Simultaneous Measurement of Monoamine, Amino Acid, and Drug Levels, Using High Performance Liquid Chromatography and Coulometric Array Technology: Application toIn VivoMicrodialysis Perfusate Analysis

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Abstract
An automated HPLC coulometric array-ECD method is described for the simultaneous analysis of monoamines, their metabolites, derivatized amino acids, and pharmacological agents. This method has been used with in vivo microdialysis in urethane-anesthetized animals to examine extracellular fluid levels of endogenous and exogenous analytes after the peripheral administration of drugs. An aliquot of dialysate was initially analyzed for the monoamines, their metabolites and drugs by isocratic elution and detection on eight serial coulometric electrodes (0 to 490 mV; 70 mV increment). The remaining sample was then derivatized, pre-column, with OPA/ßME 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 their retention time and electrochemical profile across the arrays. This method had a limit of detection of 0.125 pg/μl for the monoamines and 0.75 pg/μl for amino acids (both with a signal to noise (S/N) ratio of 3:1). The detector response was linear over several orders of magnitude (0.25 to 20 pg/μl) for monoamines, their metabolites and the 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/μl (Phe and M3G respectively) with a S/N ratio of 3:1. The detector response was linear from 0.5–500 pg/μl and the linear regression correlation coefficient (r) varied from 0.9969 to 0.9998 (H and M3G respectively). The peripheral administration of H (10 mg/kg i.v.) produced a 40% decrease in blood pressure (BP) and caused an almost immediate 220 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) increased 30–120 fold. Striatal levels of H reached a maximum of 9 pg/μl (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%, increased striatal DA levels by 100 fold, and decreased levels of DOPAC and HVA by 40–50%. Although the amino acids were also affected, their levels began to increase only 140 min after.the start of drug administration. NPr could not be detected using this method. In a separate experiment, hippocampal perfusate levels of M were found to reach a maximum of 12.6 pg/μl (567 pg/collection), 40min after its peripheral administration (10 mg/kg i.p.). Although M decreased hippocampal ECF levels of GABA and GLY, it appeared to have little effect on the other analytes measured. This method not only makes it possible to study the interaction between different neurotransmitter pathways but also offers a more detailed inspection of the mechanism of drug action, a direct measure as to whether drugs pass through the blood-brain barrier (BBB) as well as direct acquisition of pharmacokinetic data.

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