| Objective Specific,sensitive and convenient methods for simultaneous quantitative levosimendan and its metabolites OR-1855 and OR-1896,pseudoephedrine and fexofenadine of multi-component drugs in plasma will be developed and validated by liquid chromatography-tandem mass spectrometry in this thesis.These methods will be applied to pharmacokinetic studies.Method one Determination of levosimendan,OR-1855 and OR-1896 in human plasma by liquid chromatography-tandem mass spectrometry: according to the different nature of compounds,two sets of liquid chromatography systems and ionization modes were used for determination the concentration of levosimendan and its metabolites OR-1855 and OR-1896 in human plasma,respectively.Following protein precipitation with methanol,the levosimendan and internal standard(rosuvastatin) were separated on a Capcell MGⅢC18 column(35×2.0 mm I.D.,3μm) with the mobile phase consisted of methanol-15 mmol·L-1 ammonium acetate-formic acid(55:45:0.02,v/v/v).A tandem mass spectrometer equipped with electrospray ionization source was used as the detector and operated in the negative ion mode.Its metabolites OR-1855,OR-1896 and internal standard doxofylline were extracted from plasma by liquid-liquid extraction with ethyl acetate.Chromatographic separation was performed on a Zorbax Extend C18 column(150×4.6 mm I.D.,5μm) with the mobile phase consisted of methanol-15 mmol·L-1 ammonium acetate-formic acid (65:35:0.1,v/v/v).A tandem mass spectrometer equipped with electrospray ionization source was used as the detector and operated in the positive ion mode.Method two Simultaneous determination of pseudoephedrine and fexofenadine in human plasma by liquid chromatography-tandem mass spectrometry:samples spiked with the analytes and the internal standard, diphenhydramine were processed using liquid-liquid extraction.The extract was chromatographed on a Diamonsil C18 column(200×4.6 mm I.D.,5μm) with the mobile phase consisted of methanol-water-formic acid(75:25:0.2,v/v/v).A tandem mass spectrometer equipped with a electrospray ionization source was used as the detector andwas operated in positive ion mode.Selected reaction monitoring(SRM) using the precursor/product ion combinations of m/z 166→m/z 115;m/z 502→m/z (171,466) and m/z 256→4 m/z 167 were used to quantify pseudoephedrine, fexofenadine and diphenhydramine,respectively. Results The linear concentration ranges of the calibration curves for levosimendan and OR-1855 and OR-1896 were 0.10-50.0 ng·mL-1, 0.20-100 ng·.L-1,0.20-100 ng·mL-1,respectively.The lower limits of quantification of levosimendan and OR-1855 and OR-1896 were 0.10 ng·mL-1,0.20 ng·mL-1,0.20 ng·mL-1,respectively.The linear concentration ranges of the calibration curves for pseudoephedrine and fexofenadine were 0.50-800 ng·mL-1 and 1.00-800 ng·mL-1,respectively.The method has a lower limit of quantification(LLOQ) of 0.50 ng·mL-1 and 1.0 ng·mL-1 for pseudoephedrine and fexofenadine,respectively.Conclusion The methods proved to be sensitive,simple and rapid,and suitable for the pharmacokinetic studies of levosimendan injection, pseudoephedrine and fexofenadine sustained release tablets.
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