electrochemistry

Methods in neurotransmitter and neuropeptide research S.K Parvez, M. Naoi, T. Nagatsu, S. Parvez (Eds.) © 1993, Elsevier Science Publishers Β. V. All rights reserved

41 CHAPTER 2

Determination of biogenic amines, their metabolites, and other neurochemicals by liquid chromatography/electrochemistry CHESTER T. D U D A 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

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

42 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.

COLUMN

TEMPERATURE CONTROLLED OVEN

CELL

POTENTIOSTAT

C

c

I

I to V C O N V E R T E R

I

DATA P R O C E S S I N G

3 D

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

43 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 4 the ease of oxidation of tyrosine and tryptophan metabolites might recognize that 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.

44 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 16 substances. Reactions and reliable approach to the determination of redox-active at an electrode can be followed at a rate as low as 1 0 " equivalents per second! 8 Electrochemists can now make measurements at electrodes with a radius below 1 jLtm on a time scale of 1 0 ~ 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 'physical' approach (such as U V absorption or fluorescence) the medium always has an important influence on the experiment. Third, unlike some high-resolution gas-phase techniques (such as GCMS), electrochemistry has relatively poor molecular selectivity among those classes of compounds which are redox active. These three factors must be taken

45 into account in the design and use of thin-layer amperometric transducers for liquid chromatography detection. Most amperometric transducers are based on 'thin-layer hydrodynamic chronoamperometry', which, loosely translated, means the measurement of current at controlled potential as a function of time. The various electrodes are placed in a flowing stream configured as a thin film. The potential difference applied to the electrochemical cell between the reference and working electrodes is the excitation signal, analogous, for example, to wavelength in spectrophotometry or fluorescence. The potential difference serves as the driving force for the electrochemical reaction to be monitored. In a solution with sufficient electrolyte concentration, nearly all of the potential is applied across a very thin interfacial region (typically less than 50 A thick) between the working electrode surface and the solution (Fig. 2). 5The6resulting electric field in this zone is therefore very large, of the order of 1 0 - 1 0 V / c m . The magnitude of the applied potential, to a first approximation, determines the magnitude of the interfacial electric field. The more positive the potential, the better an electron 'sink' (oxidizing agent) the working electrode

SOLUTION (IONS)

°

\ ^ - E L E C T R I C

H

FIELD

Fig. 2. The bulk of the applied potential is impressed across a very thin interfacial zone termed the electrical 'double layer', a loosely ordered arrangement of ions which acts as a capacitor. The field strength is varied by changing the potential of the solution relative to the working electrode. Note that it is the potential difference across the interface which determines the field, rather than the absolute potential of either phase alone.

46 becomes. Conversely, its ability as an electron 'source' (or reducing agent) improves as the applied potential becomes more negative. The current measured at the working electrode surface is the response to the applied potential and results from whatever redox processes occur at the electrode surface, under the conditions selected. If a reduction takes place, electrons flow from the electrode to the molecule in a heterogeneous transfer; conversely, an oxidation is the transfer of electrons in the opposite direction. Under steady-state conditions, the current measured is contributed from three sources: (1) the background electrolyte (mobile phase in LCEC), and (2) the electrode material itself, and (3) the analyte (eluted peak in LCEC). The medium and electrode are chosen so that the contributions of (1) and (2) are as small as possible and the small 'residual current' from these two sources is electronically removed prior to quantitation of the analyte. To minimize solution volume for liquid chromatography detection it is practical and convenient to configure the electrochemical cell as a thin-layer sandwich. This type of flow cell can faithfully reproduce the shape of concentration profiles eluting from high-efficiency LC columns. Fig. 3 is an illustration of such an amperometric

OUTLET

INLET

Fig. 3. Thin-layer amperometric cell for LCEC. The 'sandwich' is assembled without screws, making it possible to quickly accommodate a variety of working-electrode configurations. Dual-parallel and dual-series electrodes are illustrated. (Reproduced with permission of Bioanalytical Systems, Inc.)

47 transducer. The thin-layer channel is defined by a gasket held between a stainlesssteel block and a polymeric block. The stainless-steel block is by itself an auxiliary electrode and is also the holder for the reference electrode. The polymeric block contains the working electrodes. This low dead volume (0.1-1 μ,Ι) configuration is needed when two or more types of detectors are used in series and it permits the collection of fractions for subsequent analysis, isolation, etc. What determines the applied potential necessary to force the oxidation (or reduction) of the analyte as it passes through the thin-layer cell? Several factors are involved. Of primary importance is the structure of the molecule in question. Many organic functional groups are not electrochemically active at usable potentials. These include hydrocarbons, primary aliphatic amines and alcohols, aldehydes, ketones, etc. This 'drawback' to its use as a universal detector gives electrochemistry an advantage when it comes to maintaining adequate selectivity for a complex environmental or biochemical specimen. Such samples may contain hundreds or even thousands of components, far beyond the resolving power of modern, state-of-the-art liquid chromatographic columns. In neurochemical experiments, the selectivity of the detector in screening out many potential interferences is usually more relevant than its ability to sense all components. Fortunately, the functional groups that are accessible to LCEC are important from either industrial, environmental a n d / o r biochemical standpoints. Phenols and aromatic amines undergo 1- or 2-electron oxidation reactions; their electrochemical reaction products may be simple free radicals and associated coupling products, imines, quinones, or quinoneimines. Aromatic nitro compounds may be reduced to hydroxylamines or anilines. Related to this group are nitrate esters, various C- and N-nitroso substances, and diazo compounds. Just as there are characteristic absorption bands in ultraviolet spectroscopy, electroactive functional groups have similar zones of characteristic redox potentials. Analogous substituent effects may be predicted. To ascertain redox potentials in general, several options are available. In many cases, already published research papers may indicate the proper applied potential. Also, tabulations of substituent effects for a given type of functional group are available. Finally, the compound, dissolved in an appropriate mobile phase, may be subjected to cyclic voltammetric analysis. Analogous to generating an ultraviolet spectrum, cyclic voltammetry (CV) discerns the redox potential(s) of the species in the mobile phase. CV is also useful for suggesting the mechanism of chemical reactions which are often coupled to the initial electron transfer reaction. The CV experiment involves scanning potential in a linear sweep and measuring the current that arises as a function of the potential at any point along the sweep (Figs. 4 and 5). The peak potential may be used as an approximate indication of the voltage required for the amperometric detector. Space does not permit a thorough discussion of cyclic voltammetry, however. Neurochemists with a serious interest in LCEC a n d / o r in vivo electrochemistry should definitely consult other sources (Bard and Faulkner, 1980; Kissinger and Heineman, 1984; Heineman and Kissinger, 1989).

48

Fig. 4. Excitation (potential vs. time) and response signal (current vs. potential) for cyclic voltammetry.

