then be measured by fluorescence spectroscopy. In this situation, since one is essentially measuring the same fluorescent molecule, albeit "tagging" different amino acids, a single optimal excitation and emission combination is appropriate, and so a fixed wavelength detector is adequate.

Electrochemical detection depends on the redox equilibrium reaction of molecules (e.g., A ^ A+ + e_). Application of a positive potential drives the oxidation reaction, and application of a negative current drives the reduction reaction. In electrochemical detection, a positive potential is applied and the current produced by the oxidation is measured at the working electrode. Although many compounds will oxidize if a high enough potential is applied, only a few oxidize at usable potentials. Notable among these are amine transmitters, such as dopamine, serotonin, and noradrenalin, and many of their metabolites. Most amino acids are not electrochemically active within a useful potential range, but their oPA derivatives (see above) do show electrochemical activity. Therefore it is also possible to use electrochemical detection for amino acids. While this has not been widely used for most amino acids, it is useful for GABA, which has often proved difficult to measure fluorometrically.

Enzymatic Modification

Some neuroactive compounds, notably ► acetylcholine, are difficult to detect using any of these methods. However, enzymatic treatment yields an electrochemically active product, which can be detected. Thus for acetylcholine, an additional column is placed after the analytical column, which has the enzymes acetylcholine esterase and choline oxidase immobilized onto the stationary phase. These enzymes break acetylcholine down, and produce hydrogen peroxide as a bi-product: hydrogen peroxide can be measured with an electrochemical detector. Any choline in the sample will also produce hydrogen peroxide, but can be distinguished from acetylcholine because the two have first been separated chromatographically on the analytical column.

The output from the detector feeds to a recording device, such as a chart recorder, or more commonly, an integrator: changes in current, representing the presence of a detectable entity, are displayed as peaks on a current versus time plot (the chromatogram). The area under the curve of these peaks is proportional to the concentration of the compound, and can be quantified by reference to standards of known concentration, run under identical conditions. Similarly, for a given ► mobile phase and ► stationary phase combination, individual compounds will have a constant retention time, and thus component compounds in a mixture can be identified by comparison with standards.

Thus, HPLC is an extremely versatile technique, and methods can be devised for separation, identification and quantification of a wide variety of chemical compounds. It is widely used in psychopharmacology to measure endogenous compounds, such as neurotransmitters, and metabolites, and exogenous compounds such as drugs, in post mortem brain tissue, in vitro, and in vivo procedures. It can also be used to measure these compounds in CSF and blood, as indicators of brain levels, and to measure the pharmacokinetic and pharmacodynamic profile of drug action.

Further Reading

Hanai T (1999) HPLC, a practical guide. Royal Society of Chemistry, Cambridge

Hancock WS (1984) HPLC analysis of biological compounds: a laboratory guide. Dekker, New York Kromidas S (2000) Practical problem solving in HPLC. Wiley-VHC,

Weinheim, Chichester Lim CK (1986) HPLC of small molecules: a practical approach. IRL, Oxford oliver RWA (1998) HPLC of macromolecules: a practical approach.

oxford university Press, oxford Robards K, Haddad PR, Jackson PE (1999) Principles and practice of modern chromatographic methods. Academic, London

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