Investigation of far red dyes for use in peroxyoxalate chemiluminescence detection and analysis of the cy5 derivative of amantadine hydrochloride in human plasma.

Peroxyloxalate chemiluminescence is well established as a tool for improvement of selectivity and sensitivity for chemiluminophores and their derivations in HPLC eluates. Chemiluminescence in the far-red spectral region was investigated in this work to further enhance the sensitivity of chemiluminescence through more efficient singlet excitation energy transfer and to enhance the selectivity of the approach through a reduction in matrix and scatter interference. A number of fluorescent compounds that can be excited in the UV, visible and far red spectral regions were investigated for chemiluminescence yield using the bis(2,4,6-trichlorphenyl) oxylate reaction. It was found that a trend of increasing chemiluminescence with increasing excitation wavelength could be observed with cy5 providing the most efficient chemiluminescence. The succinate ester of cy5 was used to derivatize amantadine hydrochloride, an antiparkinsons drug, to form the derivative. The derivative was separated from reaction by products by C18 reversed phase HPLC and detected using a Soma S-3400 chemiluminescence detector. The detection limit for the diluted derivative was 200 femtomoles on column and sufficient for plasma analysis. Selectivity in plasma was demonstrated through derivatization of extracts of plasma that had been spiked with amantadine hydrochloride.