3. Instrumentation The basic structure of an LCEC analyzer has been shown in Fig. 1. The first portion of the instrument is a high-resolution separation system. Mobile phase is provided by a constant-flow pump to an injection valve where samples are introduced. Most applications have employed isocratic separations (mobile-phase composition invariant with time), since gradient elution can cause baseline drift at high

49

Fig. 5. Instrumentation for cyclic voltammetry (courtesy of Bioanalytical Systems, Inc.)

detector gain. The flow should be as smooth and pulseless as possible, to minimize baseline noise. E C detectors will respond to pump pulsations. Dual-piston pumps with inexpensive pulse dampers are most commonly used. Syringe pumps are also satisfactory. Flow rates from 0.05 to 3.0 m l / m i n are typical. This will vary greatly depending on the length and the internal diameter of the LC column. Recently, columns of 1-3.2 mm have gained in popularity over the more traditional 4.6 mm i.d. columns, which dominated the field for 15 years. Smaller-diameter columns are advantageous when the amount of sample is limited since they cause less dilution of such samples during separation. Several recent papers demonstrate this with respect to LCEC of small neurotransmitter samples (Mefford et al., 1986) and microdialysis (Wages et al., 1986; Kendrick, 1990b; Huang et al., 1990a). Since an amperometric analysis necessarily involves a surface reaction between the electrode and the mobile phase and analyte, it is not surprising that the choice of geometry of the detector cell, the electrode material, and the mobile-phase composition are all crucial to success. The requirements for the mobile phase are straightforward: (1) it must have low electrochemical activity (i.e., low background currents), and (2) it must contain a dissociated electrolyte, usually at 0.01-0.1 M in ionic strength, to provide adequate conductivity and convey charge. Impurities from solvents (especially water) and salts are a frequent source of problems associated with high background currents and drifting baselines. Reagents of the highest purity are required. The mobile phase must be filtered! Particles will shorten the life of pump seals, check valves, injectors, and columns. They are very

50

Current Response

/ /

'BACKGROUND 100% Conversion

Area (Length) Fig. 6. Amperometric signal, background current, noise, and signal-to-noise ratio (SNR) as a function of electrode surface area with all other factors kept constant. The curves illustrate functional dependence; absolute magnitudes will depend on many factors besides electrode area.

damaging to LC systems and will frustrate the user who does not rigorously exclude them. Gas dissolved in the mobile phase can also contribute to poor performance. It is desirable that the pump receives the mobile phase with gases below saturation. This will minimize the likelihood of small air bubbles forming during the pump refill stroke or in the detector cell. Such bubbles can greatly upset the chromatographic baseline, limiting performance at low currents (high gain). Frequently, the amount of analyte reacted in a thin-layer cell is less than 5 femto-equivalents. For example, in the case of a molecule with a molecular weight of 200 undergoing a two-electron transfer, only 0.5 pg of sample may be converted into product while as much as 15 pg passes through the cell. Initially, it would seem worthwhile to increase electrode surface area and thereby increase the conversion efficiency. Unfortunately, the conversion efficiencies of both the analyte and the background electrolyte increase with surface area. The concomitant improvement in signal-to-noise ratio is not realized, due to the fact that each increment of surface area added contributes less and less to the total signal (Fig. 6). Eventually the increase in surface area contributes nothing to the signal amplitude while the noise continues to increase. A s a result the signal-to-noise ratio decreases. This is the reason that so-called 'coulometric detectors' do not give improved detection limits. All popular electrochemical detectors are amperometric in that current is measured vs. time. Coulometry is not an appropriate term for such experiments (Kissinger, 1986).

51 The choice of material for the electrode surface is critical to successful LCEC operation. Obviously the surface should be physically and chemically inert to the mobile phase at the chosen applied potential. Four electrode surfaces have found greatest utility: glassy carbon, carbon paste, platinum, and mercury. The most versatile choice is glassy carbon. It has excellent resistance to nearly any solvent used in liquid chromatography and may be used over a wide potential range, both positive and negative. Carbon paste, a mixture of graphite powder in a viscous organic binder, has for many years been a reliable surface for the determination of catecholamines and related substances. It has poor solvent resistance however, and finds application only in those situations involving low concentrations of methanol (e.g. 20-30%) or acetonitrile (less than 10%). A recent modification of the carbon-paste concept is the polymeric binder, which greatly enhances solvent resistance. Platinum is primarily used in combination with post-column enzymatic reactors. This allows the easy determination of such hard-to-detect analytes as glucose, acetylcholine and choline through the mediation of an appropriate enzyme. The end product of the reaction, hydrogen peroxide, is readily detected by the platinum electrode. A mercury electrode provides an extended negative range of potential for reducible substances, but is very limited in the positive direction. Conventional dropping mercury electrodes are not amenable to the low-dead-volume thin-layer design. A better alternative is to employ a mercury amalgam on a polished gold substrate. The mercury film can be made quite thin and very smooth. Mercury is advantageous compared to glassy carbon when dealing with substances which are difficult to reduce (Bratin and Kissinger, 1981). It is also the electrode of choice for determination of thiols and disulfides. 3.1. Multiple electrode

detectors

While most practical LCEC experiments continue to be carried out with transducers incorporating a single working electrode, it is a relatively simple matter to simultaneously monitor the current at several working electrodes. Multiple-electrode LCEC experiments have been reviewed by Roston et al. (1982). The electronics needed for multiple-electrode work is very straightforward and in principle any number of electrodes could be used. In practice, the use of more than three or four electrodes is rather awkward and expensive. Fig. 3 schematically depicts a thin-layer cell with two working electrodes in parallel, with a corresponding reference and auxiliary electrode across the channel. Using this arrangement one can monitor the amperometric current at two different applied potentials. This is perfectly analogous to the so-called 'dual-wavelength U V absorbance detector' and the output is typically plotted using a dual-pen recorder or dual-channel data-acquisition system. This arrangement is particularly useful when one wishes to quantitate a very easily oxidized (or reduced) substance in the presence of others which react at higher

52 energies (e.g. serotonin in the presence of tryptophan or dopamine in the presence of tyramine). This can often simplify sample preparation and save time by avoiding the need to repeat an injection at a different detector potential. The selectivity for the easily reacted substance can thus be excellent* and other compounds can be detected as well. Fig. 3 also illustrates another arrangement, the series dual-electrode transducer. The products of the upstream electrode are monitored at the downstream electrode. This can be useful to enhance selectivity when the electrolysis products can be detected in a more favorable potential region than was necessary to carry out the original reaction. In addition, some electrochemical reactions are 'chemically irreversible' (oxidation of tyramine) and some are 'chemically reversible' (oxidation of dopamine). The series dual-electrode arrangement can eliminate the response to irreversible processes while enhancing the response to reversible reactions. An application of this idea to determination or urinary catecholamines has been reported (Goto et al., 1981), as has a more general description of the technique (Roston and Kissinger, 1982). Fig. 7 illustrates a third dual-electrode arrangement, which permits enhancement of the response for reversible redox cycling. Many more electrons are therefore transferred than would be the case with a single electrode, and the current is amplified dramatically. This concept does not work well with conventional LC columns, because the volume flow rate is too great to permit a significant number of redox cycles within a cell of adequately small dead volume and reasonable cost. Nevertheless, the concept is certainly interesting, and as reverse-phase capillary columns are developed it may well have some practical value for detection of catecholamines. It would not be a useful approach for most tryptophan metabolites, because their redox reactions are chemically irreversible. The performance characteristics of the series arrangement is dependent on the spacing between the upstream and downstream electrodes. Their surface area is also important. If relatively small electrodes are placed far apart, they will behave independently. This has been used in an analyzer designed to monitor many neurochemicals simultaneously (Turk and Blank, 1990). Each electrode is designed to operate as an independent detector. Peak ratioing helps to confirm the identity of individual substances. Four electrodes can also be configured in two parallel sets of series electrodes. In this scheme, the upstream electrodes are 'generators' for

»wwwwwwwwwwwwww ». ///////////////////////////// Fig. 7. dual-electrode amperometric detection. Parallel-opposed configuration.

