An LC-MS/MS method for simultaneous determination of three Polygala saponin hydrolysates in rat plasma and its application to a pharmacokinetic study.
BACKGROUND
Radix Polygala has a long history of use as a sedative in traditional Chinese medicine and its major ingredients are saponins, which are recognized effective in memory improvement but highly toxic to gastricintestinal mucosa. Polygala saponin hydrolysates (PSH), an alkaline hydrolysis product and also the intestinal metabolites of the saponins, exhibited stronger effects in improving memory of mice and had less toxicity than its original saponins. The present study aims to develop a sensitive LC-MS/MS method for simultaneously determining PSH three major active components, 3,4,5-trimethoxycinnamylic acid (TMCA), p-methoxycinnamylic acid (PMCA) and tenuifolin (TF), in rat plasma and apply the method to a pharmacokinetic study.


METHODS
The acidic plasma (100μl) was treated by liquid-liquid extraction with ethyl acetate and reconstituted sample was analyzed on a C18 column eluted with acetonitrile-water (50:50) containing 0.2% formic acid at 0.4ml/min. The mass detection in negative electrospray ionization was used. The ion pairs for multiple reaction monitoring were set at m/z 237.0/103.0, 177.0/116.6 and 679.5/425.3 for TMCA, PMCA and TF, respectively. Their pharmacokinetic profiles were studied in rats after intravenous and oral dose of PSH at 20 and 100mg/kg, respectively.


RESULTS
The calibration curves had good linearity (r(2)>0.99) for TMCA, PMCA and TF within the tested concentration ranges. The limits of detection and quantification were 1, 10, 0.5ng/ml and 10.0, 20.0, 1.0ng/ml, respectively. The intra-day and inter-day precisions were less than 18.9% and accuracies between 93.2% and 113.3%, and the extraction recovery ranged from 91.2% to 112.1% for all analytes. The pharmacokinetic study showed that TMCA, PMCA and TF could be rapidly absorbed into the circulation and reached their peak concentrations at about 9.1, 9.0 and 24.0min, respectively. TF had a lower oral bioavailability (2.0%) than TMCA (90.1%) and PMCA (96.5%), but it remained in the body much longer (t1/2, λz, 4.8h, oral dose) than TMCA (0.6h) and PMCA (0.9h).


CONCLUSIONS
A sensitive LC-MS/MS method was developed and applied to a pharmacokinetic study of TMCA, PMCA and TF of PSH in rats. The three components are proved to be bio-available active components of PSH and might display their in vivo pharmacological activities at different levels and different time periods after oral administration.
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, PR China.
Qian
Q
Wang
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, PR China.
Bing-Xin
BX
Xiao
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, PR China.
Rui-Le
RL
Pan
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, PR China.
Xin-Min
XM
Liu
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, PR China.
Yong-Hong
YH
Liao
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, PR China.
Li
L
Feng
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, PR China.
Fang-Rui
FR
Cao
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, PR China. Electronic address: qchang@implad.ac.cn.
Qi
Q
Chang
D000818:Animals; D008297:Male; D051381:Rats; D002851:Chromatography, High Pressure Liquid; D002934:Cinnamates / Q000097:blood; D031801:Polygala / Q000737:chemistry; D012503:Saponins / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics; D053719:Tandem Mass Spectrometry
C031619:3,4,5-trimethoxycinnamic acid; D002934:Cinnamates; D012503:Saponins; C407311:tenuifolic saponin
Bioavailability; Glycyrrhizin acid (PubChem CID: 14982); Hydrolysates; LC-MS/MS; Pharmacokinetics; Polygala saponins; Tenuifolin (PubChem CID: 71307577); p-Methoxycinnamylic acid (PubChem CID: 699414)