SIMULTANEOUS MEASUREMENT OF MONOAMINE, AMINO-ACID, AND DRUG LEVELS, USING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND COULOMETRIC ARRAY TECHNOLOGY - APPLICATION TO IN-VIVO MICRODIALYSIS PERFUSATE ANALYSIS

An automated HPLC coulometric array-ECD method is described for the si multaneous analysis of monoamines, their metabolites, derivatized amin o acids, and pharmacological agents. This method has been used with in vivo microdialysis in urethane-anesthetized animals to examine extrac ellular fluid levels of endogenous and exogenous analytes after the pe ripheral administration of drugs. An aliquot of dialysate was initiall y analyzed for the monoamines, their metabolites and drugs by isocrati c elution and detection on eight serial coulometric electrodes (0 to 4 90 mV; 70 mV increment). The remaining sample was then derivatized, pr e-column, with OPA/ss ME and, after column switching, was analyzed on a parallel isocratic system with detection on four electrodes (set at 250, 450, 550 and 650 mV respectively). Compounds were identified by t heir retention time and electrochemical profile across the arrays. Thi s method had a limit of detection of 0.125 pg/mu l for the monoamines and 0.75 pg/mu l for amino acids (both with a signal to noise (S/N) ra tio of 3:1). The detector response was linear over several orders of m agnitude (0.25 to 20 pg/mu l) for monoamines, their metabolites and th e amino acids. The analysis was completed within 25 min. A variety of drugs were also measured including: apomorphine (Apo), hydralazine (H) , isoproterenol (Iso), methoxamine (Mx), morphine sulfate (M) and its metabolite morphine-3-glucuronide (M3G), and phenylephrine (Phe). The limit of detection for these compounds varied from 0.215 to 10.65 pg/m u l (Phe and M3G respectively) with a S/N ratio of 3:1. The detector r esponse was linear from 0.5-500 pg/mu l and the linear regression corr elation coefficient (r) varied from 0.9969 to 0.9998 (H and M3G respec tively). The peripheral administration of H (10 mg/kg i.v.) produced a 40% decrease in blood pressure (BP) and caused an almost immediate 22 0 fold increase in striatal dopamine (DA) levels. Levels of DOPAC and HVA decreased by 80-90% and those of the amino acids glutamate (GLU), aspartate (ASP), taurine (TAU) and gamma amino-butyric acid (GABA) inc reased 30-120 fold. Striatal levels of H reached a maximum of 9 pg/mu l (405 pg/collection) 40 min after its administration. Nitroprusside ( NPr) infusion (0.06-0.3 mg/min/kg i.v.) also decreased BP by 30%, incr eased striatal DA levels by 100 fold, and decreased levels of DOPAC an d HVA by 40-50%. Although the amino acids were also affected, their le vels began to increase only 140 min after the start of drug administra tion. NPr could not be detected using this method. In a separate exper iment, hippocampal perfusate levels of M were found to reach a maximum of 12.6 pg/mu l (567 pg/collection), 40 min after its peripheral admi nistration (10 mg/kg i.p.). Although M decreased hippocampal ECF level s of GABA and GLY, it appeared to have little effect on the other anal ytes measured. This method not only makes it possible to study the int eraction between different neurotransmitter pathways but also offers a more detailed inspection of the mechanism of drug action, a direct me asure as to whether drugs pass through the blood-brain barrier (BBB) a s well as direct acquisition of pharmacokinetic data.