53 species detected downstream. Many such combinations are possible, but whether they really improved the situation for a given assay is not often obvious. Such schemes are always subject to the problem that all electrodes do not behave identically and can vary in their properties as a function of time. We cannot currently predict such changes very well. Therefore artifacts can be expected when ratio data between electrodes is critical to analysis over long periods of time. On the other hand, multiple electrodes used and calibrated independently function just as well as single electrodes and are in widespread use. 3.2. Temperature

control

Temperature may be the most neglected success factor in LC. Even today, many chromatographs are used with columns dangling in the air. In the early days, no attempt was made to control either column or detector temperature. The attitude developed that temperature is important to GC, but not so important to LC. Refractive index is quite temperature-dependent. Because of this, U V detector noise and drift is seriously impaired by small temperature changes, which can make it very difficult to do good work at 0.001 absorbance units full scale (a.u.f.s.). Diffusion coefficients have an even higher temperature dependence. Thus, electrochemical detectors are adversely influenced by temperature changes. A few degrees change can compromise quantitation statistics in a significant way. The redox kinetics of background processes in electrochemistry are even more dependent on temperature than the diffusion coefficients. The oxidation of water and methanol at glassy carbon is a good example. This results in serious baseline drift with ambient temperature changes. Finally, the LC process itself is quite dependent on thermodynamics (retention) and kinetics (peak width). Anyone doing chromatography without controlling temperature will not get numbers as good as they might! Both the column and detector cell should be isothermal and homeothermal. The best approach is to have them both in the same temperaturecontrolled environment. A n oven is best, although 'block heaters' can also help. Considering the very high cost of animal experiments, it is ill-advised to neglect controlling the LC temperature and thus to not obtain the highest-quality data. Many researchers carefully design their animal protocols and then neglect the instrumentation on which their data depend.

4. Tyrosine metabolites 4.1 Sample preparation

strategies

The goal of any bioanalytical method is to selectively extract information from a complex sample matrix containing a great deal of uninteresting 'noise'. At some point the method will most likely have to differentiate between compounds of very

54 similar structure. This is particularly true if one is exploring the metabolites of tyrosine. Fig. 8 illustrates one strategy which has been used in the determination of these compounds. They can be conveniently classified as bases, neutrals and acids. A simple p H adjustment followed by solvent extraction or liquid-solid adsorption will permit isolating these classes for individual determination (examples of such extraction schedules may be found in Westerink, 1983; Oka et al., 1984; Herregodts and Michotte, 1987). It is important to recognize that the procedure used in any particular case will depend to a great extent on the type of specimen and the ultimate analytical objective. For example, screening body fluids for highly elevated concentrations of biogenic amines or metabolites associated with tumors or severe stress can be a very simple matter, whereas determining the same substances in a healthy individual at rest may require more elaborate procedures. Of the many applications that have made use of LCEC, none has been more successful than that of 'mapping' neurotransmitter concentrations in brain tissue. Brain tissue is a remarkably clean matrix and is amenable to rapid sample work-up. Electrochemical detection permits the analysis of specific brain regions using small punch-outs from a thin tissue slice. One early example profiled the concentration of norepinephrine in the human thalamus (Oke et al., 1978). Recently microdialysis sampling of the living brain has become quite popular among diverse groups of neuroscientists. It is now possible to directly link an awake animal (including human patients) to an LCEC system using an automated syringe pump coupled to a sampling valve (Fig. 9). This is perhaps the ultimate in sample

TISSUE SAMPLE

Solvent Extraction

Small Column Isolation

Enzymade HydnDlysis

Small Column Isolation

Liquid-Solid Adsorption

Small Column Isolation

Small Column Isolation, TLC

Liquid-Solid Adsorption

LCEC M, NM, 3-MT

LCEC DA, EPI, NE

LCEC MHPG

LCEC VMA, HVA

LCEC DOPAC

BASIC Fig. 8. Isolation of tyrosine metabolites.

NEUTRAL

Solvent Extraction

ACIDIC

55

Fig. 9. Automated microdialysis/LC system for neurochemical studies in a conscious rat (courtesy of Bioanalytical Systems, Inc.).

preparation, with the molecular-size cut-off of the dialysis probe protecting the LC column from cells, cell fragments, and high molecular weight substances. 4.2.

Catecholamines

The basic compounds (amines) of primary interest are the catecholamines and the metanephrines. At low pH these protonated amines can be adsorbed to cation-exchange isolation columns. Additional selectivity, if desired, can be achieved by a solvent extraction step, as in the case of urinary metanephrines. Separation of these two groups of biogenic amines can be accomplished by using the reactivity of the catechol moiety. The amines are eluted from the columns into small collection vials. The pH is raised to 8.5 and a small amount (typically 100 mg or less) or alumina is added (Ganhao et al., 1991). Catecholamines selectively adsorb onto alumina at high pH, forming a stable complex. The alumina can be washed, removing the undesired compounds from the sample. The catechols can then be released from the alumina by the addition of dilute acid. The wash and desorption steps are most conveniently carried out using a centrifugal microfiltration device. Boric acid forms a similar complex with catechols, and a successful isolation procedure has been developed using boric acid gel (Koike et al., 1982), or ion-pair extraction using diphenylborate (Smedes et al. 1982). Although the few initial reports utilizing this isolation technique looked promising, it was never widely adopted. Alumina extraction with or without ion-exchange pretreatment still remains the method of choice (Elrod, 1984; Koch and Polzin, 1987). This approach has been extensively used for determining catecholamines in a variety of biological tissues and fluids (Fig. 10). Although urine and plasma require extensive extraction procedures, some tissues may simply be homogenized, deproteinated and clarified prior to injection. Micro-

56 UJ

ζ

I Û

Ο

0.1 nA

Fig. 10. Sample chromatogram for an actual plasma sample (courtesy of Bioanalytical Systems, Inc.).

dialysates can be directly injected, whereas CSF must be deproteinated and clarified. The relatively more complex tissue supernatant places a greater demand on the chromatography, but allows for the determination of catecholamines and metabolites and in some circumstances the indoleamines. A widely used LCEC method for N E and D A in rat brain parts employs a reverse-phase column modified by an anionic ion-pair reagent (Felice et al., 1978). The ion-pair reagent increases the capacity factors for the catecholamines and improves resolution. Modern ion-exchange columns can also be useful, as has been demonstrated for

57 many years. With respect to organic separations, ion exchange is really a special case of reverse-phase chromatography involving both electrostatic and hydrophobic interactions. In most cases the hydrophobic interactions predominate.

4.3.

Metanephnnes

As noted above, an important step often used in the isolation of the metanephrines is cation exchange. The strength of the cation-exchange resin can provide some degree of selectivity. Weak cation-exchange resins appear to work better for the metanephrines. At this point, some means of separating the catecholamines from the metanephrines must be employed. One approach would be to pass boric acid through the columns as a wash, complexing the catechols (Trouvin and BillaudMesguich, 1987). The metanephrines would remain on the column under these conditions. Another approach is to elute all of the amines with base. The catecholamines are very unstable to oxidation in base and would be eliminated from the final samples by this process. Although the original LCEC method was applied to urine samples (Shoup and Kissinger 1977), tissue (Davies and Heal, 1986) and plasma (Pagliari et a l , 1991) are also applicable matrices.

