Determination Of Paclitaxel For Poly Dipeptide Paclitaxel

Objectives:Paclitaxel (PTX) is a potent anticancer drug that extracted from Yew. PTX is poorly water-soluble. Poly dipeptide PTX (PDP) is a macromolecular drug conjugate that links PTX-C13 with poly dipeptide. The molecular weight of PDP is range from 10 kDa to 40 kDa. PDP showed good antitumor activity and water solubility. The determination of PTX concentration is very necessary for the quality control of PDP. The approximate PTX concentration can be measured by the yield of the polymerization. But the concentration of PTX cannot be measured accurately, due to the complex MWs and structures of PDP. No paper, offered a chromatogram method for the analysis of PTX polymers, was published. The aim of the survey is to establish a method for the determination of PTX in PDP, both in vivo and in vitro. This study may also provide reference for the analysis of PDP in pharmaceutical researches, pharmacology researches, toxicology researches and clinical researches, etc.Methods:PTX in PDP is composed of free and conjugated formulations. Free PTX was extracted through a liquid-liquid extraction (LLE) method and measured by HPLC. Conjugated PTX was hydrolyzed by solutions with different pH and different kinds of proteases. The hydrolysis ratio was also estimated by compared with the theoretical value of PTX in PDP. Both PDP and PTX were unstable under the condition of pH> 13.0. The hydrolysis product was purified and identified. The concentration of PTX in PDP was calculated by its hydrolysis product, indirectly.Results:1. Determination of free PTX in PDP and the validation of the HPLC method.Free PTX of PDP was extracted with diethyl ether. HPLC method was established according to the Ch.p. The separation was achieved on a Thermo Hypersil GOLD column (4.6×150 mm,5μm). Elution was carried out using a mobile phase consisting of Acetonitrile-Water (50:50, v/v). UV detection wavelength was performed at 227 nm. The method is specificity, reproducible and suitable for the analysis of free PTX in PDP preparation. Free PTX in different batches of PDP were range from 21.6-79.2 μg per vial.2. The hydrolyze assay for conjugated PTX.Conjugated PTX was hydrolyzed by solutions with different pH (1.9-13.0) and different kinds of proteases (Bacillus thermophilus protease and Subtilisin protease). After compared with theoretical value, the enzymolysis ratio of Bacillus thermophilus protease and Subtilisin protease were defined as 10.57% and 12.49%, respectively. The hydrolysis ratio of conjugated PTX was up to 23.29% after hydrolyzed under the condition of pH 9.2. These results suggested that the reaction was not thoroughly or PTX was unstable under these conditions. After dissolved in solution with pH of 13.0, no PTX was detected in PDP which hint PTX was rapidly hydrolyzed.3. The purification and characterization for the hydrolysis product of PTX.The hydrolysis products were separated and concentrated.5.7 mg product was obtained and identified as (2R,3S)-N-Benzoyl-3-phenyl Isoserine, C13 chain of PTX (PTX C13). The molecular formula and MW were determined as C16H14NNa04 and 307.08, respectively.4. The establishment and validation of UPLC method for determination of PTX in PDP.PDP was dissolved in water and hydrolyzed by 0.2 mol·L-1 sodium hydroxide solution. The separation was achieved on a Waters BEH C18 column (4.6×150 mm,5μm). Elution was carried out using a mobile phase consisting of Acetonitrile-Water (10:90, v/v). UV detection wavelength was performed at 227 nm. The area deviation between PTX hydrolysis product and PTX C13 was-0.70%(nPTX=nPTx C13), which indicated that the hydrolysis reaction was thoroughly proceeded. This UPLC method is accurate, rapid and suitable for the analysis of PTX in PDP. PTX in different batches of PDP were range from 31.80-37.38 mg per vial.5. The development of UPLC-MS/MS methods for determination of PDP in plasma specimen.After mixed with human plasma, PDP and PTX were hydrolyzed by 0.2 mol-L’1 NaOH. The spiked samples were deposited by acetonitrile and detected by an UPLC-MS/MS method. The content of PTX in PDP was also measured by its hydrolysis product. The LLOQ is 10 ng·-mL-1, which is more sensitive than the UPLC method. PTX in different batches of PDP were range from 31.85-37.54 mg per vial.Conclusions:The HPLC, UPLC and UPLC-MS/MS methods established in this study were accurate, specific, rapid, and suitable for the determination of free and conjugated PTX in PDP. The PTX content in PTX polymeric formulations was calculated by its specific hydrolysis product. These methods may also be used for the determination of other PTX polymeric formulations.