Greener bioanalytical approach for LC/MS-MS assay of enalapril and enalaprilat in human plasma with total replacement of acetonitrile throughout all analytical stages.

Green bioanalytical approaches are oriented toward minimization or elimination of hazardous chemicals associated to bioanalytical applications. LC/MS-MS assay of enalapril and enalaprilat in human plasma was achieved by elimination of acetonitrile from both sample preparation and chromatographic separation stages. Protein precipitation (PP) by acetonitrile addition was replaced by liquid-liquid extraction (LLE) in 1-octanol followed by direct large volume injection of the organic layer in the chromatographic column operated under reversed phase (RP) separation mechanism. At the mean time, acetonitrile used as organic modifier in the mobile phase was successfully replaced by a mixture of propylene carbonate/ethanol (7/3, v/v). Three analytical alternatives ((I) acetonitrile PP+acetonitrile based chromatographic elution; (II) 1-octanol LLE+acetonitrile based chromatographic elution; (III) 1-octanol LLE+propylene carbonate/ethanol based chromatographic elution) were validated and the quality characteristics were compared. Comparison between these alternative analytical approaches was also based on results obtained on incurred samples taken during a bioequivalence study, through application of the Bland-Altman procedure.

Rapid and sensitive liquid chromatography/tandem mass spectrometry method for simultaneous determination of enalapril and its major metabolite enalaprilat, in human plasma: application to a bioequivalence study.

A rapid and most sensitive method for simultaneous determination of enalapril (ENP) and its metabolite, enalaprilat (ENPT), in human plasma using ESI-LC-MS/MS (electrospray ionization liquid chromatography tandem mass spectrometry) positive ion multiple reactions monitoring (MRM) mode, was developed and validated. The procedure involves a simple solid-phase extraction (SPE) followed by evaporation of the sample. Chromatographic separation was carried out on a Hypurity C(18) column (50 mm × 4.6 mm, 5 µm) with an isocratic mobile phase and a total run time of 2.0 min only. The MRM of ENP and ENPT is 377.10 → 234.20 and 349.20 → 206.10 respectively. The standard calibration curves showed excellent linearity within the range of 0.064 to 431.806 ng/mL for ENA and 0.064 to 431.720 ng/mL for ENPT (r ≥ 0.990). This is the only method which can quantitate upto 0.064 ng/mL for both ENP and ENPT in a single run with the shortest analysis time. In matrix effect experiment, this method shows a % CV (% coefficients of variation) of less than 5, which means that the proposed method is free from any kind of irregular ionization process. This method was successfully applied to a pharmacokinetic study after oral administration of enalapril maleate 20 mg tablet in Indian healthy male volunteers.

Determination of the cis-trans isomerization barrier of enalaprilat by dynamic capillary electrophoresis and computer simulation.

Dynamic capillary electrophoresis (DCE) and computer simulation of the elution profiles with the stochastic model has been applied to determine the isomerization barriers of the angiotensin converting enzyme inhibitor enalaprilat. The separation of the rotational cis-trans isomeric drug has been performed in an aqueous 20 mM borate buffer at pH 9.3. Interconversion profiles featuring plateau formation and peak broadening were observed. To evaluate the rate constants k(cis-->trans) and k(trans-->cis) of the cis-trans isomerization from the experimental electropherograms obtained by dynamic capillary electrophoresis, elution profiles were analyzed by a simulation with iterative convergence to the experimental data using the ChromWin software which requires the total migration times of the individual isomers t(R), the electroosmotic break-through time t(0), the plateau height h(plateau), the peak widths at half height of the individual isomers w(h), as well as the peak ratio of the isomers as experimental data input. From temperature-dependent measurements between 0 degrees and 15 degrees C the thermodynamic parameters Delta G, Delta H and Delta S, the rate constants k(cis-->trans) and k(trans-->cis) and the kinetic activation parameters Delta G*, Delta H*, and Delta S* of the cis-trans isomerization of enalaprilat were obtained. From the activation parameters the isomerization barriers at 37 degrees C were calculated to be Delta G* (trans-->cis) = 87.2 kJ.mol(-1) and Delta G*(cis-->trans) = 91.9 kJ.mol(-1).

A simple HPLC method for quantitation of enalaprilat.

A reversed-phase high performance liquid chromatography (HPLC) method with UV-detection has been developed for the determination of enalaprilat. The method produced linear response over the wide concentration range of 1-200 microg/ml, with an average accuracy of 97.35 +/- 4.93%, as well as average intra- and iter-day variations of 3.72 and 5.18%, respectively. The limits of detection and quantitation of the method were 0.125 and 0.5 microg/ml, respectively. The method was selective with respect to resolution of the peaks of enalaprilat and enalapril maleate.