| M0,as a novel compound synthesized by the Fourth Military Medical University,have been testified to treat the ischemic stroke and to improve the neurological symptoms after stroke by pharmacodynamic experiments.Preliminary studies suggested that M0 rapidly metabolized into M3 in vivo.To characterize the pharmacokinetic characteristics of M0,it is necessary to establish a method for simultaneous determination of M0 and its metabolite M3 to reveal its metabolic law,which can provide experimental support for M0 new drug research.Hence,in th is study,a sensitivity and selectivity LC-MS/MS method was developed and validated to determine the concentration of M0 and its metabolite M3 in various biological samples including the whole blood,plasma and tissue.The result which we obtained was used to calculate the related preclinical pharmacokinetics parameters,investigate the relationship between dosage and parameters of pharmaeokinetics for M0 and M3 after intravenousinjection,evaluate cumulative effect of multiple dosesand observe the distribution of the M0 and M3 in the tissues,which provided the reference for clinical rational drug use.1.The development and validation of analytical method for M0 and M3 in biological matrixIn this paper,a quick,specific and sensitive liquid chromatography-tandem mass spectrometry(LC-MS / MS)method was used to determine the concentration of M0 and its metabolites M3 in biological samples.Due to the instability of M0 in biological samples,samples pretreatment were carried out by immediate treatment.Acidified biological samples were extracted using tert-butyl methyl ether.The samples were eluted on a CNW Athena C18 column(3?m,2.1×100mm)by using a gradient mobile phase system of methanol and water(containing 0.2% formic acid).The mobile phase entered the mass spectrum directly at a flow rate of 0.3 m L/min.The mass spectrometric detection was achieved using negative ion electrospray ionization in multiple reaction monitoring modes with the precursor-to-product ion transitions m/z 323.00?263.00(M0),m/z 197?135(M3),m/z 285.00?186.10(IS,4-Hydroxytolbutamide)and m/z 169.00?125.05(IS,Gallic acid)used for quantitation.There were no significant matrix effects in the quantitative analysis.The calibration curves were linear in each sample range with correlation coefficients above 0.99.The intra-and inter-day precision(RSD)ranged from 2.08%~12.19%.The accuracy(RE)was between-0.82%~9.60% at all quality control levels.The validated method was successfully applied to the pharmacokinetics study of M0 and M3 in biological matrix after intravenous injection of M0.2.The pharmacokinetics study of M0 and M3 in SD ratsThe validated LC-MS/MS method was used to investigate the quantification of M0 and M3 after a single intravenous dose of 30,60,120 mg/kg and a multi-dose intravenous of 60 mg/kg in SD rat.The pharmacokinetic parameters,based on the measured density data,were calculated with the non-compartmental model.The results indicated that the estimated elimination half-life(t1/2)of M0 was 0.073±0.01 h?0.268±0.10 h?0.194±0.11 h for 30,60 and 120 mg/kg doses,respectively.The AUC0~? of the three doses were 10694.432 ±2044.92 h×ng/m L?20402.950±2187.52 h×ng/m L?44609.515±8719.27 h×ng/m L,respectively,which was positive correlated with the doses and the correlation coefficient was 0.8894.M0 did not accumulate in the body after multiple doses of M0.As for M3,the estimated elimination half-life(t1/2)was0.20±0.03 h?0.34±0.07 h?0.65±0.63 h for 30,60 and 120 mg/kg doses,respectively.The AUC0~? of the three doses were 228.60 ±84.14 h×ng /m L?631.31 ±327.78 h×ng /m L ?1285.81±197.20 h×ng /m L,respectively,which was positive correlated with the doses and the correlation coefficient was 0.8111.M3 did not accumulate in the body after multiple doses of M0.3.The pharmacokinetics study of M0 and M3 in Beagle dogsThe validated LC-MS/MS method was used to investigate the quantification of M0 and M3 after a single intravenous dose of 8.9 mg/kg,17.8 mg/kg,35.6 mg/kg and a multi-dose intravenous of 17.8 mg/kg in Beagle dogs.the pharmacokinetic parameters,based on the measured density data,were calculated with the non-compartmental model.The results indicated that the estimated elimination half-life(t1/2)of M0 was 1.86±0.78 h?1.50±0.13 h ?2.97±0.49 h for 8.9,17.8 and 35.6 mg/kg doses,respectively.The AUC0~? of the three doses were 1459.67±422.45 h×ng /m L?4167.87±484.11 h×ng /m L?8686.83±1562.90 h×ng /m L,respectively,which was positive correlated with the doses and the correlation coefficient was 0.9137.M0 did not accumulate in the body after multiple doses of M0.As for M3,the estimated elimination half-life(t1/2)of M3 was 0.57±0.09 h?0.34±0.11 h?0.47±0.14 h for 30,60 and 120 mg/kg doses,respectively.The AUC0~? of the three doses were 53.35±18.18 h×ng /m L,89.17±27.76 h×ng /m L,246.04±32.01 h×ng /m L,respectively,which was positive correlated with the doses and the correlation coefficient was 0.9189.M3 did not accumulate in the body after multiple doses of M0.4.The tissue distribution study of M0 and M3 in SD ratsSD rats were killed by exsanguination at 5 min?10 min?15 min?30 min?1 h?1.5 h?2 h?3 h and 4 h after a single intravenous dose of 60 mg/kg.Tissues and blood were taken out immediately,washed with physiological saline to wipe off the blood on the surface.Then 0.2 g of tissues was weighed immediately and quickly.The tissues were placed into liquid nitrogen to frozen before transferring to-80 ? refrigerator.The concentrations of tissues of SD rats were determined by the validated LC-MS/MS method.The results showed that M0 was rapidly metabolized,and could not be detected at the first blood spots(5 min).Metabolites M3 rapidly reached the maximum plasma concentration and were widely distributed to blood flow-rich tissue organs with blood circulation.The concentration of M3 in kidney was the highest. |
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