LC-MS/MS法测定人血浆、尿液中米屈肼的浓度及其药动学研究

Determination of Mildronate Concentration in Human Plasma and Urine by LC-MS/MS and Pharmacokinetics Study

目的：建立测定人血浆、尿液中米屈肼浓度的方法,研究其在健康人体内的药动学特征。方法：血浆、尿液样品经沉淀法处理后,采用液相色谱-串联质谱（LC-MS/MS）法进样测定。色谱柱为Dikma Diamonsil C18,流动相为甲醇-水（含0.2%甲酸、0.3%醋酸铵）（31∶69,V/V）,流速为0.6 ml/min;采用电喷雾离子源（ESI）,以多重反应监测（MRM）方式扫描,负离子方式检测。用于定量分析的离子分别为m/z 147.10→58.20（米屈肼）和m/z 152.00→110.10（内标,对乙酰氨基酚）。采用DAS 2.1软件分别计算单次、多次给药的药动学参数,并比较其差异。结果：米屈肼血药、尿药浓度分别在0.02~20 ng/ml（r=0.999 3）和0.05~40 ng/ml（r=0.998 2）范围内线性关系良好,定量限分别为0.02、0.05 ng/ml;精密度、回收率符合生物样品测定要求,内源性杂质不干扰测定。血浆中米屈肼低、中、高剂量（250、500、750 mg）单次给药组t1/2分别为（3.39±0.81）、（5.52±0.57）、（5.32±0.96）h,tmax分别为（0.80±0.45）、（1.38±0.43）、（1.10±0.36）h,cmax分别为（4.17±1.46）、（8.08±1.04）、（15.04±1.86）ng/ml,AUC0-36 h分别为（24.55±5.81）、（45.50±7.07）、（85.60±13.09）ng·h/ml,单次给药在250~750 mg剂量范围内,cmax、AUC0-36 h与剂量呈线性关系（R2分别为0.974 5、0.968 3）;米屈肼低剂量多次给药组达稳态时,cmin为（0.28±0.10）ng/ml,AUCss为（38.78±4.18）ng·h/ml,css为（1.62±0.17）ng/ml,DF为（3.81±1.14）,t1/2为（6.17±1.46）h,tmax为（1.20±0.33）h,cmax为（6.46±1.96）ng/ml,AUC0-36 h为（40.33±4.65）ng·h/ml;cmax、AUC蓄积因子分别为（1.73±0.90）和（1.64±0.40）。多次连续给药达稳态时与单剂量相比,t1/2、cmax、AUC0-36 h均有所改变,tmax无显著性差异。各剂量组单次给药后,26 h尿累积排泄率分别为（0.004 009±0.001 1）%、（0.004 026±0.001 01）%、（0.003 858±0.000 68）%。结论：该试验建立的方法灵敏度好、准确度高、专属性强,适用于人体中米屈肼血药及尿药浓度的测定和药动学研究。米屈肼胶囊在健康人体内具有一定的蓄积作用,且具有线性药动学特征。

OBJECTIVE： To establish the method for the determination of mildronate in human plasma and urine, and to study the pharmacokinetic characteristics in healthy volunteers. METHODS： After precipitating plasma and urine sample, LC-MS/MS method was adopted. Dikma Diamonsil C18 column was used with mobile phase consisted of methanol-water （containing 0.2% for- mic acid, 0.3% ammonium acetate） （31 ： 69, V/V） at the flow rate of 0.6 ml/min. ESI was adopted in MRM mode, by using nega- tive ion. The ion for quantitative analysis were m/z 147.10±58.20 （mildronate） and m/z 152.00→110.10 （internal standard, acet- aminophen）. The pharmacokinetic parameters of mildronate with single administration and multiple administration were calculated by using DAS 2.1 software and compared. RESULTS： The linear range of mildronate in plasma were 0.02-20 ng/ml（r=0.999 3） and in urine were 0.05-40 ng/ml （r=0.998 2）. The lowest limits of quantitation were 0.02 and 0.05 ng/ml. Precision and recovery met the requirements of biological specimen determination, and endogenous impurities hadn＇t effect on the determination. The main pharmacokinetics parameters of low-dose, medium-dose and low-dose （250, 500, 750 mg） of mildronate in plasma with single ad- ministration were as follows： t1/2 were （3.39 ± 0.81）, （5.52 ± 0.57） and（5.32 ±0.96）h; tmax were （0.80± 0.45）, （1.38± 0.43） and （1.10 ± 0.36）h; Cmax were （4.17 ± 1.46）, （8.08 ± 1.04） and （15.04 ±1.86）ng/ml; AUC0-36h were （24.55±5.81）, （45.50 ± 7.07） and （85.60± 13.09）ng.h/ml. In the dose range, Cmax, AUC036 h h had a linear relationship with dose （R2 were 0.974 5 and 0.968 3）. The main pharmacokinetic parameters of low-dose of mildronate with multiple administration after keeping stable were as follows： cmax was（0.28 ± 0.10）ng/ml; AUCss was（38.78 ±4.18）ng.h/ml; Css was（1.62± 0.17）ng/ml; DF was（3.81± 1.14） ; t1/2 was（6.17 ± 1.46）h; tmax was（1.20 ± 0.33）h; Cmax was（6.46 ± 1.96）ng/ml; AUC0-36 h was（40.33 ± 4.65）ng. h/ml; accumulation factor of cmax and AUC were（1.73 ±0.90）and （1.64±0.40）. Compared with single administration, t1/2, Cmax and AUC of mildronate with multiple admin-istration after keeping stable all changed, and tmax had no signifi- cant difference. After single administration, 26 h accumulative excretion rate of those groups were （0.004 009 ± 0.001 1）%, （0.004 026± 0.001 01）% and（0.003 858 ±0.000 68）% respec-tively. CONCLUSIONS： Established method is sensitive, accurate and specific, and suitable for the determination of mildronate concentration in human plasma and urine and pharmacokinetics study. Mildronate capsule shows certain accumulation effect in healthy volunteers, and linear pharmacokinetic characteristics.