4.4. Acid and neutral

metabolites

To the neuroscientist interested in determining the actual rate of catecholamine turnover, monitoring the degradation products is just as important as monitoring the parent amines themselves. In brain tissue the metabolites of principal interest are 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3methoxy-4-hydroxyphenylglycol (MHPG), and 3,4-dihydroxyphenylethyleneglycol (DHPG). The latter two compounds are usually determined in the free neutral form following acid or enzymatic hydrolysis of sulfate and glucuronide conjugates (Filser et al., 1989). Vanillylmandelic acid (VMA) is thought to represent a very minor pathway in the brain and is currently of little interest for determining norepinephrine activity. However, V M A is a major metabolite of peripheral norepinephrine metabolism and may be useful in identifying changes in catecholaminergic release outside the CNS. As is the case with all catecholamine metabolites, some form of group isolation is generally advisable subsequent to protein precipitation and prior to LCEC. At low pH both the acidic and the neutral metabolites can be extracted into a polar organic phase with very high efficiency. Then, depending on the amount of information required from the sample, further isolation of acidic from neutral components may not be required and this extract can be injected directly on the LC. Hefti (1979) has shown that by choice of a less polar organic extraction solvent, D O P A C and H V A can be isolated with few other interferences. A similar pre-LCEC isolation can be achieved for the primary norepinephrine catabolites, but without the solvent extraction step. The clean-up is accomplished using a small gravity-fed

58 column that eliminates all LCEC interferences for the determination of free D H P G and MHPG. The importance of measuring the acidic and neutral catechol catabolites vs. the vanil catabolites is still an unresolved question with regard to norepinephrine and dopamine activity. It has been suggested that the catechol molecules are more indicative of interneuronal activity and the vanil compounds of extra-neuronal activity. The catechol acids and neutrals can be selectively adsorbed onto alumina just as for the catecholamines. The procedure calls for protein precipitation following homogenization in acid, hydrolysis of sulfate and glucuronide esters, solvent extraction, and finally adsorption onto alumina at high pH. The molecules are eluted off the alumina with an acid/methanol solution and injected into the LC. A great deal of selectivity is achieved due to the adsorption step, but additional selectivity accrues from the ease of electrochemical oxidation of o-hydroquinones as compared to the vanil compounds. An extended discussion on the determination of sulfated biogenic amines and metabolites is beyond the scope of this chapter. However, an excellent review has been published (Elchisak, 1987).

5. Tryptophan metabolites 5.1.

Overview

Neurochemical investigations focusing on tryptophan require the determination of one or more compounds from the list of serotonin, 5-HTP, 5-HIAA, and tryptophan. In brain tissue, significant interferences can exist, often making a sample clean-up step beneficial. The method we have found to be most useful is isolation on small gravity-fed extraction columns (Koch and Kissinger, 1979). The resin selected depends on the compound preferred. Each compound can be determined independently, although a desirable feature of this approach is the ability to assay several concurrently. The following brief sections present a strategy for each metabolite and a general method for determining two or more in series. A number of reports on these compounds have appeared in the literature. However, it is likely that any particular sample will require some procedural modifications. In some cases, no sample preparation is required beyond ultrafiltration, microdialysis, or protein precipitation. The low oxidation potential of the 5-hydroxy indoles provides very high selectively compared to most other neurochemicals. There are many procedures in the literature and the situation is evolving quite rapidly. 5.2.

Tryptophan

A strong cation-exchange resin (e.g. Dowex AG-50) is employed for liquid-solid isolation of tryptophan after homogenization and centrifugation of tissue samples. Elution is followed by injection onto a reverse-phase liquid chromatograph. Am-

59 perometric detection occurs at an oxidizing potential of about + 1 . 0 V. With little alteration of this method, tryptophan from cerebrospinal fluid, plasma or urine can be determined. 5.3.

5-Hydroxytryptophan

Exactly the same procedure is followed for 5-HTP as for tryptophan. Due to the 5-hydroxy group, detection requires a potential if only + 0.5 V, enhancing selectivity. The amounts of 5-HTP in tissue are small, placing extreme demands on the analytical method. 5.4.

Serotonin

Isolation of 5-HT can be successfully achieved on a weak cation-exchange resin (Amberlite CG-50). The detector potential is + 0 . 5 0 V. Applicability to tissue, serum, CSF and urine has been established. The three-fold isolation, combining liquid-solid extraction, chromatographic separation and electrochemical detection, provides this method with maximum selectivity so that serotonin is typically the only compound which will oxidize at the chosen potential. 5.5. 5-Hydroxyindoleacetic

acid

A gel resin (Sephadex G-10) selectively adsorbs 5-HIAA. The assay for tissue samples generally functions best when a protectant (reducing agent) such as cysteine is added. The same reverse-phase chromatograph is used as for 5-HT with a detector potential of + 0.50 V. Urine, serum and CSF can be assayed as well as tissue samples. 5.6. Concurrent assay of tryptophan

metabolites

When desired, two or more of the above metabolites can be determined in a sequential process. In all cases, the step that is unnecessary may be skipped. For example, when an investigation requires determination of 5-HT and 5-HIAA, the method begins with the Amberlite isolation. The effluents from these columns are applied directly to the Sephadex resin, and the adsorption of 5-HIAA takes place at the same time the elution of 5-HT is carried out. In some cases a very simple sample preparation, such as homogenization and clarification, can be used without an extraction step. 5.7. Precolumn

sample enrichment

of 5-HT and

5-HIAA

The approach noted above for the four tryptophan metabolites is well suited for routine service in the analysis of whole brain or large regions of brain tissue. The technique is relatively rapid and the technology is rather inexpensive.

60 The desire to determine serotonin and 5-HIAA in localized regions of the rat brain, as in punch-outs from some brain slices, requires a method with extremely good sensitivity. If needed, the small, gravity-fed extraction columns used for the cleanup step in the methods above are quite convenient, but do not interface well with liquid chromatography. The minimum volume needed to elute the compound(s) of interest is much larger than the usual LC injection volume of approx. 20 μ,Ι. Thus, without some adaptation, more than 90% of the isolated compound is wasted, decreasing the overall sensitivity. A precolumn sample-enrichment system conveniently solves this problem (Koch and Kissinger, 1980a; Mailman and Kilts, 1985). Nearly 100-fold enhancement in the detection limit is obtained using this technology, allowing the determination of serotonin and 5-HIAA from small brain regions and punch-outs. Applicability to the assay of small amounts of serum and plasma has also been demonstrated (Koch and Kissinger, 1980b). The effect of /7-chloroamphetamine on the serotonin content of rat pineal gland was studied with an LCEC method (Fuller and Perry, 1977). Serotonin determinations based on LCEC have been used to quantitate concentrations in tissue (Hansson and Rosengren, 1978; Ponzio and Jonsson, 1979), synaptosomes (Dayton et al., 1979), CSF, plasma and urine (Table 1). The relative merits of LCEC and LC-fluorescence (LCF) have been compared (Anderson et al., 1981, 1990; Chin, 1990). The LCF technique can work well for certain tryptophan metabolites and for derivatized catecholamines. LCEC is generally considered to be superior to LCF for the neurochemical laboratory because it is a far more versatile tool. Virtually all of the aromatic biogenic amine metabolites (as well as many cofactors and drugs) can be detected electrochemically without derivatization. There is always the desire to acquire as much information from one sample or study as is possible. Although the resolving power of most reverse-phase columns is such that greater than a dozen compounds can be separated (Lin et al, 1984), from an analytical point of view the data may be of limited value. The polar compounds (NE for example) elute rapidly and are generally obscured by the void volume response or, if retained sufficiently, then the hydrophobic compounds (5-HT for example) are retained inordinately long. Chromatographic runs of 4 5 - 6 0 min would not be unusual if the catecholamines, indoleamines and their metabolites are to be separated (Morier-Teissier and Rips, 1987; Yi and Brown, 1990). Under isocratic conditions, this usually results in the late eluting peaks (analytes) being broad and rather difficult to quantitate. The more practical approach would be to optimize the chromatographic and detector conditions for the quantitation of a few select compounds. The advantages gained in lower detection limits (higher signalto-noise) and faster sample throughput will outweigh the necessity of running two or more assays (chromatography systems) simultaneously.

