| Paclitaxel is a widely used taxane anticancer drug. Due to its poor water solubility, paclitaxel is generally formulated for clinical use in a mixture of medicinal solvents such as Cremophor EL and ethanol, which affect the curative effect and safety of the drug. It is becoming a hotspot to develop new preparations of paclitaxel with higher potency and lower toxicity in recent years and it also initiating various demands for qualitative and quantitative analysis, as well as identification of metabolitesIn this thesis, analytical methods of paclitaxel in human plasma were studied by LC-MS and LC-MS/MS as technology platform, separately. The quantitative methods of high sensitivity and specificity were optimized, developed and validated.Based on LC-MS technique, paclitaxel and norethindrone (used as internal standard, IS) were extracted from human plasma by a one-step liquid-liquid extraction with methyl-t-butyl ether. Separation on a Zorbax SB-C18column (100mm×2.1mm,3.5μm, Agilent) was achieved by gradient elution with methanol and0.2mmol/L ammonium formate containing0.1%formic acid. The selected-ion monitoring (SIM) targeted ions of [M+Na]+at m/z876.5for paclitaxel and [M+H]+at m/z299.4for IS. The assay was validated in the range1.0-400ng/mL (r>0.998) with LLOQ of1.0ng/mL, and no significant matrix effects. Intra-and inter-day precisions were all less than8.8%, with accuracies of±6.8%. The method was successfully applied to evaluate the pharmacokinetics of paclitaxel liposome for injection in patients.Based on LC-MS/MS technique, docetaxel was used as the internal standard and separation was achieved on a Shimpack XR-ODS column (75mm x3.0mm,2.2μm, Shimadzu) with a mobile phase of methanol-1.0mmol/L ammonium formate (73/27, v/v). Mass spectrometric detection in positive ion electrospray mode was performed by multiple reaction monitoring of the transitions at m/z854.2→286.1for paclitaxel and m/z808.2→226.0for docetaxel, respectively. The lower limit of quantitation was0.5ng/mL using50μL plasma. A linear dynamic range of0.5-500ng/mL was established. The intra-and inter-day precisions were within7.5%, while the accuracy was within±3.6%of nominal values. The method showed high throughput (within4min each sample) and greater sensitivity (0.5ng/mL) based on50μL sample volume and was suitable for the analysis of a large number of samples. The established method has been successfully applied to the analysis of samples from a phase I clinical trial of polymeric micelle formulated paclitaxel.The chromatographic and mass spectrometric conditions were carefully optimized. Besides, various additives were added to the solution to investigate the ionization way of paclitaxel and formation of adduct ions. It contributed to the enhancement of ionization efficiency and stability of MS response, satisfing the need for trace determination.The MS behavior of paclitaxel was collected by systemic study of the ionization and fragmentation in MS and MS2mode. The fragmentation procedure and characteristic fragment ions of paclitaxel were clarified. The structure of the metabolites in human body and metabolic pathway were proposed. The study provides solid academic evidence for qualitative and quantitative analysis by mass spectrometry and lays foundations for research on drug metabolism. |
PDF Full Text Request