Pharmacokinetic Study Of W198 And Its Metabolites

Part I Analysis method study of the determination of W198 in plasma and urinePurpose: To develop a sensitive, accurate and simple RPHPLC-MS/MS method for the quantitative analysis of W198 in human plasma and urine.Method: The plasma and urine sample was extracted with 3.5ml isopentanol-hexane(2:98), then separation on the Diamonsil C₁8 columns (150×4.6mm, 5μm). The mobile phase was methanol containing 0.02% triethylamine (formic acid to adjust pH to 7.4) with the flow rate 0.7 ml/min. A mass spectrometer equpied with electrospray ionization source was used as detector and operated in the positive ion mode. The analysis was performed using the peak area ratio of drug to internal standard.Result: The retention times of W198 and internal standard were 4.3 and 4.0 min, respectively. The standard curve in both plasma and urine was linear over the concentration range of 0.25?256μg/L . The lowest concentration of detection in plasma and urine were 0.25μg/L .The recovery of method in plasma was 100.11%-112.8%, both the intra-day and inter-day RSD were less than 6%. The recovery of method in urine was 101.08%-109.60%, both the intra-day and inter-day RSD were less than 6%.Conclusion: This method is simple, rapid, sensitive and accurate for determination of Wl98 in human plasma and urine Part II Pharmacokinetic of W198 in healthy volunteersPurpose: To study the pharmacokinetics of W198 in healthy volunteers.Method: Following iv administration, HPLC-MS/MS was used to determine W198 concentration in plasma and urine. Then make the concentration-time curve. Use DAS 2.0 program to choose the pharmacokinetic model,calculate the pharmacokinetic parameters and complete the pharmacokinetic investigation of W198.Result: The parameter of W198 were as follows : T_max were 0.5-2h, C_max were 24.793±8.749μg/L, 39.59±9.92μg/L, 64.313±13.93μg/L, t_1/2β was 52.199±10.263?62.882±6.977 h and AUC_0?t were 365.832±78.119μg/L·h, 688.152±159.91μg/L·h, 1096.284±271.849μg/L·h, for 10, 20 and 30 mg/m² iv administration of W198 respectively. The urine cumulative excretion rate of W198 was only 0.612±0.191%.Conclusion: The pharmacokinetics of W198 after single intravenous injection was fitted as a two compartment open model, and revealed a linear kinetic absorption probably in the range of 30mg/m² dose.Partâ…¢Study of W198 's metabolitesPurpose: To study the possible metabolites of W198 using HPLC-MS/MSMethod: The rats bile and urine samples were extracted and concentratedusing ethyl acetate. The possible metabolites were isolated by HPLC and theninfused into the API-3000 LC-MS/MS System for Q1 and MS2 scanning.Analyzing Q1 and MS2 figure, fragment ions were used as diagnostic ions toidentify structures of possible metabolites. When conditions permit,synthesize reference substance to elucidate its structure.Result: The construction of DW198 was confirmed. However, the structureof ion pairs (m/z 715/717, m/z 730/732) has not confirmed, pending furtherstudy. Conclusion: DW198 was found to be one of W198's metabolites.Part VI Pharmacokinetic of W198's metabolitesPurpose: To study the determination of W198's metabolites using a sensitive, accurate and simple RPHPLC-MS/MS method.Method: The plasma and urine sample was extracted with ethyl acetate after basic purification, then separation on the Diamonsil C₁₈ columns (150×4.6mm, 5μm). The mobile phase of 50mmol/L ammonium acetate-methanol (5:95, adjust to pH 6.6 with formic acid) was pumped at 0.6 ml/min through the column. A mass spectrometer equpied with electrospray ionization source was used as detector and operated in the positive ion mode. The analysis was performed using the peak area ratio of drug to internal standard. Use DAS 2.0 program to choose the pharmacokinetic model, calculate the pharmacokinetic parameters and complete the pharmacokinetic investigation of demethyl W198(DW198).Result: The retention times of DW198 and internal standard were 3.7 and 5.7 min, respectively. The standard curve in both plasma and urinewere linear were over the concentration range of 0.0625?16μg/L. The recovery of method in plasma were 98.66%-101.59%, intra-day RSD less than 7% and inter-day RSD less than 14%. The recovery of method in urine were 96.10%-103.30%, intra-day RSD less than 6% and inter-day RSD less than 7%, respectively. The maximum plasma concentration of DW198 appears within 50-98 h. Average C_max were 1.11±0.46μg/L. Statistical moment calculate AUC_0?t,, MRT_0?t,, VRT(0?t,,) were 166.07±57.50μg/L·h, 100.96±5.22hå’Œ2717.18±272.80h respectively. Compartment model T(1/2α,) T(1/2β,) V/F and CL/F were 58.96±16.99h, 69.32±0.00h, 13159.18±6520.88L/m² and 64.53±38.88L/h/m² respectively.Conclusion: This method is simple, rapid, sensitive and accurate for determination of DW198 in human plasma and urine. The pharmacokinetics of DW198 after single intravenous injection of W198 was fitted as a two compartment open model.