6. Enzyme activity The extension of LCEC to the measurement of the activity of the enzymes central to the biosynthesis of catecholamines and serotonin is a natural one (Boulton et al.,

61

TABLE 1

Specimen

Sample 3 handling

15 Chromatography

Reference

5HIAA,

Brain punches

Freeze thaw

RPIP

Renner and Luine, 1984

DA DA DA

Urine Urine Urine

RPIP RPIP RPIP

Peaston, 1988 Elrod, 1984 Huang et al., 1988

RPIP RPIP

Ganhao et al., 1991 Smedes et al., 1982

RPIP RPIP

Koike et al., 1982 Pagliari et al., 1991

Analyte

NE, DA, 5 HT NE, Epi, NE, Epi, NE, Epi,

NE, Epi, DA NE, Epi, DA

Plasma Plasma

NE, Epi, DA Free and total NMN, MN 3-MT NMN NMN, MN, 3-MT

Brain Plasma

Affinity column Comparison Ion exchange + affinity columns Alumina Ion-pair diphenyloborate Boric acid gel Ion exchange

Brain Urine Urine

Homogenization Ion-exchange Ion-exchange

RPIP RPIP RPIP

NMN, MN, 3-MT Free and conj. MHPG Free MHPG, 5HIAA, HVA Free MHPG Free and conj. MHPG Free MHPG Free and total MHPG, DHPG DHPG, DOPA DHPG, DOMA HVA, VMA, MHPG VMA, HVA VMA, HVA VMA HVA HVA HVA HVA Tyrosine

Urine Urine CSF

Ion-exchange Liq.-liq. extn. Protein pptn.

RPIP RP RP

Davies and Heal, 1986 Brown et al., 1986 Trouvin and Billaud-Mesguich, 1987 Shoup and Kissinger, 1977 Filser et al., 1989 Elrod and Mayer, 1985

Plasma Plasma Plasma Urine

Liq.-liq. extn. SFE SFE Ion-exchange

RP RPIP RP RPIP

Schinelli et al., 1985 Candito et al., 1988 Yang et al., 1988 Julien et al., 1988

Plasma Plasma Plasma Urine Urine Plasma Plasma Plasma CSF Plasma Plasma, brain

Alumina Alumina Liq.-liq.extn. Ion-exchange Direct Liq.-liq.extn. SFE SFE Liq.-liq.extn. Liq.-liq.extn. Direct homogenization Direct Liq.-liq.extn.

RPIP RPIP RP RP RP RP RP RP RPIP RPIP RPIP

Eisenhofer et al., 1986 Eriksson and Perrson, 1987 Gergardt et al., 1986 Ong et al., 1987 Hanai et al., 1987 Schinelli et al., 1988 Semba et al., 1988 Lambert et al., 1991 Szabo et al., 1988 Zumârraga et al., 1987 Edwards et al., 1986

RPIP RPIP

Protein pptn. Homogenization Homogenization, protein pptn. Trace enrichment

RPIP RPIP RP

Anderson et al., 1990 Narasimhachari and Landa, 1986 Munoz et al., 1989 deVries and Odink, 1991 Martin and Aldegunde, 1985

RPIP

Mailman and Kilts, 1985

5HT 5HT NE, Epi, 5HT 5HIAA, 5HT 5HTP, 5HT, 5HIAA

CSF Amniotic fluid, serum, urine Plasma Brain Brain, plasma

5HTP, 5HT, 5HIAA

Tissue

62 TABLE 1 (continued) Analyte

Specimen

Sample 3 handling

5 Chromatography

Reference

5HT 5HIAA 5HIAA 5HT, 5HIAA, Tryp 5HTP, 5HIAA, 5 HT, Tryp

Urine Urine Urine CSF Pineal

Ion-exchange SFE Direct Protein pptn. Protein pptn.

RP RP RP RPIP RPIP

Jouve et al., 1986 Chou and Jaynes, 1985 Elrod et al., 1986 Baig et al., 1991 Chin, 1990

a bSFE,

solid-phase extraction. RPIP, reverse phase ion-pair; RP, reverse phase.

1986; Nagatsu, 1991). The methods noted for the quantitation of tyrosine and tryptophan metabolites can often be modified to measure the activity of related enzymes. This subject will be discussed in a later chapter in this volume. One LCEC approach used to assay dopamine-/3-hydroxylase (DBH) demonstrates a unique application of automated column-switching technology. Dopamine, the natural substrate of D B H , is incubated with the required cofactors for a fixed period of time. The reaction is stopped by precipitating the protein with acid. The product, norepinephrine, is isolated by the alumina isolation procedure described earlier, without (Davis and Kissinger, 1979) or with a prior ion-exchange step (Racz et a l , 1986). A problem arose in this method that is frequently encountered with enzyme activity assays. One must use an excess of substrate to ensure zero-order kinetics with respect to substrate. When the reaction is stopped, a sizable quantity of unreacted substrate remains. Norepinephrine and dopamine cannot be isolated separately by the alumina method; consequently the total amount of recovered dopamine is extremely large in comparison to the enzymatically generated product. Fortunately, the reverse-phase ion-pair conditions employed in the assay eliminate resolution of the two compounds as a problem. Typical capacity factors for norepinephrine and dopamine using reverse-phase columns modified with an ion-pair reagent are 2.5 and 21 respectively. The hydroxyl group at the β position on the side chain has a large effect in reducing the hydrophobicity (and thus the retention) of norepinephrine relative to dopamine. The desired information from the chromatogram is available after norepinephrine elutes, but one is forced to wait for dopamine to elute before another sample can be injected. Another negative aspect of this situation is that the large amount of dopamine (substrate) injected easily overloads the column and saturates the outputs of the detector. This is undesirable because it can reduce the lifetime of the electrode. This problem can be solved by utilizing the chromatographic technique of split-column chromatography. The idea here is to use two short columns instead of a single longer one (Davis and Kissinger, 1979). A valve is positioned between the two columns and the chromatographic conditions adjusted so that the nore-

63 pinephrine passes through both columns and the detector prior to the time dopamine exits the first column. The valve is then switched, shunting the dopamine to waste. In the case of D B H , there is no value derived from measuring the unreacted substrate. Split-column chromatography decreases the times between injections and can increase the number of samples processed per day by 500%. The split-column scheme also avoids changing mobile-phase composition, as might occur if a step gradient has been used. In this case (reverse-phase ion-pairing conditions) it is not possible to instantly re-equilibrate the column due to the large capacity factor of the modifying ion-pair reagent. The split-column technique requires careful adjustment of the mobile phase and column lengths, but the substantial saving in analysis time is well worth the effort. An LCEC method developed for the measurement of rat brain and liver catechol-o-methyltransferase activity also uses dopamine as the substrate (Shoup et al., 1980). The earlier problem of isolating a small amount of product in the presence of enzyme-saturating concentrations of substrate is avoided in this method. The product and substrate are cations and both can be isolated on small cation-exchange columns. Using a strategy described earlier, boric acid is added to complex the catechol and 'wash' it from the column bed. A significant fraction of the dopamine is thereby eliminated. The residual amount present in the final eluate poses no problem under the reverse-phase ion-pair conditions employed to separate the metanephrines. Enzyme activity assays for the other enzymes in the tryosine metabolic pathway have been developed using LCEC, including tyrosine hydroxylase, dopa decarboxylase and phenylethanolamine-AT-methyltransferase. Since 5-HTP is readily oxidized, tryptophan hydroxylase is an obvious candidate for an assay based on LCEC. In addition, 5-HT and 5-HIAA are also easily oxidized, so that it is possible to monitor changes in all three metabolites simultaneously. As described, separation of tryptophan metabolites is easily achieved on a reverse-phase column. LCEC is useful for determination of N A D H and pterins (oxidized and reduced) and it appears likely that methodology will be developed based on using redox cofactors as the analytical 'handle' on enzyme activity.

