Detection Methods For Eight Drugs In Vivo And Their Application In Pharamcokinetic Research

Nowadays liquid chromatography-tandem mass spectrometry (LC-MS/MS), due to its high sensitivity and selectivity, has become a valuable technique in the determination of biological samples and in the pharmacokinetic studies. Ultra Performance Liquid Chromatography (UPLC) which was first made by Waters corporation(USA), this new category of analytical separation science retains the practicality and principles of HPLC while increasing the overall interlaced attributes of speed, sensitivity, and resolution. Eight drugs including batifiban, ilaprazole, mepta, nicorandil, sulindac, silibin, pseudoephedrine and cetirizine in human plasma were developed and validated by LC-MS/MS and UPLC in these thesis. The methods have been successfully applied to pharmacokinetic studies.1. Determination of batifiban in plasma by LC-MS/MSA sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of batifiban in human plasma was developed. After a solid phase extraction, the post-treated samples were analyzed on a Thermo HyPURITY C18 column(150*2.1mm, 5μm) interfaced with a triple quadruple tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitril: water: formic acid(60:40:0.1, v/v/v). Selected reaction monitoring (SRM) using the precusor→product ion combination of m/z 819→m/z (623+159 ) and m/z 833→m/z(645+159) was used to quantify batifiban and IS (eptifibatide), respectively. The linear calibration curves were abtained in the concentration range of . The method has a lower limit of quantification (LLOQ)of for batifiban.The method was applied to a PhaseⅠclinical trial of batifiban.After oral administratin of increasing(low, medium, high) dose and multidose of batifiban, the plasma concentrations of batifiban were monitored by the developed sensitive and fast LC-MS/MS method.2. Direct determination of nicorandil in human plasma by LC-MS/MS A sensitive and selective LC-MS/MS method for direct determination of nicordil in human plasma was developed and was used to study the pharmacokinetics of nicorandil. After a single dose intravenous injection aminstration of nicorandil 1mg to 12 healthy Chinese volunteers, the plasma concentration of nicorandil was determined. Nicorandil and internal standard buflomedil were extracted from plasma using liquid-liquid extraction, then separated on a Hypurity C18 column. The mobile phase consisted of acetonitrile-water-formic acid(60:40:0.1,v/v/v), at a flow-rate of 0.25mL/min. A Waters QuattroMicro API tandem mass spectrometer equipped with electrospray ionization source was used as detector and was operated in the positive ion mode. Selected reaction monitoring(SRM) using the precursor to product ion combinations of m/z 212 to (79+136) and m/z 308 to (140+237) was performed to quantify nicorandil and internal standard . The pharmacokinetic parameters of nicorandil were calculated by non-compartment model statistics. The linear calibration curves were obtained in the concentration range of 0.51-520 ng/mL. Each plasma sample was chromatographed within3.0min. The intra- and inter-day relative standard deviation(RSD) across three validation runs over the entire concentration range was less than 15%. Accuracy determined at three concentrations (1.02, 16.25 and 260 ng/mL for nicorandil) ranged from 98.8% and 100.6%. The method was applied to study the pharmacokinetics of 12 healthy volunteers after intravenous injection adminstration 1 mg of nicorandil. The method is sensitive and conventient, and is proved to be suitable for clinical investigation of nicorandil pharmacokinetics.3. Determination of metazinol in plasma by LC/MS/MSA sensitive and selective liquid chromatography-tandem spectrometry method for the determination of metazinol was developed and validated over the linearity range 0.29-292.5ng/mL with 0.5 mL of plasma using acetaminophen as the internal standard. Liquid-liquid extraction using MTBE was used to extract the drug and the internal standard from plasma. The mass spectrometer was operated under the selected reaction monitoring mode using the electrospray ionization technique. The instrument parameters were optimized to obtain 3.0 min run time. The mobile phase consisted of acetonitril-10mM ammonium formate water solution(70:30, v/v), at a flow rate of 0.25mL/min. In positive mode, metazinol produced a protonated precursor at m/z 234 and a corresponding product ion at m/z 234. And internal standard produced a protonated precursor ion at m/z 152 and a corresponding product ion at m/z 110. The inter- and intra-day precision(%RSD) were less than 15% and accuracy (%error) was between±4.5. The method has a lower limit of quantification of 0.29 ng/mL for metazinol, which offered increased sensitivity and selectivity of analysis, compared with existing methods. The method was successfully applied to a pharmacokintiec study of metazinol after an oral administration of 100 mg metazinol tablet to 12 healthy volunteers.4. Simulantance quantification of pseudoephedrine and cetirizine in plasma by LC-MS/MSA sensitive and selective liquid chromatography-tandem spectrometry method for the simulantance determination of pseudoephedrine and cetirizine was developed and validated over the linearity range 5.0-1000 ng/mL with 0.2 mL of