Determination of glutamate and aspartate in microdialysis samples by reversed-phase column liquid chromatography with fluorescence and electrochemical detection¹

Abstract

Five different systems for fast determination of aspartate and glutamate in microdialysis samples are described: (I) a high-speed HPLC using a gradient pump with a sharp elution profile, (II) a column switching technique, (III) an isocratic pump with a low-pressure switching valve for one-step gradients, (IV) microbore chromatography using injections of acetonitrile as a wash-out step, (V) on-line connection of microdialysis and HPLC/derivatization. In all cases, automated precolumn derivatization with o-phthalaldehyde-2-mercaptoethanol reagent were used. Both fluorescence and electrochemical detection techniques were evaluated in terms of reproducibility, sensitivity, interference, maintenance and troubleshooting. The electrochemical detection method required a second derivatization step with 0.2 M iodoacetamide to remove excess of a thiol moiety and regular recalibrations after each six to ten injections. Under these conditions the correlation coefficients for electrochemical vs. fluorescence detectors were 0.918 for Asp and 0.988 for Glu for 65 microdialysis samples. Coefficients of variation for six analyses between calibrations were below 3% for both detectors. The limits of detection for both amino acids were about 0.4 pmol for electrochemical detection with a thiol scavenger step, 50 fmol for fluorescence detection using conventional columns and about 20–30 fmol for the microbore system. All systems are suitable for detecting basal levels of Asp and Glu in 5–10 μl microdialysis samples from a rat brain where typical concentrations lie around 1–10 pmol or more. It is concluded that a microbore setup with one isocratic pump and an autosampler optimized for injections of washing solvent between samples is the most practical and economical. The system allows analysis of minute sample volumes down to 1–2 μl.

Introduction

Glutamate (Glu) is well recognized as a major excitatory neurotransmitter in the central nervous system 1, 2. It is hypothesized that the massive efflux of Glu and aspartate (Asp) observed in different neuropathological models of brain injury 3, 4, 5causes an uncontrolled excitotoxic stimulation of postsynaptic (mainly NMDA) receptors, membrane depolarization and energy depletion, which in turn leads to neuronal cell death [6]. However, recent studies indicate that besides Asp and Glu, other neurotransmitters or their metabolites can also be involved in the mechanisms of ischaemic brain damage [7]. Furthermore, Asp and Glu play important roles in cell metabolism. For instance, Glu is an intermediate in energy metabolism, a precursor of the main inhibitory neurotransmitter γ-aminobutyric acid and detoxifies ammonia via the formation of glutamine.

The technique of in vivo microdialysis 8, 9allows sampling and continuous monitoring of extracellular pools of neurotransmitters and other small molecules.

Microdialysis has been shown to be an elegant tool in studies of amino acid outflow [10], ischaemia [11], hypoglycaemia [12], epilepsy [13]and brain lesions [14].

In most cases, amino acids were separated after precolumn derivatization with an o-phthalaldehyde 2-mercaptoethanol (OPA-MCE) reagent using reversed-phase liquid chromatography as originally described by Lindroth and Mopper [15]. The method was recently modified for an automated derivatization procedure [16]and for microdialysis samples [17]. It was shown that, besides fluorescence (FL), electrochemical (EC) detection of OPA derivatives is possible 18, 19, although with some precautions. A typical separation of 15–25 physiological amino acids found in microdialysis samples can be achieved by gradient elution in 30–40 min. In this mode, Asp and Glu elute at the beginning of the chromatogram with retention times of some 5–10 min. This means that elution can occur almost under isocratic conditions. Thus, if just these two amino acids are of interest, there is no need for additional separation. On the contrary, a fast and simple analysis would be more beneficial, especially due to the high sampling frequency during a microdialysis experiment. A simple sharpening of the gradient profile will reduce the analysis time down to approximately 10 min, including reequilibration. However, a more rapid and economical separation is possible by omitting the gradient pump and using different switching techniques instead. The use of microbore columns allows detection of Asp and Glu in volumes of microdialysates as low as 1–2 μl which corresponds to approximately 1 min sampling intervals.

In the present paper, methods for fast automated Glu and Asp determination, based on column switching (a second high-pressure valve), a one-step gradient (3-way low-pressure valve) and flushing by a second injection of an organic solvent (for microbore columns) are described. Furthermore, the possibility of an on-line connection of microdialysis to the liquid chromatograph and corresponding on-line derivatization with OPA is discussed. The sensitivities of fluorescence and electrochemical detectors are compared.