STEREOSELECTIVE DETERMINATION OF UNCHANGED AND GLUCUROCONJUGATED ELIPRODIL, A NEW ANTIISCHEMIC DRUG, IN HUMAN PLASMA AND URINE BY PRECOLUMNDERIVATIZATION AND COLUMN-SWITCHING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH FLUORESCENCE DETECTION

An HPLC method was developed and validated for the determination in hu man plasma and urine of the enantiomers of eliprodil, enyl-4[(4-fluoro phenyl)methyl]piperidine-1-ethanol hydrochloride, a new anti-ischaemic agent administered as a racemate. Both enantiomers are present in hum an plasma in unchanged and glucuroconjugated form, whereas only the gl ucuroconjugated form is excreted into urine; as a consequence, such me tabolites in human plasma and urine should be submitted to enzymatic d econjugation with beta-glucuronidase (Escherichia coli) before being e xtracted. The general method involves a liquid-liquid extraction of el iprodil and internal standard from alkalinized plasma or urine with n- hexane, evaporation of the organic phase and derivatization with (S)-( +)-naphthylethyl isocyanate to give carbamate diastereoisomeric deriva tives of (S)-(+)and (R)-(-)-eliprodil and internal standard; after eva poration of the derivatizing mixture and dissolution of the residue in a small volume of phosphate buffer-acetonitrile (60:40, v/v), an aliq uot is injected into a column-switching HPLC system. The derivatized s ample extract is purified on a precolumn filled with C-8-bonded silica material, which is flushed with acetonitrile-water, then diastereoiso mers of eliprodil and the internal standard are automatically transfer red by the mobile phase to the analytical column. The analytical colum n is a C-8 type, specially deactivated for basic compounds, the mobile phase is 0.025 M phosphate buffer (pH 2.6)-methanol-acetonitrile (42: 2:56) at a flow-rate of 1.2 ml min(-1) and a fluorimetric detector ope rating at lambda(ex) = 275 nm and lambda(em) = 336 nm is used. The ret ention times, under these conditions, are about 16 and 17 min for (S)- (+)- and (R)-(-)-eliprodil diastereoisomers, respectively, and about 1 9 min for the first-eluted diastereoisomer of the internal standard. D uring the analysis time, the precolumn, reset in a different path from that of the analytical column, is back-flushed with different solvent s, then re-equilibrated with acetonitrile-water before the next inject ion. Linearity in plasma, for unchanged eliprodil enantiomers, was ass essed in the range 0.15-10 ng ml(-1) and for total eliprodil enantiome rs (unchanged + conjugated) in the range 0.75-500 ng ml(-1); the limit of quantitation (LOQ) is 0.15 ng ml(-1) for each unchanged enantiomer and 0.75 ng ml(-1) for each total enantiomer. Linearity was also asse ssed in urine for total (conjugated) eliprodil enantiomers in the rang e 50-25 000 ng ml(-1); the LOQ is 50 ng ml(-1) for each enantiomer. Th e intra- and inter-day precision and accuracy of the method were inves tigated in plasma and urine and found to be satisfactory for pharmacok inetic studies. The method has been extensively used in pharmacokineti c studies in man treated with a 20-mg dose of eliprodil racemate and s ome results of this application are reported.