Enantiomeric separation of metoprolol and #alpha#-hydroxymetoprolol by liquid chromatography and fluorescence detection using a chiral stationary phase

摘要

A sensitive and stereoselecti.vehigh.perlormance liquid chromatographic assay for the determination of the enantiom~rsof metoprolol (R- and S-J and the diastereoisomers of #alpha#-hydroxymetoprolo1 (llA.llB )in plasma is reported. Chromatography invoJveddirect separation ofenantiomersus.ing aChirobioticT bonded phase column (250x4.6 mm) and atnobile phase consistIng ofaceto:nitri!e-meihario)C:methylene chloride-glacial aceticacid-triethylamine (56:30:14:2:2,v/.v).Solid-phase extraction using sil.icabonded with ethyl group (C_2) was used to extract the compounds of interestfroinplasma arid atenolo1 was used as the internal standard. The column effluent was moni.tored using fluorescence detection with excitation and emission wavelengths of225 and 310 ntn, res~ijvely.S-Metoprolo). R-metoprolollIB and IIAeluted at about 5.9, 6. 7, 7.3 and 8.2 rnin without any interfering peaks, The calibration curve was linear over the range of. 0.5 to I 00 ngl ml for each Isomer ofmeloprolol and 1 to loo ng/ml for each Isomcr ofa-hydroxytnetoprolol (llA & lIB). The mean intra-run accuracies were in the range of. 96.2 to 1 }.4% for R -metoprolol.. 94.0 to 111% for S-metoprolol 90..2 to 110% for IIA. and 94.6 to l06o/(!forIIB. The mean intra-run precisions werea.ll in theTal1ge of 2..2to.l2.00/Qfor R-metoprolol,2.1 to 11.1% for S-metroprolol,1.9- to 14.5% for IIA,and 3.2 to 11.0% for IIB. The lowest level of quantitation for theenantiotners of metoprolol was 0;5ngfml and].Ong/mIfor a-hydro~ymetoprolol (llAandIIB1:The absolute recoveries foreachanalyt~ was >=95%. The validatcd method accurately quantitated the enantiomers of parent drug and metabolite after a single dose of an extended releasemetoprolol formulation.