The Method For The Determination Of Ecabet In Human Plasma And The Pharmacokinetic Study Of Ecabet Disodium Tablet In Healthy Volunteers

AIM: To develop a rapid and sensitive method for the determination of ecabet in human plasma by liquid chromatography-mass spectrometry (LC-MS/MS). The essay has been successfully applied to a pharmacokinetic study of ecabet in healthy volunteers after oral administrations of ecabet disodium tablets containing 1.0 g and 2.0 g ecabet, respectively.STUDY METHOD: Ecabet and the internal standard, valsartan, were precipitated from the matrix by methanol, then acidified with acetic acid and separated on a Venusil MP C18 column employing an isocratic mobile phase consisting of methanol and 10mM ammonium acetate (75:25, v/v) adjusted to pH 3.0 using formic acid. Detection was carried out by multiple reaction monitoring (MRM) on an API 4000 LC-MS/MS system with an ESI interface in the negative ion mode. MRM was performed at low resolution using the mass transition ion-pairs m/z 379.1→m/z 277.1 and m/z 434.3→m/z 350.1 for ecabet and valsartan, respectively. The specificity, matrix effects, linearity, sensitivity, precision, accuracy, extraction recovery and stability were estimated for the validations of the assay.The plasma concentrations of ecabet in healthy volunteers were measured after oral administrations of ecabet disodium tablets (Aikang Pharmaceutical Co.) containing 1.0 g and 2.0 g ecabet, respectively. After the plasma concentration–time profile was shown, the area under the curve (AUC0-t) was determined by the linear trapezoidal rule. SPSS version 11.0 software was used to determine statistically significant differences between results. Differences were considered statistically significant at P < 0.05.RESULTS: A highly selective, sensitive, rapid and reproducibility method for the determination of ecabet in human plasma by using LC-MS/MS has been developed and validated in this study. The standard curve of ecabet was linear over a working range of 10-6000 ng/mL with the lower limit of quantitation (LLOQ) of 10 ng/mL and the limit of detection (LOD) of 2 ng/ mL. The accuracy was in the range 96.7-105.4% and the intra- and inter-day precision was <2.55%. It showed relative high and stable recovery for the sample preparing procedure. The samples were stable in storage at ?20°C for 42 days, in three freeze-thaw cycles test, and in the autosampler at room temperature for 24 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 ecabet.This validated assay was applied to a clinical pharmacokinetic study in 8 healthy volunteers following oral administrations of ecabet disodium tablets containing 1.0 g and 2.0 g ecabet, respectively. The pharmacokinetic parameters were as follows: Tmax: 0.88±0.23 h (mean±SD) and 0.81±0.26 h; Cmax: 4998±769.5 ng/mL and 10977±1556 ng/mL; t1/2: 7.84±1.25 h and 7.74±2.11 h; CL/F: 0.416±0.127 L/h/kg and 0.378±0.090 L/h/kg; V_d/F: 4.8±2.1 L/kg and 4.1±1.1 L/kg; AUC_0-t: 42214±8713 ng·h/mL and 93061±24943 ng·h/mL; AUC_0-∞: 43606±8942 ng·h/mL and 97369±27211 ng·h/mL, respectively.The results of phamarcokinetic study of ecabet showed that: there was no significant difference in T_max, t_1/2, CL/F or V_d/F between the doses of 1.0 g and 2.0 g (P>0.05); the pharmcokinetics of ecabet was proven to be linear with dosage over the range of 1.0-2.0 g; there was no significant difference in T_max, C_max, AUC_0-t, CL/F or Vd/F between the male and female groups (P>0.05).