| AIM: To develop a rapid and sensitive method for the determination of ctahydroaminoacridine (OHA) in human plasma by liquid chromatography-mass spectrometry (LC-MS/MS). The assay has been successfully applied to the pharmacokinetic study of OHA in healthy volunteers after oral administrations of OHA succinate tablets containing 2.0,4.0,8.0 mg OHA , respectively. The effect of food on the pharmacokenitic parameters (PK) properties of OHA in the dose of 4.0 mg was also assessed.STUDY METHOD: OHA in human plasma using tramadol as internal standard (I.S.). Sample preparation involved pH adjustment with sodium carbonate followed by solvent extraction with dichloromethane:ethyl ether (40:60, v/v). Chromatographic separation was achieved on a Venusil MP-C18 column (5μm, 100×4.6 mm) using acetonitrile:10 mM ammonium acetate:formic acid (30:70:0.7, v/v/v) as mobile phase. Detection utilized an API 4000 system operated in the positive ion mode with multiple reaction monitoring of the analyte at m/z 203.1→175.1 and of the I.S. at m/z 264.1→58.0. The specificity, matrix effects, linearity, sensitivity, precision, accuracy, extraction recovery and stability were estimated for the validations of the assay.The plasma concentrations of OHA in healthy volunteers were measured after oral administrations of OHA succinate tablets containing 2.0,4.0,8.0 mg OHA, respectively. The effect of food on the pharmacokenitic parameters (PK) properties of OHA in the dose of 4.0 mg was also assessed. The main pharmacokenitic parameters including AUC0-t, Cmax, Tmax, t1/2 for OHA in each subject were analyzed by noncompartmental analysis using Topfit 2.0 ( Thomae GmbH,Germany ) Pharmacokinetic parameters (Tmax, t1/2) were compared among dose levels using analysis of 1-way variance (ANOVA) using SPSS version 13.0 software. . The effect of food on the pharmacokenitic parameters (PK) properties of OHA in the dose of 4.0 mg was also assessed. Differences were considered statistically significant at P < 0.05. The linearity of octahydroaminoacridine pharmacokinetics was assessed by examining Cmax, AUC0-t in the range of 2-8 mg.RESULTS: A rapid and sensitive LC–MS/MS method for the determination of OHA in human plasma after a therapeutic oral dose has been developed and validated. The method allows efficient analysis of the large numbers of samples involved in clinical studies. The method was linear in the range 0.01-10 ng/mL with a lower limit of quantitation of 0.01 ng/mL. Intra- and inter-day precisions measured as relative standard deviation were < 3.15 % and < 5.01 %, respectively.. It showed relative high and stable recovery for the sample preparing procedure. The samples were stable in storage at ?20°C for 30 days, in three freeze-thaw cycles test, and in the autosampler at room temperature for 12 h. The method, which kept the conformance to the relevant standards of Pharmacopoeia of People's Republic of China, was ideally suited for the study of pharmacokinetics of OHA. .The assay has been successfully applied to a pharmacokinetic study of OHA in healthy volunteers after oral administrations of OHA succinate tablets containing 2.0,4.0,8.0 mg OHA , respectively. The pharmacokinetic parameters were as follows: Tmax: 0.866±0.653 (mean±SD),0.869±0.42 and 0.801±0.477 h; Cmax: 0.817±0.486,1.373±0.374 and1.555±0.371 ng/mL; t1/2: 2.466±0.473,2.799±0.555 and 2.364±0.517 h; CL/F: 22719±13063,21320±7484 and 32420±13158 mL/min; Vd:4881±2963,5147±2020 and 6547±2615 L; AUC0-t: 2.037±1.245 ng?h/mL,3.396±0.981ng?h/mL和4.276±1.939ng?h/mL, respectively. The pharmacokinetic parameters of the effect of food on the pharmacokenitic properties of OHA in the dose of 4.0 mg include Tmax1.20±0.592 h, Cmax1.166±0.398 ng/mL, t1/2 2.362±0.505 h, CL 24020±9609 mL/min, Vd 4820±1874 L, AUC0-t,3.14±1.096 ng?h/mL.The results of phamarcokinetic study of OHA showed that: there was no significant difference in Tmax, t1/2, among the doses of 2.0,4.0 and 8.0 mg (P>0.05); the pharmcokinetics of OHA was proven to be linear with dosage over the range of 2.0-8.0 mg; there was no significant difference in Tmax, t1/2, Cmax, AUC0-t between the food fed and fasted groups (P>0.05).
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