HPLC-UV And UPLC-Q-TOF Analysis And Metabolism Study On Celastrol In Rats

Celastrol (Tripterine), presents in the root of Tripterygium wilfordii Hook. f. , a Chinese traditional medicine of Celastraceae plants. It has many biological activities such as anti-inflammatory, immunity suppression and anticancer. Scientists all over the world are paying close attention to its pharmacological action. In this study, high performance liquid chromatography (HPLC) method, ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) method were used to determine the concentration of celastrol in rat plasma after intravenous injection through caudal vein and intragastric administration. Its pharmacokinetics and biotransformation in rats'body were initially studied as well.Concretely, this dissertation is divided into four section:1. An HPLC method was established for the determination of celastrol in rat plasma. After iv administration and liquid–liquid extraction of the plasma with ethyl acetate, analytes were analyzed on a Unimicro C₁₈ column with an isocratic elution consisting of methanol–10% acetate acid (92:8, v/v), at a flow rate of 1 ml·min^-1. Ultraviolet detection was set at 425 nm, using trioxymethylanthraquinone as an internal standard. The assay was linear over the concentration range of 0.048 6 1.62 mg·L^-1. The main pharmacokinetic parameters of celastrol after i.v. (1 mg·kg^-1) were as follow: C_max 0.48μg·ml^-1, T_max 5 min, AUC_0-τ 23.44μg·ml^-1·min. This validated method for the quantification of celastrol in rat plasma is sensitive, accurate, precise, and is suitable for the pharmacokinetic studies of celastrol in rats.2. A rapid, sensitive UPLC-Q-TOF-MS assay has been developed to accurately measure the concentration of celastrol in rat plasma after intravenous injection through caudal vein and intragastric administration, which has been further used to study its pharmacokinetics. The quantitative and qualitative analyses were performed by UPLC-Q-TOF in negative mode, using trioxymethylanthraquinone as an internal standard (I.S.). The assay was linear over the concentration range of 0.024 3 0.81 mg·L^-1 in rat plasma with a limit of quantification of 0.024 3 mg·L^-1. This validated method for the quantification of celastrol in rat plasma is sensitive, accurate, precise, and is suitable for the pharmacokinetic studies of celastrol in rats.3. Under the proper chromatographic and mass spectrographic condition, celastrol dissolved in methanol was analyzed by UPLC-Q-TOF/MS/MS. By inspecting the fragments information, referring to the literatures of the fragmentation mechanisms of friedelane, a type of pentacyclic triterpenes, study the?fragmentation regularity from electrospray ionization mass spectra of celastrol with positive and negative mode.4. Using UPLC-Q-TOF/MS/MS instrument, the biotransformation and metabolite identification in the rat plasma after intravenous injection through caudal vein were initially studied. Rat blood samples were collected at 0, 15 min, 5 h and 24 h intravenous injection of 1 mg·kg^-1 celastrol, extracted with ethyl acetate and mixed solvent of methanol and acetonitrile, then analyzed by UPLC-Q-TOF with MSE function. Performed by comparing their changes in molecular masses (ΔM) , retention times and full scan MSE spectra with those of the parent drug and blank plasma, the metabolites and their structural eluciution were identified.To summerize, modern liquid chromatography and liquid chromatography-tandem mass spectrometry were utilized to analyze celastrol in rat plasma and to study its pharmacokinetic and biotransformation in rat body, so as to provide useful information in the course of its exploration as a potential medicine.