Determination of Morphine and Its Metabolites in Human Urine by Capillary Electrophoresis with Laser Induced Fluorescence Detection Employing On-Column Labeling with a New Boronic Acid Functionalized Squarylium Cyanine Dye

Abstract

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.

Novel monofunctional and bifunctional boronic acid‐functionalized squarylium dyes as precolumn and on‐column labels for protein analysis by capillary electrophoresis with laser‐induced fluorescence

“…According to the results obtained for both dyes, we can conclude that SQ‐BA4 showed to be a more stable dye either with or without HSA protein than SQ‐DBA2. These results are consistent with LOD results in the last section and also with the stability constant studies, which showed values of 3.67 × 10 6 M −1 and 1.17 × 10 5 M −1 for SQ‐BA4 and SQ‐DBA2, respectively. However, these stability constant results are shown to be superior to other reported stability constants for some squarylium dye–protein complexes, having higher concentrations of both the dye and HSA …”

“…The limits of detection (LODs), calculated based on the signal to noise ratio, for HSA labeled in the case of on‐column mode with both SQ‐BA4 and SQ‐DBA2 dyes were relatively lower (more sensitive) than those for precolumn labeling. Also, the relatively low LOD for the protein complex with SQ‐BA4 is likely an indication of stronger protein–dye interactions than with SQ‐DBA2, which is in agreement with the higher value of the stability constant as determined previously for SQ‐BA4 with HSA . Also, limits of quantification (LOQs) in the case of on‐column mode with both dyes were one order of magnitude lower than those for precolumn labeling.…”

“…Fluorimetric studies that were previously conducted helped to determine the suitability of the boronic acid‐based squarylium dyes as protein labels. As a continuous work, CE‐LIF was employed to study the noncovalent interactions between the dyes and various model proteins.…”

“…To determine the sensitivity of on‐column vs. precolumn‐labeling methods, a protein titration was initially performed for each dye. In these studies, citrate buffer (pH 3.5) was used based on our former studies and the amount of both SQ‐BA4 and SQ‐DBA2 dyes remained constant at 1.00 × 10 −6 M, while the concentration of HSA in the injected sample was increased due to additional complex formation.…”

“…By understanding the nature of noncovalent interactions between the dye and protein, it is possible to develop a more efficient analytical method for detection as well as a more sensitive probe protocol. As such, absorbance and fluorimetric studies previously conducted in our previous work helped to determine the suitability of the squarylium dye series as protein labels using human serum albumin (HSA) as a model protein. To further asses their utility as protein labels, CE‐LIF was employed to study the noncovalent interactions between the dye and various model proteins.…”