HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF THE FLUORESCENT DYES FURA-2AND MAG-FURA STABILITY IN ORGANIC-SOLVENTS

The fluorescent dye indicators Fura-2/AM and Mag-fura are used to esti mate changes in intracellular concentrations of calcium and magnesium, respectively. HPLC, coupled to fluorescence and ultraviolet detectors , indicated that these dyes are unstable when dissolved in methanol an d are especially unstable in glass containers compared to plastic. Bot h dyes were very stable in acetonitrile and dimethyl sulfoxide. The gl ass vials used in these studies appear to contain an unidentified subs tance which causes changes in the spectral intensities of these fluore scent dyes in the presence of methanol but not other solvents. With Fu ra-2/AM, there was an initial two fold increase in fluorescence during the first four hours in methanol and glass with no change in retentio n time, peak shape or ultraviolet absorption. With Mag-fura, no increa se in fluorescence was observed but instead there was a reduction of m ore than 90% in both fluorescence and ultraviolet absorption within 30 min, indicating that significant decomposition occurs under these con ditions. These results confirm previous studies which suggest that ace tonitrile is preferable to methanol for sample preparation and chromat ographic analysis of Fura-2 and Mag-Fura. These data also indicate tha t glass vials contain an extractable substance which markedly enhances the spectral intensities of Fura-2/AM.