7. Amino acids Direct E C detection of amino acids is generally limited to tyrosine, tryptophan, L-DOPA, and cysteine. The detection of a mixture of amino acids requires some type of pre-or post-column derivatization in order to achieve the necessary detection limits for neurochemical purposes. The chemistries that have been used are varied (for an excellent recent review, see D o u et al., 1990), and in many cases have to be modified to fit a particular sample matrix. Derivatization of amines with aromatic nitro reagents is by now an ancient practice to biochemists. This is convenient for LCEC because all aromatic nitro groups can be easily reduced electrochemically. The measurement of the in vitro

64 release of endogenous γ-aminobutyric acid (GABA) from a caudate mince utilizing precolumn derivatization with 2,4,6-trinitrobenzenesulfonic acid has been reported (Caudill et al., 1982). In another study with a similar goal and derivatization technique, G A B A was determined using two working electrodes in series; deoxygenation of mobile phase and sample was not required (Yamamoto et a l , 1985; see also Jacobs, 1982). The reaction between primary alkyl amines and o-phthalaldehyde (ΟΡΑ) in the presence of an alkyl thiol is a widely used derivatizing chemistry for determination of amines and amino acids by LC. The normal products of this reaction, 1-alkylthiol-N-alkylisoindoles, are formed rapidly in high yield and can be detected with good sensitivity using either fluorescence or electrochemical oxidation (Joseph and Davies, 1982, 1983). Poor stability of the isoindoles, due to further reaction with excess Ο Ρ Α in the derivatization mixture, has been a problem (Jacobs et a l , 1986).

SR

Reaction 2. Formation of ΟΡΑ-amino acid derivatives using the ΟΡΑ/ί-butyl thiol reagents.

Derivative stability can be markedly improved by alterations in the structure of the thiol used as coreagent. Replacing mercaptoethanol (usual coreagent for LCF) by teri-butylthiol (tBT) or substituting sulfite for the thiol results in half-lives for the derivatives of several hours (Allison et a l , 1984; Jacobs et a l , 1986; Jacobs, 1987a). While such changes in derivative structure have relatively little impact on electrochemical reactivity, they have been shown to often degrade the quantum yield of fluorescence. O P A / t B T derivatives of a mixture of neurotransmitter amino acids in microdialysis samples and brain homogenates have been separated by linear gradient (Shea and Jacobs 1989a; Globus et a l , 1991) or step gradient elution (Zielke, 1985; Hikal et a l , 1988). In cases where one or a few amino acids are of interest, simpler separation schemes may prove more appropriate. A n isocratic method designed expressly to allow determination of G A B A in microdialysis samples has been reported (Shea and Jacobs, 1989b). Aspartic and glutamic acid in microdialysis samples have also been determined using an isocratic separation. The more hydrophobic amino acid derivatives must be flushed out with a step (or gradient) to a higher organic content if sample throughput is to be optimized (Jacobs and Shea, 1989). Not only is LCEC an attractive alternative to LCF for the detection of OPA-derivatized amino acids, it augments the usefulness of ΟΡΑ chemistry by adding flexibility and expanding

65 the range of applicability. Of particular interest is the reported ability to determine peptide derivatives with good sensitivity (Jacobs, 1986). Further research on alternative thiols is under way to further enhance performance and reduce the odor which currently requires the use of sealed autosampler vials for both reagent storage and reaction.

NDA

CBI-derlvative

Reaction 3. Reaction of N D A / C N with primary amines.

A relatively new reagent designed for detection of primary amines has been used for the determination of amino acids and peptides (Lunte and Wong, 1990a,b). The reagent, naphthalene-2,3-dicarboxaldehyde (NDA), reacts with primary amines in the presence of cyanide to yield l-cyano-2-substituted-benz[/]isoindole (CBI) derivatives. The CBI products of the amino acids are substantially more stable than their OPA/mercaptoethanol counterparts. In addition, the CBI derivatives are electrochemically active, fluorescent, and absorb in the U V . In very recent work, Lunte and coworkers have demonstrated the determination of amino acids in brain microdialysates using N D A derivatization prior to capillary electrophoresis. ΟΡΑ chemistry has been used for the determination of other endogenous biological amines as well as exogenously introduced amines. The polyamines (spermine, spermidine, putrescine, and cadaverine) (Morier-Teissier et al., 1988) and the sympatomimetic drugs (Leroy et al., 1983) have been determined using this derivatization chemistry coupled to LCEC. Determination of secondary amine drugs via LCEC requires utilization of other derivatization chemistries (Leroy and Nicolas, 1984; Nakahara and Takeda, 1988).

8. Choline and acetylcholine The neurochemical acetylcholine (ACh) and its precursor, choline (Ch), are refractory to electrochemical, absorbance, and fluorescence detectors, under reasonable conditions. Although there are numerous methods for their determination, including bioassay, gas chromatography-mass spectrometry and radioenzymatic assay, most are complicated and time-consuming, lack sensitivity or specificity, require expensive equipment or require disposal of hazardous waste.

66 Using enzymes as specific and selective derivatizing agents in liquid chromatography was first reported in the early 1970's. For ACh and Ch the initial report (Potter et a l , 1983) utilized post-column addition of soluble enzymes (acetylcholinesterase and choline oxidase) to the effluent from the analytical column. Endogenous Ch and Ch formed by the enzymatic hydrolysis of ACh were both hydrolyzed by choline oxidase to betaine (non-EC-active) and hydrogen peroxide. The peroxide generated was then detected downstream using oxidative EC. The specificity of the method was based on LC (separating ACh from Ch, and both from interferences), two specific enzyme-catalyzed reactions, and EC detection of hydrogen peroxide on a Pt electrode. The homogeneous procedure, as outlined by Potter et al. (1983), does not allow for utilization of the full catalytical potential of the enzymes. After a single use enzyme is directed to waste. A simpler assay, conserving enzyme and requiring less equipment, was described by Meek and Eva (1984). Acetylcholinesterase and choline oxidase were adsorbed to a weak anion-exchange cartridge. Conversion of ACh and Ch to peroxide is quantitative during the residence time in the cartridge. However, separation conditions are limited since the quasi-immobilized enzymes are easily washed off by moderate ionic strengths. Furthermore, the pH required for maximum catalytic activity of choline oxidase (pH 8.5) is detrimental to the silica-based matrix of the anion-exchange cartridge. With these factors in mind, the next logical step was to covalently immobilize the enzymes onto a polymeric matrix and use a polymeric analytical column. This results in a more rugged enzyme reactor and analytical column under the high-pH conditions required of assay and allows for greater flexibility in separation conditions (Shoup, 1989a). Several covalent attachment methods of acetylcholinesterase and choline oxidase have been reported (Damsma et a l , 1985; Asano et a l , 1986; Shoup, 1989a). Most immobilized enzyme reactors (IMERs) have utilized columns of various lengths and internal diameters (2.1 to 4.6 mm) with detection limits around 1 pmole. Utilizing 1 mm microbore columns (Fig. 11) (Huang, 1991) or high-speed columns (Damsma et a l , 1987), detection limits of 5 0 - 7 5 fmoles/injection have been reported. ACh-Ch has been determined in a wide variety of biological tissues and fluids: in heart (Nomura et a l , 1990); in plasma (Fujiki et a l , 1990); in CSF (Okuyama and Ikeda, 1988; Matsumoto et a l , 1990); in microdialysates from spinal cord and brain (Tyrefors and Gillberg, 1987; Damsma et a l , 1987; Shoup, 1989a); cultured neurons (Takei et a l , 1988, 1990) and brain tissue (for example, Kaneda et a l , 1986). The LCEC detection of ACh and Ch has been extended to the measurement of enzymes associated with the metabolism of ACh. Assays for choline acetyltransferase (Kaneda and Nagatsu, 1985) and acetylcholinesterase (Kaneda et a l , 1985) activities have been reported. In both reports less than a milligram of tissue could be utilized and the concentration of enzyme was determined from the amount of product formed (ACh and Ch respectively). As mentioned above, excess substrate can be a problem in enzyme activity assays, but not so in this instance. The

