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A Unique Two-Phase
Derivatization Using a Novel Fluorescent Labeling Reagent
for Carboxylic Acids
Kazumi
Sasamoto
Director, Research & Development Department,
Dojindo Laboratories
- A
new coumarin-based amine, 4-aminomethyl- 6,7- dimethoxycoumarin,
was synthesized as a precolumn fluorescence derivatization
reagent for carboxylic acids, and the derivatization using
the amine in a two-phase medium of water and ethyl acetate
was examined.
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Two-phase derivatization of carboxylic acids with 4-aminomethyl-
6,7- dimethoxycoumarin.
-
The
compound was designed to enhance its fluorescence upon
derivatization due to the elimination of intramolecular
fluorescence quenching of the fluorophore by the benzylic
amino group. The two-phase derivatization technique with
the amine allows simple and rapid HPLC assays of carboxylic
acids with a reasonable sensitivity and separability.
The relatively small peak intensities of early-eluting
components are characteristic to this method, which suggests
that the unreacted amine, being protonated at a neutral
pH, remains in the aqueous phase. The detection limit
was determined to be 20 - 50 fmol per 10-ul injection
volume at a signal-to-noise ratio of 3 under partially
optimized conditions. The method was also applied to the
assay of fatty acids in human serum.
A typical chromatogram of human serum.
A 50 ul of human serum was vortex-mixed with 200 ul
of ethyl acetate and 100 ul of dilute HCl (pH 3.0) for
5 min and the supernatant was separated. To this was
added 50 ul DMF solution containing 4-aminomethyl- 6,7-
dimethoxycoumarin (4 mM) and HOBt (25 mM), 40 ul of
phosphate buffer (0.5 M, pH 7.0), 100 ul of EDC (2 M
in water), and 10 ul of nonadecanoic acid (1 mM in methanol)
as an internal standard; the mixture was again vortex-mixed
at 25 C for 5 min and a 10-ul aliquot of the organic
layer was injected to HPLC.
HPLC analyses were carried out using a Wakosil-II 5C18
HG reversed-phase column with an excitation wavelength
of 348 nm and an emission wavelength of 429 nm, at an
oven temperautre of 40 C. The mobile phase was aqueous
acetonitrile, which was pumped at a flow rate of 1.0
ml / min. Peaks: 1 = C14:1; 2 = C18:3; 3 = C14:0; 4
= C16:1; 5 = C20:4; 6 = C18:2; 7 = C16:0; 8 = C18:1;
9 = C18:0; 10 = C19:0.
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