Quantification Of Adefovir, Dextrorphan, Zolmitriptan And Erdosteine In Plasma By Liquid Chromatography-tandem Mass Spectrometry

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. Four rapid and sensitive methods for quantitative analyses of adefovir, dextrorphan, zolmitriptan and erdosteine in plasma were developed and validated by liquid chromatography-tandem mass spectrometry in this thesis. The methods have been successfully applied to pharmacokinetic studies.1. Determination of adefovir in plasma by LC/MS/MSA sensitive and selective liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for the determination of adefovir in human plasma was developed. After a simple protein precipitation, the post-treatment samples were analyzed on a Diamonsil-C₁₈ column interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of methanol-water-formic acid (20: 80: 0.25, v/v/v). Selected reaction monitoring (SRM) using the precursor → product ion combination of m/z 274.1 → m/z (162.1+226.0) and m/z 288.1 → m/z 176.1 was used to quantify adefovir and PMPA, respectively. The linear calibration curves were obtained in the concentration range of 0.25-100 ng/mL for adefovir. The method has a lower limit of quantification (LLOQ) of 0.25 ng/mL for adefovir.The method was applied to a Phase I clinical trial of adefovir dipivoxil. After oral administration of increasing (low, medium, high) dose and multidose of adefovir dipivoxil, the plasma concentrations of adefovir were monitored by the developed sensitive and fast LC/MS/MS method. Mean plasma concentration-time curves of adefovir were best fitted to two-compartment models. The absorption of the drug was not significantly influenced by concomitant food intake.2. Direct determination of dextrorphan in human plasma by LC/MS/MSA sensitive and selective LC/MS/MS method for direct determination of dextrorphan in human plasma was developed and was used to study the pharmacokinetics of dextrorphan. After a single dose oral administration of dextromethorphan hydrobromide 60 mg to 18 healthy Chinese male volunteers, the plasma concentration of dextrorphan, an active metabolite of dextromethorphan, was determined. Dextrorphan and internal standard chlorpheniramine were extracted from plasma using liquid-liquid extraction, then separated on a Zorbax Extend Cig column. The mobile phase consisted of methanol-water-formic acid (70:30:1), at a flow-rate of 0.5 mL/min. A Finnigan TSQ 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 258 to 157 and m/z 275 to 230 was performed to quantify dextrorphan. The pharmacokinetic parameters of dextrorphan were calculated by non-compartment model statistics. The linear calibration curves were obtained in the concentration range of 0.2-80.0 ng/mL. Each plasma sample was chromatographed within 3.0 min. The intra- and inter-day relative standard deviation (RSD) across three validation runs over the entire concentration range was less than 8%. Accuracy determined at three concentrations (0.5, 6.0 and 70 ng/mL for dextrorphan) ranged from 98.8% to 100.6%. The method was applied to study the pharmacokinetics of 18 healthy volunteers after oral administration of 60 mg of dextromethorphan hydrobromide test formulation or reference formulation. Mean peak plasma levels (Cmax) of 13.5 ± 7.6 ng/mL and 13.6 ± 7.7 ng/mL and rmax of 2.1 ± 0.7 h and 2.0 ± 0.7 h were observed. The mean tin value of 3.8 ± 1.8 h and 3.8 ± 1.5 h were obtained, AUCo* values were calculated to be 60.3 ± 37.3 ngh/mL and 65.9 ± 44.3 ng-h/mL. The method is sensitive and convenient, and is proved to be suitable for clinical investigation of dextrorphan pharmacokinetics and bioequivalence evaluation of formulations containing dextromethorphan.3. Determination of zolmitriptan in plasma by LC/MS/MSA sensitive and selective liquid chromatography-tandem spectrometry method for the determination of zolmitriptan was developed and validated over the linearity range0.05-30 ng/mL with 0.5 mL of plasma using diphenhydramine as the internal standard. Liquid-liquid extraction using a mixture of diethyl ether and dichloromethane 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-water-formic acid (70:30:0.5), at a flow rate of 0.5 ml/min. In positive mode, zolmitriptan produced a protonated precursor ion at m/z 288 and a corresponding product ion at m/z 58. And internal standard produced a protonated precursor ion at m/z 256 and a corresponding product ion at m/z 167. The inter- and intraday precision (%RSD) were less than 8.5% and accuracy (%error) was between ±2.5%. The method had a lower limit of quantification of 0.05 ng/ml for zolmitriptan, which offered increased sensitivity and selectivity of analysis, compared with existing methods. The method was successfully applied to a pharmacokinetic study of zolmitriptan after an oral administration of 5 mg zolmitriptan to 22 healthy volunteers. After oral administration of 5 mg of zolmitriptan test formulation or reference formulation, mean peak plasma levels (Cmax) of 10.3 ± 5.8 ng/mL and 10.6 ± 5.4 ng/mL and Tmm of 2.1 ± 1.3 h and 2.6 ± 1.6 h were observed. The mean t\n value of 9.7 ± 2.9 h and 9.5 ± 3.7 h were obtained, A UCo-t were calculated to be 75.5 ± 35.8 ng ? h/mL and 76.9 ± 39.4 ng ? h/mL.4. Quantification of low level erdosteine in dog plasma using a reduced sample volume with a liquid-chromatography/tandem mass spectrometryErdosteine is an acidic compound, which can be used as a functional acid radical to form new salt, ambroxol erdosteinate. When the new salt used as a mucolytic drug, it showed better mucolytic effect and the dosage was much lower than erdosteine used singly.A sensitive and selective liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for the determination of erdosteine in dog plasma was developed and validated. Plasma proteins were precipitated by acetonitrile, followed by vortex mixing and centrifugation. Chromatographic separation was performed on a Cs column with a mobile phase consisting of 0.5% ammonia (aqueous), 1% formic acid (aqueous)and methanol at a flow-rate of 0.5 ml/min. MS detection using negative electrospray ionization (-ESI) as an interface was used in selective reaction mode (SRM) to monitor transition m/z 248 -> m/z 110 (erdosteine) and m/z 404 -> m/z 114 (lisinopril, internal standard). The method has a lower quantitation limit of down to 0.20 ng/ml and a lower consume of plasma only 25 \i\. Validation experiments have shown that the assay has good precision and accuracy over a wide concentration range (0.20²000 ng/ml), and no interferences caused by endogenous compounds were observed by matrix effect test. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 10.0%. The accuracy determined at three concentrations (0.5,25.0 and 1800 ng/ml for erdosteine) was within ±3.0% in terms of relative error.To investigate the pharmacokinetics of erdosteine in dogs, the LC/MS/MS method was applied for the determination of erdosteine in dog plasma. The absorption of erdosteine in dogs was rapid and complete. Individual plasma-concentration data were analyzed by compartmental analysis. Mean plasma concentration curves of erdosteine were best fitted to two-compartment models. The absolute bioavailability of erdosteine in dogs was 67.5%.