High-performance liquid chromatographic analysis of (S)-alpha-amino-5-phosphonomethyl[1,1'-biphenyl]-3-propanoic acid (EAB 515) in brain and blood microdialysate (on-line) and in plasma ultrafiltrate of freely moving rats.
(S)-alpha-Amino-5-phosphonomethyl[1,1'-biphenyl]-3-propanoic acid (EAB 515, I), a competitive antagonist of the N-methyl-D-aspartate receptor, has significant 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, II) in the microdialysate (MD) and plasma ultrafiltrate (UF) of rats. Microdialysis 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 microdialysis probes to estimate the recovery of I. Compound I, being extremely hydrophilic with a high degree of ionization at the physiological 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 separated (within 5 min) by reversed-phase HPLC on a 250 x 4.6 mm I.D. Maxsil 5 microns RP-2 column, and fluorescence of the eluent was monitored at 255 nm (lambda ex) and 320 nm (lambda em). A 0.09% (v/v) aqueous solution 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/ml 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 binding of I (fu = 0.68) was determined to be linear from 0.1 to 10 micrograms/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 freely moving rats, with continuous microdialysate sampling of multiple tissues and simultaneous on-line HPLC analysis. Pharmacokinetic parameters for I, as determined from concentrations in blood MD samples with on-line analysis, were in good agreement with those estimated from concentrations in the UF of plasma samples obtained by conventional sampling.
Malhotra
B K
BK
Department of Pharmaceutics, University of Minnesota, Minneapolis 55455, USA.
Lemaire
M
M
Brouillard
J F
JF
Sawchuk
R J
RJ
D000818:Animals; D001713:Biphenyl Compounds / Q000008:administration & dosage / Q000032:analysis* / Q000097:blood / Q000493:pharmacokinetics; D001921:Brain / Q000378:metabolism*; D002550:Cerebral Veins / Q000601:surgery; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D018691:Excitatory Amino Acid Antagonists / Q000008:administration & dosage / Q000032:analysis* / Q000493:pharmacokinetics; D066298:In Vitro Techniques; D007262:Infusions, Intravenous; D007601:Jugular Veins / Q000601:surgery; D016014:Linear Models; D008297:Male; D017551:Microdialysis / Q000379:methods; D009862:Online Systems; D011169:Portal Vein / Q000601:surgery; D011422:Propionates / Q000008:administration & dosage / Q000032:analysis* / Q000097:blood / Q000493:pharmacokinetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014462:Ultrafiltration / Q000379:methods
D001713:Biphenyl Compounds; D018691:Excitatory Amino Acid Antagonists; D011422:Propionates; D016194:Receptors, N-Methyl-D-Aspartate; C077642:2-amino-5-phosphonomethyl(1,1'-biphenyl)-3-propanoic acid