Effect Of Sarpogrelate Hydrochloride On The Pharmacokinetics Of Propafenone Hydrochloride In Rats

Objective : To develop a HPLC method for the determination of propafenone hydrochloride in rat plasma. The rats were randomly divided into two groups including control group in which the rats were given distilled water + propafenone hydrochloride and treatment group where the rats received sarpogrelate hydrochloride + propafenone hydrochloride. After administration, the concentration ofpropafenone hydrochloride in plasma was measured. Then the pharmacokinetic characteristics of propafenone hydrochloride in two groups were profiled and compared, the impact of sarpogrelate hydrochloride on the pharmacokinetics of propafenone hydrochloride in rats was studied.Methods:1 Drug delivery route and plasma samples collecting: 38 male Wistar rats were randomly divided into two groups. The experiment group was given 10mg/kg of sarpogrelate hydrochloride for consecutive 5 days, 3 times a day. While, equal volume of distilled water was given to the control group, 3 times a day continuously for 5 days. On the sixth day, 9mg/kg of propafenone hydrochloride was given. Plasma samples were collected in different time points and saved under-40℃.2 Plasma samples processing: 200μl plasma was added 20μl internal standard(6μg/ml of voriconazole) and 100μl borax buffer solution(p H 9.0). After mixing thoroughly, 800μl ether was added, then vortex for 1minute. After centrifugation at 10900 rpm, the supernatant was dried under the nitrogen gas flow in 40℃water bath. The residue was dissolved in 100μl methanol solution and 20μl was injected into HPLC for analysis.3 Chromatographic conditions: The samples were separated by Symmetry Shield RPC18 chromatographic column(250 mm×4.6 mm,5 μm)and detected by Waters 486 UV detector;Mobile phase consisted of methanol, Potassium dihydrogen phosphate and phosphate buffer solution,and triethylamine(64:36:0.144,V/V); Flow rate: 1 ml/min; Detection wavelength: 248 nm;Column temperature: 30℃;Sample: 20μl;Internal standard: voriconazole.4 The lowest limit of quantification(LLOQ) and limit of detection(LOD): LLOQ and LOD were detected by double dilution methods.5 The preparation of standard curve: The simulated plasma samples of propafenone hydrochloride were respectively made at the concentrations of 0.125, 0.25, 0.5, 1, 2, 4, 8 μg/ml, then processed for injection. The standard curve was constructed by the regression of the peak area ratio of propafenone hydrochloride and IS(Y) and plasma drug concentration(X).6 Recovery: The QC samples at concentration of 0.25, 1, 6.4μg/ml(n=5) were prepared and processed for injection. The extraction recovery rate of propafenone hydrochloride was the peak area ratio of propafenone hydrochloride in plasma to the equal amount of propafenone hydrochloride in methanol. The extraction recovery rate of internal standard was also investigated as above. The ratio of the measured concentration and theoretical concentration was calculated as the method recovery.7 Precision: The intraday and interday variations(RSD) were determined by analysis the QC samples in one day or consecutive 5 days.8 Stability: The placement stability after treatment was measured at 0, 1, 2, 3, 4h after processing of the 1 μg/ml propafenone hydrochloride simulated plasma samples. The low temperature preservation stability was measured by comparing the concentrations of 1ug/ml propafenone hydrochloride simulated plasma samples after immediately processing or after 15days’ preservation in-40 ℃without light. The repeated freeze-thaw stability was measured by comparing the concentration of propafenone hydrochloride simulated plasma samples after immediately processing and repeated freeze-thaw 3 times.9 Pharmacokinetics: Process the rat plasma samples according to the plasma sample treatment method, then detect and calculate the plasma drug concentration according to the standard curve equation. Plot the rat drug dose-time curves and calculate pharmacokinetic parameters with DAS2.1.1 pharmacokinetic software. Statistical analysis was carried out on the parameters of the two groups to investigate whether there was significant difference.Results:1 The retention time of propafenone hydrochloride was 8.1 min and the retention time of voriconazole was 11.3 min. Plasma-derived substances had no interference on the determination.2 Standard curve equation: Y = 1.1789 X + 0.0685, correlation coefficient was R2=0.9998. Propafenone hydrochloride had a good linear relationship in the range of 0.125~8.0 μg/ml concentrations. The lowest limit of quantitation was 0.125μg/ml, the detection limit was 0.05μg/ml.3 Recovery: Method recovery of the low, middle and high concentrations of propafenone hydrochloride plasma samples were(96.50±6.75)%,(102.33±2.81)% and(106.70±0.78)%, RSD values were 7.0%, 2.8% and 0.7%; extraction recovery of the low, middle and high concentrations of propafenone hydrochloride plasma samples were(73.71±6.59)%,(82.45±5.86)% and(87.37±2.63)%, RSD values were 8.9%, 7.1% and 3.0%; extraction recovery of internal standard was(69.71±1.18)%, RSD values was 1.7%.4 Precision: The intraday precision results showed the RSD values in low, middle and high concentrations of plasma samples were 7.0%, 2.8% and 0.7%; the RSD values of interday precision were 5.9%, 1.1% and 4.3%.5 Stability: Plasma samples stay stable under the conditions of freezing for 15 days or repeated freeze-thaw three times, the RSD values were 1.9% and 5.7%; treated plasma samples were stable after placement for 4h, it had no effect on the determination, RSD is 7.1%.6 The pharmacokinetic parameters of propafenone hydrochloride in control group and experimental group were as follows: t1/2 were(2.50±1.41) h and(2.02±0.77) h; AUC(0-t) were(4.03±0.74) mg·h/L and(5.40±2.20) mg·h/L; AUC(0-∞) were(5.36±1.73) mg·h/L and(6.61±2.81) mg·h/L; CLz were(1.85 ± 0.58) L/h/kg and(1.57 ± 0.56) L/h/kg; Cmax were(3.31±0.76) mg/L and(3.95±1.78) mg/L; Vz were(6.00±2.92) L/kg and(4.31±1.68) L/kg.Conlusions:1 A HPLC method was developed for the determination of propafenone hydrochloride in rat plasma. This method enabled to detect the propafenone hydrochloride and internal standard with good separation. Endogenous impurities in plasma had no effect on the determination results. Plasma samples can keep stable after preservation for 15 days or through repeated freeze-thaw three times. Plasma samples placed 4h after treatment had no effect on the determination. The method was simple, accurate, reproducible and suitable for determination of the concentrations of propafenone hydrochloride in rat plasma.2 Rats were treated with placebo or drug hydrochloride ester reaches a steady state given propafenone hydrochloride drug pharmacokinetic parameters AUC(0-t), AUC(0-∞) and Cmax of 90% confidence intervals are exceeded, there was a significant difference. Therefore, The experimental results of sarpogrelate hydrochloride of propafenone hydrochloride in rats in vivo pharmacokinetics had an impact, the metabolism of propafenone hydrochloride slowed down.