A novel method for the labeling and rapid separation of morphine, morphine-3-beta-Dglucuronide (M3G) and morphine-6-beta-D-glucuronide (M6G) in human urine employing a new boronic acid functionalized squarylium dye (SQ-BA3) and capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is described. The spectrochemical properties, solution stability, pH range, and mechanisms for interactions with morphine and its metabolites were first established for SQ-BA3, followed by optimization of an on-column labeling procedure and CE-LIF method. SQ-BA3 itself was shown to be unstable and weakly fluorescent in aqueous buffers due to aggregate formation. However, SQ-BA3 showed a relative stability and dramatic increase in fluorescence intensity upon the addition of morphine, M3G, and M6G. Because of the low background fluorescence of this dye, on-column labeling was feasible, leading to a simple and rapid analytical method with the potential for clinical applications.

This article presents a continuous capillary electrophoresis with laser‐induced fluorescence (CE‐LIF) following spectral studies of the noncovalent interactions between novel Squarylium Boronic Acid 4 (SQ‐BA4) & Squarylium Diboronic Acid 2 (SQ‐DBA2) squarylium dyes and human serum albumin (HSA). Two protocols were used wherein the on‐column‐labeling protocol was found to be more sensitive than the precolumn one by showing a better enhancement in the peak area of the HSA–dye complex besides lower limits of detection (LODs) for HSA. Also, stability studies were conducted with or without HSA using precolumn‐labeling mode over one week exhibiting the superiority of SQ‐BA4 to SQ‐DBA2. Then, a mixture containing three model proteins, HSA, β‐lactoglobulin B, and transferrin, was labeled on‐column with both dyes and completely resolved by CE‐LIF after optimization of several parameters. Both dyes provided lower LODs for HSA than those of β‐lactoglobulin B and transferrin with higher sensitivities. In addition, the SQ‐BA4 dye showed again greater sensitivities with all the three proteins than SQ‐DBA2.