Determination Of Febuxostat In Human Plasma By LC/MS/MS And The Pharmacokinetics Of Febuxostat Tablet In Chinese Healthy Adults

Objective: It is to establish a sensitive,rapid,accurate and stable LCMS/MS method for the determination of febuxostat in human plasma and investigate the pharmacokinetics of febuxostat tablet in Chinese healthy adults.Methods:According to an randomized,paralleled,open,single-cycle design,50 enrolled healthy subjects were divided into 5 groups with each group composed of 5 male and 5 female subjects.Three dose groups(A,B,C group)were orally administered a single dose of 40 mg,80mg and120 mg febuxostat tablets,and two groups(D,E)were orally administered multiple doses of40 mg and 80 mg,febuxostat tablets on seven consecutive days,respectively.Blood samples were collected from elbow vein before and after administration at designed moment.After centrifugation of blood,the plasma samples were obtained and kept for determination.The plasma samples were precipitated by acetonitrile.The supernatant was separated on a Capcell PAK MG C18(50×4.6 mm I.D.,5 ?m)analytical column connected with a C18(4×3.0 mm I.D.,5 ?m)guard column using acetonitrile-water-formic acid(67: 33: 0.1,v/v/v)as the mobile phase at a flow rate of 0.8 ml/min.The column temperature was 40?C,the injection volumn was 5?l,and the overall running time of a single sample was 2.8min.The analyte febuxostat was detected by tandem quadrupole mass spectrometor equipped with Turbo Spray source and operated in negative ion mode.Multiple-reaction monitoring(MRM)of the precursor to product ion combinations of m/z315.1?m/z271.0 were used for quantification of febuxosta,while m/z322.1?m/z170.0 for gliclazide(internal standard).Full scan time was 150 ms.Confirm the determine method under the above conditions.The method was fully validated on specificity,linearity,limit of quantification,precision,accuracy,extraction recovery,matrix effect and sample stability,et al.The pharmacokinetic parameters were calculated with the software DAS2.0 on the basis of non-compartment model,and then the main pharmacokinetic parameters were analyzed by SPSS 13 software.Firstly,the pharmacokinetic characteristics of febuxostat administered at different single dose were analyzed,tmax was analyzed by the Kruskal-Wallis H nonparametric rank sum test,t1/2 was analyzed by variance analysis,and the linear correlationship were developed between Cmax,AUC0-t or AUC0-? and dose.Differences of the main pharmacokinetic parameters in terms of gender were analyzed for each group with tmax by Wilcoxon nonparametric test and Cmax,AUC0-t,AUC0-?,t1/2 by independent-sample t test.The pharmacokinetic parameters of febuxostat administered at multiple doses(Css-max,T1/2,AUCss)were compared with those at single doses by independent-sample t test to observe the accumulation of febuxostat in the body and the effect of enzyme induction or inhibition.Results:The linear concentration ranges of the calibration curves for febuxostat was 5.00-10000 ng·mL-1,the low limit of quantitication was 5.00 ng·mL-1.A representative calibration equation was Y=0.00491C+0.0061(r2=0.9972,n= 8);For plasma samples of febuxostat at 10,400,and 8000 ng/ml,the inter-day RSDs(%)were 6.00,2.90 and 3.20,the intra-day RSDs(%)were 14.2,12.3and 12.8,the accuracies(%)were 108.1,103.5 and 97.5 and extraction recoveries were(119.2±4.7)%,(117.3%±3.0)% and(110.5±3.6)%,respectively.The recovery of the IS was(108.7±2.6)%.The matrix effect determined at two concentrations(10 and 8000 ng/ml for febuxostat)were(92.4±5.87)% and(107.1±3.31)%,the matrix effect of the IS were(93.2±3.04)% and(103.2±5.16)%,respectively.Stability tests indicated that febuxostat work solution was stable at room temperature for 6 h,at 5°C for 8d;Febuxostat and gliclazide stock solution were stable at-20°C for 109d;gliclazide work solution were stable for 105 d at 5°C.Febuxostat plasma samples were stable for 6 h at room temperature,for 104 d at-70°C and after three successive freeze-thaw cycles.The processed samples were stable for24 h in autosampler at room temperature.The main pharmacokinetic parameters of GroupA,B and C were as follows: Tmax were(1.5±1.2),(1.0±0.6)and(1.5±0.8)h,T1/2 were(7.1±1.9),(5.5±1.3)and(6.2±1.3)h,Cmax were(2580±941),(5014±906)and(9925±5211)?g ·L-1,AUC0-t were(7744±2360),(17378 ± 2406)and(34375 ±13772)h·?g·L-1;There was no statistic difference among Tmax and T1/2 of different doses,and there was a significant positive correlation between Cmax,AUC0-36 h,AUC0-? and the dose.The gender difference of pharmacokinetic parameter Cmax and AUC of febuxostat was significant for group A(40mg)and group C(120mg),while not for group B(80mg).In multiple dose test,plasma concentration of febuxostat reached to steady state after five consecutive administration of febuxostat tablet.The main pharmacokinetic parameters of group D(40mg*7d)and E(80mg*7d)were as follows: Cmax(3209±983)and(6067±1868)?g·L-1,AUCss(10601±2793)and(18830±3004)h·?g·L-1,Cav(442±116)and(785±125)?g·L-1,respectively.Conclusion:1 The developed LC-MS/MS method for the determination of febuxostat in human plasma is sensitive,accurate,simple and reproducible.It is suitable to be applied to the pharmacokinetic research of febuxostat.2 Febuxostat has first-order elimination kinetics in Chinese healthy subjects.There is a positive correlationship between Cmax,AUC and the dose.at the range of 40 mg to 120 mg.3 There is gender difference in the pharmacokinetics of febuxostat and the difference may be have dose interaction.4 For multiple doses group,febxostat pharmacokinetics reaches to steady state after five consecutive administration of febuxostat tablet.Concluded from the changes of Cmax and AUC,there is no accumulation of febuxostat in the body after multiple doses of 80 mg,but the Cmax and AUC for 40 mg group at steady state is higher than those at a single dose.This confusing results maybe related to the individual difference in different dose groups.