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Fig. 3.26.
Fluorescence labeling reagents for carboxylic acids.
(1) Bromomethyl Reagents (Fig. 3.27)

Br-MMC was first used as a fluorescence labeling reagent for carboxylic acids (Fig. 3.27A) [108]. The
application of this reagent is reviewed in detail [109]. The reagent reacts with carboxylic acids in
acetonitrile or acetone under fairly drastic conditions and the reactions are catalyzed by a crown ether
(18-crown-6, dibenzo-18-crown-6) and potassium ion (K CO , KHCO ) [110-113]. In general, the
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reaction of the acids with Br-MMC is carried out by heating at 60 °C for 30-60 min for monocarboxylic
acids and 80 °C for about 60 min for dicarboxylic acids. The MMC esters of fatty acids are determined
at 10 pmol levels. Br-MMC have the following disadvantages; the reagent has a limited sensitivity and
the fluorescence intensity of the MMC esters of fatty acids are strongly affected by their chain length
and the eluent composition [114]. In order to overcome these problems, various bromomethyl reagents
are developed. MMC esters are detected by He-Cd LIF laser, and permit the detection of the acids at the
fmol levels [115].

Br-DMC [116] and Br-MDC [117] (Fig. 3.27B) are reported as higher sensitive reagents for carboxylic
acids than Br-MMC. Br-DMC is applied successfully to the analysis of 5-fluorouracil, an antineoplastic
agent, in human serum [118]. Br-MDC is used in a pharmocokinetic study of loxoprofen in human
plasma and urine [119].

Br-MAC is reported as a highly sensitive reagent [120]. This high sensitivity is based on the strong
fluorescence intensity of 4-methyl-7-hydroxycoumarin in alkaline media. After derivatization of
carboxylic acids with Br-MAC and HPLC separation, the eluent is mixed with a 0.1 M borate buffer
(pH 11.0). The MAC esters are hydrolyzed to 4-methyl-7-hydroxycoumarin, and measured
fluorimetrically.

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