| Objective:1. To establish a sensitive and selective HPLC-MS method for the determination of M1, the main active metabolite of moguisteine in human plasma.2. To study the pharmacokinetics of moguisteine after oral administration of single-dose (200mg,400mg and600mg) and multiple-dose (200mg, three times daily [tid]) of moguisteine tablet.3. To establish HPLC-MS method for the determination of M1in human urine.4. To study excretion pharmacokinetics of Ml after oral administration of400mg moguisteine tablet.5. To analyze metabolites of moguisteine in rat and investigate the possible metabolic pathway of moguisteine.Methods:1. Plasma samples were analyzed by protein precipitation with acetonitrile. The analysis was performed on a Diamonsil(?) C18(2) column (150mm×4.6mm,5μm) with the mobile phase consisting of0.1%formic acid-acetonitrile (57:43, V/V, pH=3.0) at a flow rate of0.8mL-min-1. The pseudo-molecular ions [M+H]+(m/z312.2for M1and446.3for glipizide) were selected as the target ions for quantification in the SIM mode.2. Single-dose study:Thirty healthy Chinese subjects, were randomly assigned to three groups and to receive a single oral dose of200mg,400mg and600mg moguisteine tablet, respectively. Blood samples were collected before and0.25,0.5,0.75,1.0,1.25,1.5,2.0,3.0,4.0,5.0,6.0,8.0hours after administration. Multiple-dose study:Ten healthy Chinese subjects, were assigned to receive multiple oral doses of200mg moguisteine tablet (200mg, three times daily [tid]). Blood samples were collected on the morning of4th,5th,6th, and7th days before and0.25,0.5,0.75,1.0,1.25,1.5,2.0,3.0,4.0,5.0,6.0,8.0hours after administration on the morning of7th day, then were processed with the same method as the single-dose study. The HPLC-MS method was used to determine the concentration of M1in plasma to evaluate the pharmacokinetic characteristics and dose-dependent relationship. The main pharmacokinetic parameters were calculated by DAS2.0.3. An HPLC-MS method was established to determine the concentration of M1in urine to study the excretion pharmacokinetics of moguisteine. The HPLC-MS conditions were similar to the method in plasma. Urine samples were appropriately diluted with blank urine for analysis.4. The HPLC-MS method was used to determine the concentration of M1in urine to evaluate the excretion pharmacokinetics. The main pharmacokinetic parameters were processed by DAS2.0. Urine samples of subjects with400mg moguisteine tablet were collected at time zero (blank),0-1,1-2,2-3,3-4,4-6,6-8,8-10,10-12,12-24hours after administration.5. HPLC-QTOF-MS technology was employed to analyze the biological samples of rats after orally taking moguisteine and search possible metabolites. The Agilent Metabolite ID software was used to identify a series of metabolites. HPLC-MS/MS technology was used to study the mass fragmentation patterns of moguisteine in the positive ion mode by full scan and product ion scan in mass spectroscopy. The parent ion and fragment ion of moguisteine were confirmed. To investigate the possible positions of MS/MS fracture of moguisteine and summarize the fragmentation pattern in mass spectroscopy. After orally taking moguisteine, and then bile, blood, urine and feces samples of rats were collected. The biological samples were pretreated and analyzed by HPLC-MS/MS for the metabolites, and to detect their mass information and confirm the structures of metabolites.Results:1. The HPLC-MS method for the analysis of M1in plasma:Calibration curve was ranged from0.02to8μg·mL-1. Blank plasma didn’t produce matrix effect and ion suppression effect to M1and I.S. The extraction recovery in plasma was over90%. Both the inter-and intra-day precision values were less than10%. M1in plasma was stable after storage at-20℃for1,7or28days respectively, or after one or two freeze-thaw cycles, or after post-pretreatment sample (in plasma) placed at room temperature for8hours, with the RE of less than15%.2. After subjects taking single dose of200mg,400mg and600mg moguisteine tablet, the pharmacokinetic parameters were as follows:Tmax1.30±0.44h,1.35±0.21h and1.20±0.41h; AUC0～83.73±1.20μg·mL-1-h,7.02±1.22μg-mL-1·hand11.23±2.53μg·mL-1·h; t1/20.65±0.09h,0.88±0.20h and0.87±0.17h. The pharmacokinetic parameters of multiple-dose study were as follows:Tmax1.25±0.35h, AUC72～84.02±0.63μg·mL-1·h, t1/20.95±0.46h.3. The HPLC-MS method for the analysis of M1in urine:M1and I.S. peaks were free from any endogenous substances of urine sample. Calibration curve was ranged from0.05to8μg·mL-1. Blank urine didn’t produce matrix effect and ion suppression effect to M1and I.S. Both the inter-and intra-day precision values were less than10%. M1in urine was stable after storage at-20℃for1or7days, or after one or two freeze-thaw cycles, or after post-pretreatment sample (in urine) placed at room temperature for8hours, with the RE of less than15%.4. There was greater individual difference between ten subjects on the cumulative excretion amount of M1in urine over a24h interval, and the mean value was121.073±59.108mg. The mean cumulative excretion rate was32.990±6.106%. The urine excretion rate constant was0.433±0.037h-1and half-life time was1.613±0.132h.5. The possible metabolic pathway of moguisteine in vivo was investigated according to the speculation of metabolites. The results demonstrated that through biological transformation of moguisteine in rat, there were four possible metabolites (molecule weight as follows: M1311.1, M2327.1, M3297.1and M4325.1) in bile, blood, urine and feces of rats.Conclusion:1. After subjects oral administration of single-dose (200mg,400mg and600mg) and multiple-dose (200mg, three times daily [tid]) of moguisteine tablet, the results demonstrated that Cmax and AUC0-8of M1had good linear dose-dependent relationship. The pharmacokinetic process of M1fitted to two-compartment model with weight coefficient of1; a large amount of administered dose was excreted in urine within6h, and this suggested that moguisteine was cleared quickly in vivo; minor stomach discomfort was observed from some subjects during the study, but the trial was well done and all subjects were in good compliance.2. Moguisteine was transformed extensively in rat, and four metabolites was discovered. This method was very sensitive, specific, rapid and suitable for on-line qualitative analysis of the metabolites of moguisteine. |
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