| m-Nisoldipine (m-Nis) is a metamer of Nisoldipine,a new1,4-dihydropyridine derivative.Previous studies have shown thatm-Nis had the effect that to extend the cardio vessel and to treatcoronary heart disease,hypertension and chronic congestiveheart failure.Now,m-Nis is in the stage of preclinical-study andthe study about the pharmacokinetics of it following intravenousadministrations in dogs cann't be found.Objective: The purpose of this study is to establish aquantitative measurement for m-Nis being in the plasma ofbeagle dogs ,and the pharmacokinetics after intravenousadmistration of dogs was studied using this method.Methods: (1)The concentration of m-Nis being in the plasmawas assayed by the RP-HPLC method.Choose the bestchromatogram condition:Made sure of the ratio and the flowrate of the mobile phase and the temperature of thecolume,selected the best internal standard(IS) .(2)Thepreparation of the plasma samples: The plasma samples wereseparated by centrifugation at 3000 rpm for 5min, 1mol/LNaOH 0.4ml and the internal standard-Nimoldipine 200μl wereadded, vortexed 30s and then added extract solution(ether:hexane = 1:1 v:v) 5ml,vortexed 1min and thencentrifugated.The upper clear solution was transferred to a tubeentirely and dried by N2, 0.5ml ether was added to it and driedby N2 again. And then 60μl methanol was added to it andinjected 20μl of each sample into the instrument. (3)theselectivity of the method: Each of the m-Nis solution ,ISsolution ,blank plasma,plasma with m-Nis and IS,actual samplewas injected to the instrument and the chromatogram of themwas recorded respectively.(4) Determination of the lowestdetection limitation: A set of solution of m-Nis was injectedand the quantity of it when S/N ≥3 is the lowest detectionlimitation.(5) Preparation of standard curve : IS solution and aset of m-Nis solution was added to several blank plasma andwas injected to the instrument after treated.Standard crurve wasmade with the content of m-Nis as abscissa and the ratio of peakarea as ordinate and the regression equation was got .Make sureof the range of it.(6)to determine the absolute recoveries ofm-Nis: Fifteen shares of plasma samples containing low,middleand high concentration of m-Nis was prepared.Five sampleseach group.Injected and took record of them respectively. Theratio of plasma samples'peak area to the peak area of thesolution containing equal quantity of m-Nis was computed. (7)to determine the absolute recoveries of IS: Five shares ofplasma samples that each of them containing 200ng IS wasprepared as the above.Injected and took record of them. Theratio of plasma samples'peak area to the peak area of thesolution containing equal quantity of ISwas computed.(8) todetermine the method recoveries : Fifteen shares of plasmasamples was prepared as (5) and the concentration of them wascomputed through regression equation.Computed the tatio of thequantity assayed and the quantity added. (9)Precision: Fifteenshares of plasma samples was prepared as (5) and was assayedat different time in the same day to determine the within-dayprecision. Each of the low,middle,high concentration plasmasamples was prepared and assayed one share every dayrespectively .Prepared and assayed three days continuously anddetermined the day-to-day precision. (10)Stability: Nine sharesof plasma samples containing middle concentration of m-Niswas prepared and then they were assayed at 0,2,4,6,8h and1,3,5,7day respectively, the RSD of them was computed.(11)Freeze -melt repeatedly: Six shares of plasma samples wereprepared. Three of them were prepared as (10) and the else ofthem were prepared after freezeing and melting three times. wasThe difference of them was compared.(12)The reserve stabilityof the plasma samples: Compare the plasma sample assayed onthe first day with that assayed after reserve 45 days.(13)Pharmacokinetic experiment:Eighteen dogs which healthy andadult were divided into three groups randomly, six dogs eachgroup. A solution containing m-Nis was given through iv. todogs at the doses of 0.1 mg·kg-1 , 0.2 mg·kg-1and 0.5mg·kg-1respectively. Blood samples were collected from the vein ofhind legs after given drug at the prearranged time (5,10,15,20,30,60,90,120,180,360 min),and then they were drawninto tubes containing heparin sodium,respectively. The plasmawas Separated by centrifugation at 3000rpm for 5min. (14) Getthe pharmacokinetic parameters:The peak area of m-Nis andNim was record ,the ratio of them and the concentration ofm-Nis in the plasma were calculated.The original data wasanalysed by the 3P97 pharmacokinetics software. Make the bestcompartment model according to the least AIC and the biggestcorrelation coefficient obtained from real concentration.Results: (1)HPLC system: Waters chromatography system(a515 pump ,a 486 UV tunable absorbancdetedtor );MillenniumTM2010 chromagraphy workstation;Waters C18 column was used asan analytical column (300mm×3.9mm, 10μm); The mobilephase consisted of 64 volume of methanol and 36 volume ofwater at the flow rate of 1.0ml·min-1; The temperate of thecolumn was 30℃;The UV detection was set at 236nm; Thesensitivity was 0.05 AUFS and the volume of inject to theinstrument is 20μl. (2)Nimoldipine was choosen as the internalstandard substance.(3)suitabity test:m-Nis and Nim wereseparated good and not be interfered by the impurities.Theshape of peak were good .The retain time of m-Nis and Nimwere 18.5min ans 11.6min respectively.(4) The lowest detectionlimitation was 0.396ng.(5)The regression equation was Y =0.012525X + 0.005037 , (r=0.9999) ,and the standard curverange was 1.75~480ng·ml-1. (6)The average absolute recoveriesin three different concentrations (low,middle and high) were96.20%,84.66%,81.61% respectively and the value of RSDwere 1.6%,6.5% and 3.5% (n=5).(7)The average absoluterecoveries of Nim was 83.8% and the value of RSD of it was5.5%(n=5).(8)The average method recoveries in three differentconcentrations (low,middle and high) were 97.9%,100.4%,99.0% and the value of RSD of them were 3.2%,1.5% and 4.1%(n=5). (9)The values of RSD of within-day were 2.3%,1.0%,3.4%(n=5). The values of RSD of day-to-day were 2.6%,3.1%,1.8% ,respectively(n=5). (10)The value of RSD of the stabilityexperiment was 1.8%.(11)The average concentration of theplasma samples that melted one time and freeze-melted threetimes were 103.27±0.66ng/ml and 103.59±2.26ng/ml(n=3).(12)The average concentration of the plasma samples that detectedon the first day and detedted after 45 days were94.96±0.82ng/ml and 92.55±2.16 ng/ml, respectively(n=3).(13)The plasma concentration-time curves of m-Nis at differentdosages after intravenous administration were confirmed to beopen two compartment model in beagle dogs. The T1/2αwere6.32, 7.07and 16.0min, T1/2βwere 137, 149 and 136 min , V(c)were 4.4,6.7 and 10.3L, the CL were 0.20, 0.20, 0.30L/min,respectively. The AUC appeared to be dose-dependent.Conclusion:This study established the method to detect the... |
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