plasma using tramadol as the internal standard. Direct prodein precipitation using acetronitril was used to extract the drug and the internal standard from plasma. The mass spectrometer was operated under the selected reaction monitoring mode using the electrospray ionization technique. The instrument parameters were optimized to obtain 5.0 min run time. The mobile phase consisted of 65% methanol and 35% water (contained 0.1% formic acid, 10 mM ammonium format, at a flow rate of 0.2 mL/min. In positive mode, pseudoephedrin produced a protonated precursor at m/z 166 and a corresponding product ion at m/z 148. cetirizine produced a protonated precursor at m/z 389 and a corresponding product ion at m/z 201. And internal standard produced a protonated precursor ion at m/z 264 and a corresponding product ion at m/z 246. The inter- and intra-day precision(%RSD) were less than 15%. The method has a lower limit of quantification of 5.0 ng/mL for pseudoephedrine and cetirizine, which offered increased sensitivity and selectivity of analysis, compared with existing methods. The method was successfully applied to a pharmacokintiec study of pseudoephedrine and cetirizine after an oral administration of a cetirizine and pseudoephedrine sustained-release capsules (including 120mg pseudoephedrine and 5 mg cetirizine) to 12 healthy volunteers.5. Direct determination of silibin in human plasma by LC-MS/MSA sensitive and selective liquid chromatography-tandem spectrometry method for the determination of silibin was developed and validated over the linearity range 0.64-325 ng/mL with 0.5 mL of plasma using VBE-1 as the internal standard. Liquid-liquid extraction using MTBE was used to extract the drug and the internal standard from plasma. The mass spectrometer was operated under the selected reaction monitoring mode using the electrospray ionization technique. The instrument parameters were optimized to obtain 3.0 min run time. The mobile phase consisted of acetonitril-10mM Ammonium Formate water solution (contained 0.1%formic acid) (50:50, v/v), at a flow rate of 0.3 mL/min. In negative mode, silibin produced a protonated precursor at m/z 481 and a corresponding product ion at m/z 125. And internal standard produced a protonated precursor ion at m/z 355 and a corresponding product ion at m/z 339. The inter- and intra-day precision(%RSD) were less than 15%. The method has a lower limit of quantification of 0.64 ng/mL for silibin, which offered increased sensitivity and selectivity of analysis, compared with existing methods. The method was successfully applied to a pharmacokintiec study of silibin after an oral administration of a 140 mg silibin capsules to 22 healthy volunteers.6. Determination of ilapraozle and its two metabolites in human plasma by LC-MS/MSAn analytical method based on liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has been improved and validated for the quantitative measurement of ilaprazole and it's two metablites in human plasma. Separation of analytes and the internal standard (IS) omeprazole was performed on a Thermo HyPURITY C18 column (150×2.1mm, 5μm) with a mobile phase consisting of 10mM ammonium formate water solution- acetonitrile (50:50,v/v) at a flow rate of 0.25 mL/min. The API4000 triple quadrupole mass spectrometer was operated in multiple reaction monitoring mode via positive electrospray ionization interface using the transition m/z 367.2→m/z184.0 for ilaprazole, m/z 383.3→m/z184.1 for ilaprazole sulfone, m/z 351.2→m/z168.1 for ilaprazole thiol ether and m/z 346.2→m/z198.0 for omeprazole. The method was linear over the concentration range of 0.23 -2400.00 ng/mL for ilaprzole, 0.05-105.00 ng/mL for ilaprazole thiol ether and 0.06 - 45.00 ng/mL for ilaprazole sulfone, respectively. The intra- and inter- day prcisions were all less than 15% in terms of relativestandard deviation (R.S.D), and the accuracy was within 15% in terms ofrelative error (R.E) for ilaprazole, ilaprazole sulfone and ilaprazole thiolether. The lower limit of quantification (LLOQ) was identifiable andreproducible at 0.23, 0.05 and 0.06 ng/mL with acceptable precision andaccuracy for ilaprazole, ilaprazole sulfone and ilaprazole thiol ether,respectively. The validated method offered sensitivity and wide linearconcentration range. This method was successfully applied for theevaluation of pharmacokinetics of ilaprazole and it's two metablites aftersingle oral doses of 5 mg ilaprazole to 12 Chinese healthy volunteers.7. Using UPLC to detect sulindac and its two metabolites in plasmaA Waters AcQURITY BEH C18 (50*2.1 mm, 1.7μm) column wasselected. The mobile phase was 20 mM ammonium acetate water solution(including 1%Acetic Acid). The column temperature was 30℃, thedetection wavelength was 325 nm. The subjects received 200 mgSulindac capsules. Sulindac capsules on first day morning after fastingover night. Then blood samples were collected at 0-36 hours after takingthe drug. Sulindac and it's two metabolites pharmacokinetics wasmeasured by UPLC with ultraviolet detection. Sulindac and it's twometabolites was retented within 5min, the peaks was in a good separate,and this method was very stable. In the FMO3 E308G and E158K linkagemutation, sulindac pharmacokintics parameter has significant difference,but not in sulidac sulfone and sulidac sulfide.