Study On The Pharmacokinetics And Bioequivalence Of Compound Betamethasone Injection In Healthy Volunteers

Objectives: To establish accurate and sensitive LC-MS/MS method for the determination of betamethasone (BOH) and betamethasone 17-monopropionate (B17P) in human plasma; To calculate the pharmacokinetics of BOH and B17P after single-dose intramuscular administration of compound betamethasone injection (containing 5 mg BDP and 2 mg BSP) in 22 Chinese healthy volunteers. The method was applied to evaluate the bioequivalence of compound betamethasone injection test product and reference product in Chinese healthy male volunteers.Methods: A LC-MS/MS method for quantification of BOH and B17P has been established. The human plasma added internal standard beclomethasone dipropionate was extracted by ether/cyclohexane (4:1, v/v), subsequently, the extraction was determined by LC-MS/MS. The column was C18 (Agilent Zorbax , 2.1 mm×150 mm,3.5μm). The mobile phase was water /methanol with the volume ratio of 15:85, and the flow rate was 0.20 ml per minute. Mass spectrometer was set to the data acquisition mode of multiple reaction monitoring mode(MRM). Positive ESI (ESI+) was chosen for B17P and internal standard, and negative ESI (ESI-) was chosen for BOH. The m/z 361→307 (BOH), 471→397(B17P) and 543→433 (IS) transitions were used in the present method.The main pharmacokinetic parameters of BOH and B17P obtained from 22 Chinese healthy volunteers after single 1-ml dose intramuscular administration of compound betamethasone injection were calculated, and the bioequivalence of two kinds of compound betamethasone injection were evaluated. 22 Chinese healthy male volunteers were randomly divided into two groups, serial blood samples (4 mL at each time point) were collected from vein vessels in the antebrachium prior to dosing (0 hour) and at 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, 48, 72, 120, 192, 264, 360 hours after dosing. Blood samples were collected in sodium heparin-containing tubes and immediately (within 40 seconds) mixed with 2 mol·L-1 concentration of sodium arsenate, an esterase inhibitor. The blood samples were then centrifuged at 4000 rpm for 5 min (4°C) to obtain plasma, which were then stabilized with 50% (w/v) sodium fluoride to inhibiting plasma esterase. Treated plasma samples were kept frozen at -65°C pending analysis for BOH and B17P. The concentrations of BOH and B17P in health volunteers'plasma samples were determined by the above LC-MS/MS method. The pharmacokinetic parameters (AUC, Cmax and Tmax) were calculated and the bioequivalence of two formulations were evaluated by BAPP 3.0 program.Results: 1. The linear range of BOH and B17P was 0.1～30 ng·ml-1 and 0.05～2 ng·ml-1, respectively. The intra-day and inter-day precision were less than 15%, the absolute recovery rate were more than 65%, the relative recovery rate were 85～120%, BOH and B17P in plasma samples were stable in several kinds of situations;2. The pharmacokinetic parameters of BOH were as follows: Tmax(1.8±1.2), (1.5±1.0)h, Cmax(15.06±3.91),(15.56±4.27)ng·ml-1, T1/2(14.65±3.65),(15.08±4.11)h, AUC0-t(203.87±50.04),(200.73±47.08)ng·h·ml-1, AUC0-∞(208.96±50.07),(206.61±48.57)ng·h·ml-1;3. The pharmacokinetic parameters of B17P were as follows: Tmax(23.4±13.0),(18.0±11.7)h, Cmax(0.77±0.25),(0.74±0.19)ng·ml-1, T1/2(88.67±17.78),(78.63±18.69)h, AUC0-t(67.38±15.17),(65.84±14.99)ng·h·ml-1, AUC0-∞(76.23±16.92),(72.79±15.69)ng·h·ml-1;4. Evaluation the parameters of Cmax,AUC0-t and AUC0-∞was made by analysis of variance,two one-sided test and [1-2α] confidence interval method. The results of statistical analysis showed that the two formulations had the same biological effects.Conclusion: The LC-MS/MS method built is simple, rapid, sensitive, and accurate. It was suitable for determination of drug concentration of BOH and B17P in human plasma. The bioequivalence of the two formulations of compound betamethasone injection may be identifiable based on the above results.