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consisting of an aromatic ring [15] conjugated with a diene system (Fig. 1.1.8; excitation 375 nm;
emission 475 nm). This reaction does not require a separation of excess fluorescent reagent or reaction
by-products from the analytical fluorophore for application to biological samples. The method reported
by Tolan et al. [14] involves acetylation of the hydroxyl groups with acetic anhydride in pyridine in
advance of dehydration and this derivatization required a reaction time of 24 h.

Fink et al. [16] review the evolution of the derivatization procedure from the standpoint of
improvements in throughput and sensitivity. Connors and co-workers found 4-dimethylaminopyridine
and some Nalkylated imidazoles are superior to pyridine as a nucleophilic catalyst for aetylation by
acetic reagent [17,18]. When 1-methylimidazole was used as an acetylation catalyst, the derivatization
reduced the reaction time to 1 h [19]. Trifluoroacetic anhydride as the acetylation reagent reduced the
reaction time to less than 30 seconds. For this case, the detection limit of ivermectin was ca. 20 pg (S/N
= 2) [20]. The use of laser-induced fluorescence detection further reduced the detection limit [21].

In this case, knowledge based on organic chemistry contributes to establish a sophisticated
derivatization method without using fluorescent reagents. The reaction does not require a separation of
excess fluorescence reagent or reaction byproducts from the analytical fluorophore for application to
biological samples.

1.1.2.4—
Other Methods

In this section, some recent publications (1996 or later) on the derivatization of drugs using HPLC are
summarized.

Antiviral Agents

Cidofovir and cytosine-containing compounds in plasma [22].

Derivatization: Precolumn derivatization with phenacyl bromide to form 2-phenyl3,N4-ethenocytosine
derivative

Detection: Fluorescence

Analytical parameters: LOQ, 5 ng/ml (ca. 100 fmol on column); betweenday and within-day precision,
<17%.

Antitubercular Agents

Ethambutol in human plasma and urine [23]. Derivatization: Precolumn fluorescence derivatization
with 4-fluoro-7-nitrobenzo 2-oxa-1, [3-diazole]

Clean-up: Liquid-liquid extraction, from basified plasma samples with diethyl ether and back-extracted
into 0.01 M phosphoric acid Detection: Fluorescence
Analytical parameters: LOQ, 10 ng/ml in plasma and 10 µ/ml in urine.

Antineoplastic Agents

5-Fluorouracil in hair [24]. Derivatization: Fluorescence Clean-up: Liquid-liquid extraction Detection:
Fluorescence

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