HPLC Determination of the Cardiotonics, Dopamine and 4-Methyl-2-aminopyridine, in Serum Following Fluorescamine Derivatization

Abstract

A method for the simultaneous determination of the cardiotonics dopamine and 4-methyl-2-aminopyridine in serum, previously derivatized with fluorescamine, by high performance liquid chromatography coupled with a fluorescence detector is presented. Chromatographic and detection conditions were optimized; a LiChrospher 100 RP-18 reversed phase column (250 × 4 mm; 10 µm) and the mobile phase of methanol:water, in gradient mode, were found as the most suitable. The derivatives were eluted in 10 min with good reproducibility at a flow rate of 0.9 mL/min, being 20 µL the volume of sample injected. The detection was done at 490 nm using borax buffer solution (pH 8) for both cardiotonics. Relative standard deviations (n = 10) of 6.7% and 5.9% (concentration) and 2.4% and 3.05% (retention time), were obtained for dopamine and 4-methyl-2-aminopyridine, respectively. Recoveries of the spiked female rabbit serum ranged from 90% to 110%, and detection limits of 122 ng/mL for dopamine and 108 ng/mL for 4-methyl-2-aminopyridine, were obtained.

Keywords:

• Cardiotonics
• Dopamine
• Fluorescamine
• HPLC
• 4-Methyl-2-aminopyridine

ACKNOWLEDGMENT

This work was supported by the Ministerio de Ciencia y Tecnologia of the Spain Government (Project CTQ04-07778)

Manuscript 6396

Notes

^a n = 3. ^b Detection limit for a signal-to-noise ratio = 3. ^c Quantification limit for signal-to-noise ratio = 10.