67

ΟΙ

A

Β

c

2

D

Ε

ο

kl 8

min

Fig. 11. Acetylcholine (ACh) and choline (Ch) in rat striatum microdialysate using microbore (1.0 mm i.d.) columns. A, blank perfusion solution (Ringer's); B, dialysate collected prior to addition of neostigmine to perfusion solution; C and D, dialysate collected during perfusion of Ringer's solution containing neostigmine; Ε, 1 pmole standard (Huang, 1991).

transferase requires excess Ch as co-substrate, which can easily be removed by a pre-column (i.e. pre-analytical) IMER containing choline oxidase and catalase. The choline is oxidized to betaine and H 20 2, and the catalase oxidizes H 20 2 to 0 2 and H 20 ; the final result being that only ACh is detected (via the post-column IMER-EC combination). The esterase requires excess ACh as substrate, a much easier-to-handle situation; the post-column IMER contains only choline oxidase (Overdorf, 1991) and thus only choline is detected. The determination of ACh in biological tissues and fluids almost always requires that endogenous acetylcholinesterase be inactivated. In studies involving isolated

68 organs, this has been conveniently accomplished by focused microwave irradiation (Potter et al., 1983; Ikarashi et al., 1985; Damsma et al., 1985; Asano et al., 1986; Nomura et al 1990). In this technique, localized heating denatures the esterase in situ and also results in the death of the animal. The brain or other tissue can then be excised and handled using conventional surgical techniques. There were significant differences in ACh concentrations in tissue, dependent upon whether death was by in vivo irradiation or decapitation; in situ microwave irradiation is generally accepted as the method of choice. Another promising technique is in situ freezing, which allows blood perfusion of cerebral tissue until freezing time (Beley et al., 1987). In vivo sampling of tissue via the microdialysis technique also requires inactivation of acetylcholinesterase. Since the animal (test subject) must be kept alive, the above techniques will not apply. ACh needs to be protected only until it passes through the semipermeable membrane of the microdialysis probe into the dialysing solution. The esterase inhibitor neostigmine, added to the dialysing solution, has been used (Damsma et al., 1987; Shoup, 1989a; O'Connor et al., 1991). Neostigmine diffuses out of the probe into the surrounding fluid, reversibly inhibiting the endogenous esterase. Neostigmine, at the concentrations used in microdialysis, does not appear to affect the IMER-based determination of ACh (Damsma et al., 1987; Shoup, 1989b). Although there is little direct evidence of a neurochemical role for polyamines and glucose, their determination has been of interest. Both glucose and the polyamines (putrescine, spermidine, spermine and cadaverine) have been determined utilizing IMER technology combined with LCEC (Huang and Kissinger, 1989b; Maruta et al., 1989; Watanabe et al., 1989).

9. Microdialysis and column format Microdialysis, as an in vivo sampling technique, has come into widespread use (as demonstrated by the attendance at the second International Symposium on Microdialysis and Allied Analytical Techniques, Current Separations Vol. 10, pp. 66-119, 1991; and the number of papers using this technique presented at the 5th International Conference on in vivo Methods; Rollema et al., 1991). The technique has been applied to anesthetized and freely behaving animals, and used to monitor biogenic amines in various tissues and fluids (Bibliography of Microdialysis, Bioanalytical Systems, Inc.), release of peptides (Kendrick, 1990), drug binding, stability, and pharmacokinetics (Lunte and Scott, 1991; Lunte et al., 1991; Scott et al., 1990, respectively), glucose, purines, organic acids; microdialysis is in principle a technique which can collect virtually any low molecular weight substance from any tissue and fluid with a minimum amount of damage or fluid loss. A number of excellent reviews have appeared, the most recent being that by Benveniste and Huttemeier (1990) and a book edited by Robinson and Justice (1991).

69 LU Ζ

Α.

B.

< Û

5ηΑ

4 min

LU

I ill JL Fig. 12. Comparison between microbore and conventional columns. Same amount of catecholamines (0.5 ng in 5 μ,Ι) injected on the two columns. (A) microbore column, 100 X 1.0 mm, ODS, 3 μ,πι, flow rate 70 μΐ/min. (B) conventional column, 100 X 3.2 mm, ODS, 3 μπ\, flow rate 0.7 ml/min (Huang et al., 1990a).

Microdialysates can fit many analytical methodologies, including immunoassay, mass spectrometry with liquid interface, ion-selective electrodes, electrophoresis, and clinical analyzers. Nevertheless, the vast majority of publications from laboratories active in microdialysis suggest that liquid chromatography is the most general method of choice. The primary reason for this is that many researchers wish to determine more than one analyte at a time or one substance within a mixture. In neuroscience, there is the added convenience that many LC methods for tyrosine metabolites (catecholamines, et al,), tryptophan metabolites (serotonin, et al,), amino acids, and acetylcholine have been developed for biological fluids, tissues, and cell cultures. Often these methods, along with the apparatus already at hand, can be adapted to dialysates. Microdialysis provides a highly filtered, low volume, aqueous solution of low molecular weight polar analytes. In many situations, this defines the ideal sample for direct injection into a liquid chromatograph. An LC is a diluter! When we start with a low-concentration sample, the last thing we would want to do is dilute the sample. When a separation is required, there is little choice. We do, however, have a choice of column length and

70

0.05 nA

4 min

1

Fig. 13. Chromatogram of in vivo microdialysate collected 5 h after perfusion was initiated. Sample corresponds to a 2.5 min sampling interval; 4.3 femtomoles of serotonin are observed. Column was 1 mm i.d. (Huang et al., 1990b).

diameter. Traditional columns, 2 5 - 3 0 cm long and 4.6 mm in diameter, really have no place in most microdialysis applications. If the column length is reduced from 25 to 10 cm, we expect the contribution to dispersion to be decreased by 60%. Reducing the diameter from 3.2 or 4.6 mm to 1 mm will present the detector with a 10- or 20-fold more concentrated analyte, for columns of equal length (Fig. 12). These changes in column geometry may also require changes in the pumping system, injector, connecting tubing and detector (Kissinger and Shoup, 1990; Huang et a l , 1990a; Kissinger, 1991). The latter three components of the system will affect extra-column band broadening, which needs to be minimized. The

71 demands placed on reducing extra-column dispersion are not as great for the 'high speed' (3-4.6 mm diameter and 10-15 cm long) columns, and most of the assays for biogenic amines and metabolites, amino acids, and acetylcholine have been transferred to this column size. More recently, some of these assays have utilized microbore columns that are 1 mm in diameter (Huang, 1991; Huang et al., 1990a,b; Kendrick, 1990b; Wages et al., 1986). The electrochemical detector is compatible with both high-speed and microbore column formats, because it is a surface-based rather than a volume-based device; for thin-layer cells the dead volume is determined by gasket thickness and can easily be reduced to below 0.5 μΐ. Why then are not all LCEC determinations carried out using a microbore column? The primary determinant will be sample size. If the concentration is low but the sample size is relatively large, then a conventional or narrow-bore (2-3 mm i.d.) column can be used; amount injected can be increased by increasing the volume injected (50-200 μ,Ι). If the concentration and sample size are low then microbore columns would be of advantage (Fig. 13). A 10 μΐ microdialysate sample may otherwise be unduly diluted. In order to select an appropriate column format, it is necessary to define the sample problem in terms of meaningful chromatography and analytical parameters and take the time to reason them out (see Kissinger and Shoup, 1990; Kissinger, 1991).

