| ObjectiveTo establish a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method for the simultaneous analysis of epimedin A, epimedin B, epimedin C and icariin in rat plasma and various tissues for pharmacokinetic, tissue distribution and excretion, after a single dose oral administration of epimedium extract. This study provides informative data for understanding the in vivo disposition of epimedium flavonoids, which, in turn, help in interpreting the mechanism of its effectiveness.MethodsPlasma and tissue samples were pretreated by protein precipitation with methanol. Urinary samples were pretreated by dilution with methanol. Genistein was used as the internal standard for plasma and tissue; while ginsenoside Rgl was used as the internal standard for urine. The chromatographic separation was carried out on an Agilent Eclipse XDB-C18 (2.1×150 mm,5μm) column. The mass analytes were performed in an API 4000+ triple-quadrupole mass spectrometer via multiple reaction monitoring (MRM) with negative scan mode. The MS/MS ion transition monitored were 897.6â†'675.5 m/z for epimedin A,867.6 â†'645.4 m/z for epimedin B,881.5â†'659.4 m/z for epimedin C and 735.7â†'513.5 m/z for icariin. The concentrations of epimedin A, B, C and icariin in rat plasma, tissue and urine were detected by LC-MS/MS, and mean concentration-time curves of epimedin A, B, C and icariin and main pharmacokinetic parameters of epimedin A, B, C and icariin in rat after a single oral administration of epimedium extract were obtained with DAS 2.0 program software.ResultsFor plasma samples, calibration curves (1/X2 weighted) offered satisfactory linearity (r>0.99) over the concentration range 1-500 ng·mL-The intra-day precision of this method was less than 8.7%, and the inter-day precision was less than 14.6%. After a single oral administration epimedium extraction (corresponding to 149.1 mg·kg(-1)pimedin C), the four favonoids were rapidly absorbed with T(max) of approximately 0.6 h. The AUC(0-1)of epimedin A, B, C and icariin were 25.641±17.162 ug/L·h,24.957+15.765 ug/L·h, 262.261+174.458 ug/L·h and 44.859±26.827 ug/L·h, respectively. While t, of these four favonoids were 1.308+0.561 h,1.700+1.172 h,1.535+0.793 h and 1.361±0.766 h, respectively. As for tissue samples, calibration curves (1/X2 weighted) offered satisfactory linearity (r>0.99) over the concentration range 1-500 ng ·mL(-1) The intra-day precision of this method was less than 8.4%, and the inter-day precision was less than 14.9%. After a single oral administration, the four flavonoids rapidly distributed within 1h to most of the tissue extensively, without significant accumulation or persistence. Besides, the calibration curves of urinary samples were linear in the concentrations range of 1-500 ng·mL(-1) for epimedin A, B, C and icariin in urine, respectively. The intra-day precision of this method was less than 8.4%, and the inter-day precision was less than 14.9%. The cumulative excretion of analytes in urine within 36 h of the dose were less than 0.266‰,0.198‰, 0.297‰ and 0.425‰, respectively.ConclusionThe validated assays were successfully applied to quantify plasma, tissue and urine concentrations of epimedin A, B, C and icariin in rats after a single oral administration of epimedium extract. The main PK parameters, the mean maximum concentration (Cmax) and the mean area under the concentration-time curve (AUC(o-t)), indicated that the flavonoids were rapidly absorbed and eliminated. However, the flavonoids can be absorbed little by the gastrointestinal tract. As for tissue distribution, the four flavonoids were mainly distributed in the liver, lung and female genital organs. It is indicated that these thress organs may be the target organs of these four flavonoids. Moreover, the result of excretion indicated that the analytes is hardly eliminated through urine within 36 h in rats, which suggested that renal excretion may not be the main elimination way. Biological transformation could occur in vivo. |
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