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Fig. 3.19.
Fluorogenic reactions of catecholamines by: (A) THI, (B) ED and (C) DPE method.

simple clean-up of plasma by solid-phase extraction using a cation-exchange cartridge. Cleanup by
liquid-liquid extraction is also effective [82,83]. An on-line automated pre-column derivatization has
been devised for reproducible results [84]. An automated catecholamine analyzer system has been
constructed based on the DPE reaction applied to post-column HPLC. For the quantification of
catecholamines and their metabolites (normetanephrine et al.), an ion-pair RP-HPLC system with post-
column derivatization involving coulometric oxidation followed by fluorescence reaction with DPE has
been established.

DPE Application: HPLC Determination of Plasma Catecholamines [81]

To 0.5 ml of plasma, 25 µ1 of 10 nM isoproterenol (IS) solution and 0.5 ml of 0.2 M lithium phosphate
buffer (pH 5.8) are added. The mixture is poured into a Toyopak SP cartridge. The cartridge is washed
successively with 5 ml of water (twice) and 1 ml of aqueous 50% acetonitrile. The adsorbed amines are
eluted with 300 µ1 of 0.6 M KC1-acetonitrile (1:1, v/v) containing 0.6 mM potassium hexacyanoferrate
(III). To the resulting solution, 50 µl of 0.1 M DPE in 0.1 M HCI are added and the mixture is allowed
to stand at 37°C for 40 min. The reaction is stopped by cooling the mixture in ice-water. A 100-µl
aliquot of the mixture is applied onto RP chromatography (Fig. 3.20).

3.2.2.2—
Tryptophan and Indolamines

Tryptophan and indolamines react with formaldehyde [86], chloroacetaldehyde [86] and
methoxyaldehyde [87] to form fluorescent derivatives. The reaction conditions are fairly drastic (acidic
medium, 80-100 °C, 15-60 min) in the presence of an oxidizing agent, and they are used for precolumn
derivatization.

PGO reacts selectively with tryptophan under relatively mild conditions [88]. This reaction is applied to
the determination of free and total tryptophan in human serum [89] (Fig. 3.21).

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