HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ANALYSIS OF AMINO-5-PHOSPHONOMETHYL[1,1'-BIPHENYL]-3-PROPANOIC ACID (EAB-515) IN BRAIN AND BLOOD MICRODIALYSATE (ONLINE) AND IN PLASMA ULTRAFILTRATE OF FREELY MOVING RATS

Amino-5-phosphonomethyl[1,1'-biphenyl]-3-propanoic acid (EAB 515, I), a competitive antagonist of the N-methyl-D-aspartate receptor, has sig nificant pharmacological activity in the central nervous system (CNS). An extremely sensitive and selective analytical method was developed for the simultaneous analysis of I and its hydroxylated analog (RDC, I I) in the microdialysate (MD) and plasma ultrafiltrate (UF) of rats. M icrodialysis was used for in vivo sampling of unbound drug in the CSF, cortical extracellular fluid and in the blood of freely moving rats. Compound II was used for retrodialysis-based in vivo calibration of mi crodialysis probes to estimate the recovery of I. Compound I, being ex tremely hydrophilic with a high degree of ionization at the physiologi cal pH of 7.4, has limited access to the brain regions, This, combined with its low microdialysis recovery, made the estimation of low brain concentrations of I a challenge. The analytes in MD and UF were separ ated (within 5 min) by reversed-phase HPLC on a 250x4.6 mm I.D. Maxsil 5 mu m RP-2 column, and fluorescence of the eluent was monitored at 2 55 nm (lambda(ex)) and 320 nm (lambda(em)). A 0.09% (v/v) aqueous solu tion of trifluoroacetic acid (1 ml/min) was used as the mobile phase. The response for I in MD and UF samples was linear from 5 to 2000 ng/m l and from 20 to 10 000 ng/ml, respectively. The between-run (n=6) and within-run (n=3) variability of the assay was <15%. Plasma-protein bi nding of I (f(u)=0.68) was determined to be linear from 0.1 to 10 mu g /ml. The analytical sensitivity, precision and accuracy of this method was suitable for the characterization of the pharmacokinetics and the CNS distribution of I, following administration of intravenous (i.v.) infusion, single i.v. bolus and multiple i.v. bolus doses of I to fre ely moving rats, with continuous microdialysate sampling of multiple t issues and simultaneous on-line HPLC analysis. Pharmacokinetic paramet ers for I, as determined from concentrations in blood MD samples with on-line analysis, were in good agreement with those estimated from con centrations in the UF of plasma samples obtained by conventional sampl ing.