Determination of biogenic amines, their metabolites, and other neurochemicals by liquid chromatography/electrochemistry

Chester T. Duda and Peter T. Kissinger,     Department of Chemistry, Purdue University, W. Lafayette, IN 47907, and Bioanalytical Systems Inc. 2701, Kent Avenue, West Lafayette, IN 47906-1382, USA

Publisher Summary

This chapter focuses on the determination of biogenic amines, their metabolites, and other neurochemicals by liquid chromatography/electrochemistry. Electrochemistry has a distinct advantage compared to most analytical techniques as it involves a direct conversion of chemical information to an electrical signal without any need for intermediate optical or magnetic carriers. For example, all catechol derivatives can be readily oxidized at a graphite electrode to generate the corresponding orthoquinone, two protons, and two electrons. The combination of gas chromatography and mass spectrometry has revolutionized the ability to handle extremely complex mixtures of chemical substance. The direct conversion of chemical information into electricity gives electrochemical measurements a significant advantage when compared with many other analytical techniques. It has been recognized that considerable advantage results from the coupling of liquid chromatography (LC) with electrochemistry (EC). The chapter discusses the application of LCEC to endogenous compounds of neurologic interest.

1 Introduction

A great many problems in biomedical research involve the determination of fewer than 10 individual substances in very complex samples such as biological fluids or tissue homogenates. In many laboratories it has become routine to isolate a few microliters of perfusate from a living animal using the in vivo microdialysis sampling technique. These samples contain thousands of individual compounds and ions which are thought to be irrelevant to the problem at hand. The amount of sample is frequently limited, particularly in experiments with laboratory animals, and it is often necessary to determine amounts of individual compounds in the picomole range and below. To meet these challenges a selective analytical approach is needed, with good detection limits for substances of interest.

A combination of existing technologies can provide the desired instrumentation. For example, the combination of gas chromatography and mass spectrometry (GCMS) has revolutionized our ability to handle extremely complex mixtures of chemical substances. Unfortunately, this technique does not solve all problems equally well. Many nonvolatile and thermally labile metabolites of biomedical interest are not directly suitable for GCMS. In addition, for many laboratories the expense and complexity of the instrumentation rules it out for routine purposes. Since this chapter was first prepared in the early 1980s, powerful GCMS systems have become available at much lower cost. This trend will continue. LCMS is also gaining in capability at lower cost, but is generally insufficient for neurotransmitter studies.

For over twenty years it has been recognized that considerable advantage results from the coupling of liquid chromatography (LC) with electrochemistry (EC) (see for example Krstulovic, 1986; Kissinger, 1989). While more limited in scope, the LCEC system has many parallels with the GCMS system. In both cases a high-resolution separation technique is coupled to a measurement scheme involving the direct conversion of chemical information into electricity. Many of the compounds which cause problems for the gas-phase technique are well suited to the liquid-phase variant. The detection limits achievable with both methodologies are roughly equivalent. While GCMS is far more versatile and has the edge in molecular specificity, LCEC is less expensive and is more convenient to use for many problems. LCEC systems are sufficiently inexpensive that one laboratory will frequently use several instruments with autosamplers to handle a large sample load. The basic components of an LCEC system are depicted in Fig. 1.

Fig. 1 Basic components of an LCEC system. (Reproduced with permission of Bioanalytical Systems, Inc.)

Phenols and indoles have been known for at least 60 years to be electrochemically reactive. Nevertheless, for all practical purposes it was not until the early 1970s that this reactivity was used to advantage by analytical chemists. Professor Ralph Adams and his co-workers at the University of Kansas were the first to recognize that the ease of oxidation of tyrosine and tryptophan metabolites might provide a ‘handle’ for measurement of these substances in brain tissue. Adams was particularly intrigued by the possibility of using implanted microelectrodes to follow the release of neurotransmitters in vivo. While this revolutionary idea must still be considered to be at a very early stage of development, a number of promising results have already been published. Several excellent reviews on in vivo electrochemistry have appeared in recent years (Marsden et al., 1984; Justice et al., 1985; Justice, 1987).

Electrochemistry has a distinct advantage compared to most analytical techniques in that it involves a direct conversion of chemical information to an electrical signal without need for intermediate optical or magnetic carriers. For example, all catechol derivatives can be readily oxidized at a graphite electrode to generate the corresponding orthoquinone, two protons, and two electrons:

Reaction 1

To use this anodic oxidation analytically, it is most convenient to measure the rate at which electrons are transferred across the electrode-solution interface, in other words, the anodic current, ia. The instantaneous current is directly proportional to the number of molecules coming into contact with the interface per unit time and can therefore be used to determine the concentration of the reactant in the neighboring solution.

One of the principal problems of electrochemistry is that its molecular specificity is inadequate for many purposes. All catechol derivatives in a complex mixture react similarly and generally cannot be distinguished, one from the other, by an electrode. For this reason it is necessary to incorporate a separation step into the electrochemical experiment. Modern reverse-phase or ion-exchange chromatography is ideally suited to this purpose because ionic mobile phases are used (necessary for electrochemical detection). Modern microparticle columns are capable of rapidly separating closely related compounds in a few minutes with relatively little dilution. Minimizing the dilution inherent in chromatography requires a careful selection of the column diameter to match the volume of sample available.

Liquid chromatography has many advantages for the trace determination of polar organic substances. The number of sample manipulations can often be reduced when compared to gas-phase, fluorescence, chemiluminescence, or radioenzymatic methods. The primary disadvantages are (1) the fact that samples must be processed in series for the final quantitation, and (2) that the reliability of the instrumentation (including columns) is not perfect. While the latter problem has been dramatically improved in the last few years there remains considerable room for further progress, particularly with respect to pumps, autosamplers, and columns. LC systems do require maintenance. Like automobiles, they can last a very long time with proper care. An excellent recent book contains many good ideas on how to care for a liquid chromatograph (Dolan and Snyder, 1989).

Because electrochemistry is a surface technique, it is a simple matter to build thin-layer detector cells with microliter volumes. Such cells are capable of monitoring eluted components without distorting the chromatographic separation. The first experiments in this area were carried out in the spring of 1972 (Kissinger et al., 1973) and since that time over two thousand papers have appeared, many of which are dedicated to neurochemical measurements. The physical principles of electrochemistry will be briefly reviewed in the following section.

2 Principles

Electrochemistry is one of the most sensitive tools available to the analytical chemist. The direct conversion of chemical information into electricity gives electrochemical measurements a significant advantage when compared with many other analytical techniques. Recent advances in metal-oxide semiconductors provide an inexpensive yet effective means to measure very low electric currents. MOSFET electronics combined with an appropriate electrode provides a sensitive and reliable approach to the determination of redox-active substances. Reactions at an electrode can be followed at a rate as low as 10−16 equivalents per second! Electrochemists can now make measurements at electrodes with a radius below 1 μm on a time scale of 10−8 s!

In order to effectively utilize such ‘amperometric’ measurements, several points must be considered. First, electrochemistry is a surface technique; to optimize its use for trace analysis one must enhance the ratio of the surface area relative to the volume of the solution, while keeping the latter small. Second, because electrochemistry is a ‘chemical’ as opposed to a...