| Xiyanping injection is produced in Jiangxi Qingfeng Pharmaceutical CompanyLimted,which is andrographolid and sulfuric acid、 ethanol heating reactionpreparation of multi-component injection. The character of Xiyanping injection isfaint yellow to orange-yellow. It can invigorate the heat clearing and detoxicating,relieving cough and dysentery, clinical mainly used for bronchitis、tonsillitis、bacterialand dysentery.9-dehydro-17-hydro-andrographolide(DHA) and sodium9-dehydro-17-hydro-andrographolide-19-yl sulfate(DHAS) are main active constituent ofXiyanping injection, the concentration of DHA or DHAS is0.8mg/mL. In vitroexperiment, they have bioactivities in anti-microbial, anti-virus, anti-inflammation.There have many reports in Xiyanpimg injection for the past few years. There havebeen no reports of the pharmacokinetic and tissue distribution of Xiyanping injectionand active constituents in the relevant journal.AIM: To develop two simple and sensitive methods by HPLC-ESI-MS/MS forthe determination of9-dehydro-17-hydro-andrographolide and sodium9-dehydro-17-hydro-andrographolide-19-yl sulfate in rat plasma and tissues. The methods wereapplied to study the pharmacokinetics and tissue distribution of9-dehydro-17-hydro-andrographolide and sodium9-dehydro-17-hydro-andrograp-holide-19-ylsulfate after intravenous administrations in rats. To provide basis for using clinicalrational dug and developing new drug.METHODS:9-dehydro-17-hydro-andrographolide and diethylstilbestrol (inte-rnal standard,IS) were extracted from biosample by liquid-liquid extraction withmethanol and then separated on a Zorbax SB-C18(2.1×150mm I.D.,1.8μm）equipped with a Agilent HPLC In-Line Filter using a mobile phase of15:85(v/v)acetonitrile:water(ccontaining0.1%acetonitrile and5mM ammonium acetate). Theelution program was as follows:15%acetonitrile (initial),15-80%acetonitrile(3.5min),80-15%acetonitrile (0.1min), and15%acetonitrile (1.4min). The flow ratewas0.4mL/min and the injection volume was5μ L. Mass spectrometric detectionwas performed on a triple-quadrupole mass spectrometer (API4000). Turning the ion spray, operated in the negative ionization mode, MRM(multiple reaction monitoring)transitions were m/z349.1'm/z286.9for9-dehydro-17-hydro-andrographolide andm/z267.1'm/z236.9for diethylstilbestrol.sodium9-dehydro-17-hydro-andrographolide-19-yl sulfate and Andrographolidesodium bisulfite (internal standard, IS) were extracted from biosample by liquid-liquid extraction with methanol and then separated on a Zorbax SB-C18(2.1×150mmI.D.,1.8μm）equipped with a Agilent HPLC In-Line Filter using a mobile phase of0:100(v/v) acetonitrile:water(ccontaining0.1%acetonitrile). The elution programwas as follows:100%water(initial),100%water(2.0min),100-30%water(1.0min),30%water(3.0min) and30-100%water(0.1min). The flow rate was0.4mL/min andthe injection volume was5μ L. Mass spectrometric detection was performed on atriple-quadrupole mass spectrometer (API4000). Turning the ion spray, operated inthe negative ionization mode, MRM(multiple reaction monitoring) transitions werem/z428.9'm/z96.0for sodium9-dehydro-17-hydro-andrographolide-19-yl sulfateand m/z413.0'm/z79.9for andrographolide sodium bisulfite.Plasma、 tissue samples and internal standard solution were extracted bymethanol. After vortexing and centrifuging, the supernatant fluid was filtered into ainjected vial and the aliquot was injected into the HPLC-ES-MS/MS for analysis.After intravenous administration to SD rats through the caudal vein, serial bloodsamples were drawn in heparinized centrifuge tubes from the eye veins of rats atdifferent times and the tissues (heart, liver, spleen, lung, kidney, stomach andintestines) of the rats were harvested. Drug concentration in rat plasma and tissueswere determined by the validated HPLC-ES-MS/MS methods. The mainpharmacokinetic parameters were calculated and metabolic rules were discussed.Drug concentration were compared in different tissues and tissues distribution studywere discussed.RESULTS: Under these chromatographic conditions described above, the peaksshape of measured drug were well separated.9-dehydro-17-hydro-andrographolide:the lower limit of detection (LLOD) of was0.1ng/ml and the lower limit ofquantification (LLOQ) of was0.5ng/mL, the linearity of the method was0.5-300ng/mL, R2≥0.9974, intra-and inter-day precision (as relative standard deviation, R.S.D.) were <7%, stability were80-115%, recovery were80-110%. Afterintravenous administration of5mg/kg9-dehydro-17-hydro-andrographolide injectionto rats through the caudal vein, the pharmacokinetic parameters were calculated bydrug and statistics (DAS3.0) software. Cmax:765.8±40.5ng/mL, Tmax:5.8±2.0min,t1/2α:7.1±2.5min, t1/2β:27.5±15.1min, AUC0-t:942.302±476.648ng·h/mL, AUC0-∞:1018.227±484.677ng·h/mL. The9-dehydro-17-hydro-andrographolide underwent arapid and wide distribution in tissues. All of the analyzed tissues contained a certainamount of9-dehydro-17-hydro-andrographolide. Kidney had the highest tissueconcentration of9-dehydro-17-hydro-andrographolide in5min after administration,followed by heart, liver, lung, large intestines, spleen and stomach. Large intestineshad certain concentration in every time after administration.Sodium9-dehydro-17-hydro-andrographolide-19-yl sulfate: the lower limit ofdetection (LLOD) of was0.1ng/mL and the lower limit of quantification (LLOQ) ofwas0.5ng/mL, the linearity of the method was0.5-1000ng/mL, R2≥0.9840, intra-and inter-day precision (as relative standard deviation, R.S.D.) were <5%, stabilitywere78-100%, recovery were80-105%. After intravenous administration of5mg/kgsodium9-dehydro-17-hydro-andrographolide-19-yl sulfate injection to rats throughthe caudal vein, the pharmacokinetic parameters were calculated by drug andstatistics (DAS3.0) software. Cmax:5019.167±1046.931ng/mL, Tmax:4.98±1.0min,t1/2α:6.0±2.5min, t1/2β:18.1±9.4min, AUC0-t:4669.427±1968.581ng·h/mL, AUC0-∞:5300.826±1772.722ng·h/ml. The sodium9-dehydro-17-hydro–andrographolide-19-yl sulfate underwent a rapid and wide distribution in tissues. All of the analyzedtissues contained a certain amount of sodium9-dehydro-17-hydro-andrographolide-19-yl sulfate. Kidney had the highest tissue concentration of sodium9-dehydro-17-hydro-andrographolide-19-yl sulfate in5min after administration, followed bylarge intestines, liver, lung, heart, spleen and stomach. Kidney and large intestineshad certain concentration in every time after administration.Conclusion: Two specific and sensitive methods was fully validated for thedetermination of9-dehydro-17-hydro-andrographolide and sodium9-dehydro-17-hydro-andrographolide-19-yl sulfate in rat plasma and tissues by LC-MS/MS. Verygood accuracy, precision and recovery were obtained and no significant matrix effect was observed.The proposed methods were successfully applied to the pharmacok-inetics and tissues distribution studies of9-dehydro-17-hydro-andrographolide andsodium9-dehydro-17-hydro-andrographolide-19-yl sulfate after intravenous admin-istration. the pharmacokinetics parameters and the tissues distribution character isticsof rats provide basis for using clinical rational dug and developing new drug. |
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