Abstract
A novel method for the labeling and rapid separation of morphine, morphine-3-beta-d-glucuronide (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.
Highlights
Morphine is an opioid analgesic drug used for the treatment of moderate to severe pain.The primary metabolism pathway of morphine is by way of glucuronidation, where glucuronic acid is added at the 3- or 6-carbon of the molecule through UDP-glucuronosyltransferase-2B7 enzyme (UGT2B7) action in the liver, leading to formation of morphine-3-beta-D-glucuronide (M3G) and morphine-6-beta-D-glucuronide (M6G) [1]
In an effort to establish a broader range of possible applications for boronic acid functionalized squarylium dyes, we explored the effects of solution conditions on interactions of SQ-BA3 with morphine, M3G and M6G, as described
Spectral properties of the novel squarylium dye SQ-BA3 were determined under various solution conditions in order to assess its suitability as a fluorescent probe for morphine and its glucuronide metabolites
Summary
Morphine is an opioid analgesic drug used for the treatment of moderate to severe pain. Squarylium dyes are a particular class of cyanine dyes that were first synthesized almost 50 years ago [22,23] by way of the condensation of squaric acid with two aromatic and/or heterocyclic compounds to yield 1,3-disubstituted dye structures These dyes exhibit effective light absorption and are resistant to photodegradation, and they have been used in organic solar cells and optical recording media, and as photoconductors in copier machines [24,25,26]. Buffer composition and pH were shown to impact the stability of the dye-analyte complex and its emission properties By optimizing these experimental parameters, we could develop suitable on-column labeling protocols for the simultaneous detection of morphine and its isobaric glucuronide metabolites M3G and M6G by a rapid and efficient CE-LIF method. The sensitivity of this method does not rival that of CE-MS, it does illustrate the utility of the method as a possible screening tool for small molecule drugs and drug metabolites, with the possibility of extension to other analytes in this class
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