10. Review of general applications of LCEC to drugs and other organics 10.1 Aromatic

amines

Like phenols, aromatic amines are oxidized at a carbon electrode over a wide range of oxidation potentials. Some compounds (phenylenediames, benzidines and aminophenols) are ideal candidates due to their very low oxidation potentials, and numerous applications have been developed. For example, LCEC is commonly used for the determination of benzidine and related compounds, primarily in urine (for monitoring in the workplace) and wastewater. Detection limits of 50 pg or better are not unusual for these readily oxidized bis-anilines. Hydroxylamines, naphthylamines, toluidines and sulfanilamides are representative of other substances in this class. For example, LCEC has been used to determine the content of the suspect agent 4-methoxy-m-phenylenediamine in hair-dye preparations. Another analytical solution provided by LCEC was in quantitation of aromatic amine residues in commercial polyurethane foams. A standard manufacturing scheme involves the reaction of 2,4-diisocyanotoluene (TDI) with a long-chain polyol followed by reaction in situ with 4,4'-methylene-bis(2-chloroaniline), MOCA, as a curing agent. Both M O C A and the hydrolysis product of TDI, 2,4-diaminotoluene, may be assayed by LCEC methodology. These and related toxic amines have been purported to leach out of some materials used for medicinal implants.

72 10.2.

Thiols

Thiols ('sulfhydryls' or 'mercaptans') are very easily oxidized to disulfides in solution, but this thermodynamically favorable redox reaction occurs only very slowly at most electrode surfaces (e.g. glassy carbon). LCEC methods for thiols therefore usually depend on the unique behavior of these compounds at a mercury electrode surface at about + 0 . 1 0 V (a very low potential). The reaction involves formation of a stable complex between the thiol and the mercury surface. Formally, the mercury rather than the thiol is oxidized. This approach has been used to determine the amino acid cysteine, the tripeptide glutathione, and the pharmaceuticals penicillamine and captopril (Allison and Shoup, 1983). Disulfides can also be determined by a simple modification of this procedure. A dual-series electrode approach is used. The upstream mercury electrode functions as a post-column reactor to reduce any disulfides eluting from the analytical column; the thiols produced from these disulfides are swept downstream to the second mercury electrode where they are oxidized (detected). Endogenous thiols as well as the disulfide-derived thiols are individually detected here by virtue of their chromatographic separation (for a short review see Shoup, 1987). This scheme has been applied to the detection of disulfide bonds (and free thiols) in peptides (Jacobs, 1987b). 10.3. Miscellaneous

oxidizable

compounds

A number of unique substances have been studied by oxidative LCEC. Being an excellent reducing agent, ascorbic acid is easily detected with excellent selectivity in very complex samples (Pachla et a l , 1985; Huang and Kissinger, 1989a). Similarly, uric acid is readily detected in biological materials (Pachla et a l , 1987). The important enzyme cofactor N A D H is readily oxidized at carbon electrodes, and LCEC methods for this biochemical are beginning to appear (Davis et a l , 1979; Wright et a l , 1986; Eisenberg and Cundy, 1991). Some heterocycles of pharmaceutical interest (phenothiazines, imipramine) are also uniquely applicable (Lavrich and Kissinger, 1985; Kissinger, 1989; Kissinger and Radzik, 1991). Vitamin B 6 derivatives (pyridoxal, pyridoxic acid, pyridoxamine, etc.) are phenolic in nature and LCEC provides a selective approach for these substances at the subnanomole level. 10.4.

Quinones

Quinones are among the best behaved organic compounds with respect to redox reactions in aqueous solutions. There is a reasonably large number of synthetic and natural products containing the quinone moiety and many of these are excellent candidates for selective determination by LCEC. The antineoplastic drug adriamycin is an example of a complex quinone that may be reduced at a very

73 moderate negative potential. Naturally occurring quinones, such as a-tocopherolquinone and ubiquinone, and their respective reduced products, α-tocopherol and ubiquinol, have been determined in various biological samples (Pascoe et a l , 1987; Okamoto et a l , 1988, respectively). Vitamin Ε and α-tocopherolquinone were determined in a single injection utilizing dual-series electrodes. 10.5. Nitro

compounds

Nitrobenzene was one of the first organic compounds studied by classical polarography in the 1920's, and it is not surprising that nitro (and nitroso) compounds have been among the most extensively investigated by both organic and analytical electrochemists. Aromatic nitro and nitroso compounds are very readily reduced at both carbon and mercury electrodes, but other compounds such as nitrate esters, nitramines, and nitrosamines are often good candidates as well. Many pharmaceuticals, explosives, agricultural chemicals, and important industrial intermediates fall into these classes, and a number of LCEC methods have been developed (Bratin and Kissinger, 1981). Often the selectivity is extremely good in biological and environmental samples, because the nitro group is rare in nature and few other organic compounds are so easily reduced. Reagents containing the aromatic nitro group have frequently been used to derivatize amines, aldehydes, ketones, and carboxylic acids to improve their characteristics for determination by U V absorption spectroscopy. The same or closely related reagents are now being used to provide an electrochemically reactive handle for many of these same compounds (Jacobs and Kissinger, 1982b; Krull et a l , 1985). While it is true that aldehydes and ketones can be electrochemically reduced and alkyl amines and carboxylic acids can be electrochemically oxidized, the energy required to carry out these well-known reactions is far too great to permit development of a successful LCEC trace determination procedure without derivatization.

11. Conclusion Liquid chromatography/electrochemistry (LCEC) has in most instances become the method of choice for the determination of neurologically important biogenic amines and their metabolites. While very significant progress has been made recently, there is still room for improvement. Simplified sample work-up procedures and more reliable columns are two major areas where further work is needed. The electrochemical detection of many tyrosine and tryptophan metabolites is now routine for injection of picomole amounts isolated from biological samples. In some cases, even subfemtomole amounts have been determined. Although the present discussion has emphasized the application of LCEC to endogenous compounds of neurological interest, there have been many applica-

74 tions to other biomedical problems. Thiols, phenothiazines, ascorbic acid, morphine, uric acid, methylxanthines, pterins and a number of other compounds have been measured in biological samples by direct electrochemical

detection. Re-

ducible substances can now be readily detected at limits approaching those which have been well established for oxidizable substances. This opens the field to a wide variety of pharmaceuticals, agricultural chemicals, and industrial intermediates. Some of these compounds are neurologically active and LCEC may well play a role in future work in neurotoxicology. Recently various pre- and postcolumn reaction schemes have been devised which extend electrochemical detection to compounds which are themselves not electroactive at easily accessible potentials. Amino acids, glucose, acetylcholine, fatty acids and unsaturated lipids are among those classes of compounds which are now detectable using hyphenated amperometric methods.

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