On the basis of the Isolndole formation mechanism In the o-phthalaldehyde/2-mercaptoethanol (OPA/2-ME) derivatization of primary amines and the structure-stability relationships for Isolndoles, an Improved fluorogenic reagent, naphthalene-2,3-dlcarboxaldehyde (NDA) In the presence of cyanide ion (CN ), has been developed. Reaction of NDA/ CN' with primary amines In aqueous media results In the formation of -substKuted 1-cyanobenz[/]lsoindole (CBI) derivatives which have significantly Improved stability compared to the corresponding OPA/2-ME derivatives (for glycine greater than 50-fold Improvement was realized) and have high quantum efficiencies for fluorescence ( , = 0.54 In 60% aqueous acetonitrile for the CBI--propylamine derivative) In solvent systems commonly used In liquid chromatography. Parameters In the NDA/CN' derivatization of alanine are defined (l.e., pH and the reagent component concentrations) and used In the development of a labeling procedure for amino acid mixtures. Gradient elution fractionation of 18 CBI-amino acid derivatives was accomplished In 60 min and permitted detection limits of less than 200 fmol Injected (excitation 246 nm) or 3 pmol Injected (excitation 420 nm). The utility of the reagent In assaying amino acid mixtures resulting from the enzymatic hydrolysis of the peptides Met-enkephalln and glucagon Is demonstrated.

A separation-based biosensor has been developed that is capable of near-real-time analysis of aspartate and glutamate with a temporal resolution of less than 2 min in anesthetized or awake, freely moving animals. The instrument consists of a microdialysis sampling system, an on-line reactor, an injection interface, and a CE-LIF system. Primary amine analytes are derivatized with NDA/CN following microdialysis sampling using an on-line reactor to produce fluorescent CBI derivatives. The reaction takes approximately 1 min. The derivatized sample then travels to a microinjection valve which alternately sends CE running buffer and reacted microdialysis sample to the CE column via an injection interface. The interface allows a controllable volume of 10-20 nL to be injected onto the CE separation capillary. Separation of aspartate and glutamate from the other amino acids present in the microdialysis sample was achieved within 70 s. Detection limits for glutamate and aspartate using laser-induced fluorescence detection were 0.1 microM. The linear dynamic range was acceptable for the determination of aspartate and glutamate in dialysate samples where the levels are between 1 and 10 microM. Full automation of the system was achieved by computer control of the valve, the interface, and the data collection system. The performance of this system was demonstrated in an anesthetized rat by monitoring ECF levels of aspartate and glutamate released in brain after stimulation with high concentrations of K+.

The first applications of a novel polyanionic derivative of (S-cycIodextrin (/3-CD), sulfobutyl ether ß-CO (ß-CO-SBE(IV)), as a stereoisomer selector in capillary electrophoresis are described. Separations were developed for one and two chiral molecules, including (±)-ephedrine, (±)pseudoephedrine, and several structurally related compounds.The separations achieved were found to be superior to those possible with neutral selectors such as ß-CO or heptakis(2,6dimethyl)-/8-CD. Base line separation of the four stereoisomers of (±)-ephedrine and (±)-pseudoephedrine was achieved in a single run with /S-CD-SBE(IV). Aspects regarding structural specificity, binding magnitude, and the utility of having a stereoisomer selector with a large countercurrent mobility are discussed.

Microdialysis sampling has become an important means of continuously monitoring reactions in vivo. This sampling technique places a constraint on the analysis method because of the very small sample volume provided. On the other hand, microdialysis provides the advantage of clean samples that do not require cleanup prior to analysis. An on-line coupling of microdialysis sampling to capillary electrophoretic (CE) analysis is described that uses the advantages of microcolumn separations to overcome the small volume limitation. An interface was designed which converts the continuous microdialysis sample stream into discrete 60-nL sample plugs and then injects a portion of this plug into the CE system. The on-line interface provided precision of 2.6% with minimal band broadening or peak height loss relative to off-line sampling. Using a high-speed micellar electrokinetic chromatography (MEKC) separation, resolution of the investigational antineoplastic SR 4233 from its main metabolite SR 4317 was achieved in less than 60 s. This allowed the on-line system to achieve a 90-s temporal resolution for determining the pharmacokinetics of SR 4233 in vivo.

Objective. Intracellular methotrexate (MTX) polyglutamates (MTXGlu) have been shown to be potentially useful biomarkers of clinical response in adult patients with rheumatoid arthritis. The present study was undertaken to measure intracellular MTXGlu concentrations in a cohort of patients with juvenile idiopathic arthritis (JIA) to determine the predictors of MTXGlu variability in these patients.Methods. Blood samples were obtained from patients with JIA who were being treated with a stable dose of MTX for >3 months. Clinical data were collected by chart review. Concentrations of MTXGlu 1-7 in red blood cell lysates were quantitated using an innovative ionpairing chromatography procedure, with detection by mass spectrometry.Results