW kaempferol. And the major pharmacokinetic parameters of Tmax, Cmax AUC0-∞, MRT0-∞and T1/2 were reported. For 2500 mg·kg-1 BW dosage group, Tmax = (1.21±0.46) h, Cmax = (232.90±5.14) ng·mL-1, AUC0-∞= (1728.41±57.31) ng·h·mL-1, MRT0-∞= (13.38±3.87) h, T1/2 = (9.27±1.84) h. For 1250 mg·kg-1 BW dosage group, Tmax = (1.08±0.43) h, Cmax = (165.67±1.99) ng·mL-1, AUC0-∞= (729.01±29.44) ng·h·mL-1, MRT0-∞= (4.77±2.06) h, T1/2 = (3.30±1.50) h.Part II. Determination of phenolic compounds of Salvia miltiorrhiza in rat blood and brain by liquid chromatography with chemiluminescence detection coupled to microdialysis sampling and pharmacokinetic studyStudy I Simultaneous determination of six water-soluble components in Salvia miltiorrhiza and its preparations by liquid chromatography with chemiluminescence detectionA novel method using high-performance liquid chromatography coupled with chemiluminescence detection for the simultaneous determination of six phenolic compounds of Salvia miltiorrhiza (danshensu, protocatechuic acid, protocatechuic aldehyde, caffeic acid, salvianolic acid B, and salvianolic acid A) in Salvia miltiorrhiza crude drug and preparations. Good separations of six water-soluble components were achieved on Shim pack ODS column (250×4.6 mm I.D. 5μm) with a gradient elution using the mixture of aqueous phosphoric acid (0.1%, v/v) and methanol phosphoric acid (0.1%, v/v) within 26 min. The detection limits (S/N = 3) for six water-soluble components were in the range of 0.03?6.72 ng·mL-1. The relative standard deviations (RSDs) of precision were below 2.5%. The mean recoveries for six water-soluble components in Salvia miltiorrhiza preparations ranged from 95.3% to 103.9%. The proposed method is highly selective and sensitive, reproducible, and suitable for the quality control of six water-soluble components in Salvia miltiorrhiza crude drug and preparations. Study II Determination of phenolic compounds of Salvia miltiorrhiza in rat microdialysates from blood and brain and pharmacokinetic study after administration of danshen injectionA novel and ultrasensitive method using high-performance liquid chromatography coupled with chemiluminescence detection was developed for the simultaneous determination of six phenolic compounds of Salvia miltiorrhiza (danshensu, protocatechuic acid, protocatechuic aldehyde, caffeic acid, salvianolic acid B, and salvianolic acid A) in rat microdialysates from blood and brain. The method is based on the chemiluminenscent enhancement by phenolic compounds of the HAuCl4–luminol–H2O2 system. Good separations of six phenolics were achieved with a gradient elution using the mixture of aqueous phosphoric acid (0.1%, v/v) and methanol phosphoric acid (0.1%, v/v) within 26 min. The detection limit for the six phenolics in in Ringer's solution (for blood and brain perfusion) were in the range of 0.07?12.8 ng·mL-1.The mean accuracies for six phenolics (spiked at three levels of ng·mL-1) in Ringer's solution (for blood and brain perfusion) ranged from 92.04%?101.4% with the relative standard deviations (RSDs) of precision below 6.5%. The proposed method has been successfully applied to the continuously monitoring of unbound danshensu for the pharmacokinetic study in SD rats'blood and brain with in vivo microdialysis sampling.Blood and brain microdialysis probes were inserted into jugular vein and brain hippocampus under anesthesia and perfused with ringer's solution at the rate of 2.0 and 0.8μL·min-1 respectively. Blank microdialysates were collected after 2 hours'post-implantation equilibrium time. Danshen injection was then administrated via caudal vein and blood and brain samples were collected at 15 and 40 min time intervals for 9 and 4 samples respectively. The in vivo recoveries of danshensu were (34.89± 0.06)% and (27.48±1.20) % in microdialysis probes of blood and brain, respectively (n = 5). Danshensu could be detected in both blood and brain after single intravenous administration of Danshen Injection (danshensu 3.5 mg·kg-1 BW). The pharmacokinetic parameters of t1/2, AUC0-∞, and MRT0-∞t1/2,α, t1/2,βin blood and in brain were estimated by 3p87 using statistical moment. And brain-to-blood (AUCbrain/AUCblood) distribution ratio of danshensu was as high as 0.25±0.07, which suggests danshensu is a potential brain protective agent. It is the first time to provide the pharmacokinetic profile of danshensu in brain as well as the blood-to-brain distribution ratio by high-performance liquid chromatography–chemiluminescence method.Study III Determination of phenolic compounds of Salvia miltiorrhiza in rat microdialysates from blood and brain and pharmacokinetic study after intragastrically administration of danshen extratA novel and ultrasensitive method using high-performance liquid chromatography coupled with chemiluminescence detection was developed for the simultaneous determination of three phenolic compounds of Salvia miltiorrhiza (danshensu, protocatechuic acid and protocatechuic aldehyde) in rat microdialysates from blood and brain. The method is based on the chemiluminenscent enhancement by phenolic compounds of the HAuCl4–luminol–H2O2 system. Good separations of the three phenolics were achieved on Shim pack ODS column (250×4.6 mm I.D. 5μm) with a isocratic elution using the mixture of aqueous phosphoric acid (0.1%, v/v) and methanol within 35 min. The linear range for danshensu, protocatechuic acid, and protocatechuic aldehyde were in the range of 2.29?179.0, 3.46?345.6, and 4.80?800.0 ng·mL-1, and the detection limit (S/N = 3) were 0.29,0.52 and 0.80 ng·mL-1 respectively. The mean accuracies for the three phenolics in Ringer's solution (perfusate) ranged from 95.0 to 102.9% with the relative standard deviations (RSD) of intra-day and inter-day precision below 5.5 %. The proposed method has been successfully applied to the continuously monitoring of unbound phenolics for the pharmacokinetic study in SD rats'blood and brain with in vivo microdialysis sampling. Microdialysis probe implantation was the same as study II. Blank microdialysates were collected after 2 hours'post-implantation equilibrium time. Danshen extract (danshensu 40 mg·kg-1 BW, protocatechuic aldehyde 149 mg·kg-1 BW) was then administrated intragastrically and blood and brain samples were collected at 15 and 30 min for 4 h respectively. Danshensu and protocatechuic acid could be detected in both blood and brain microdialysates, while protocatechuic aldehyde was not detected. The pharmacokinetic parameters of t1/2, AUC0-∞, and MRT0-∞t1/2,α, t1/2,βfor danshensu and protocatechuic acid in blood and in brain were estimated by 3p87 using statistical moment and their brain-to-blood (AUCbrain/AUCblood) distribution ratio were 0.25±0.04 and 0.09±0.02 respectively, suggesting DSS can readily permeate the blood brain barrier after oral administration of danshen extract and protocatechuic acid is a potential Oxidative metabolites of protocatechuic aldehyde.