High-performance liquid chromatography of 5-fluorouracil after derivatization with 4-bromomethyl-7-methoxycoumarin. Characterization of the derivative and the use of column switching for the improvement of resolution and the enhancement of sensitivity.
The derivatization of 5-fluorouracil with 4-bromomethyl-7-methoxycoumarin has been reported previously; however, the structure of the derivative was not confirmed. The synthesis and purification of the 5-FU derivative is described along with the spectroscopic (MS and NMR) determination that it is labelled at both heterocyclic nitrogens as expected. A column switching HPLC system is also presented which consists of primary separation on a cyanopropyl column followed by a final separation on an ODS column with fluorescence detection. This system removes all interferences from the derivatization system and has a limit of detection for the pure derivative of less than 50 fmol (injection volume = 100 microliters).
Kindberg
C G
CG
Center for Bioanalytical Research, University of Kansas, Lawrence 66045.
Slavik
M
M
Riley
C M
CM
Stobaugh
J F
JF
D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation; D002853:Chromatography, Liquid; D005472:Fluorouracil / Q000032:analysis* / Q000737:chemistry; D015394:Molecular Structure; D013053:Spectrophotometry; D014468:Umbelliferones / Q000737:chemistry*
D014468:Umbelliferones; C000317:4-bromomethyl-7-methoxycoumarin; D005472:Fluorouracil