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Published on: August 21, 2019
A simple, sensitive microsample LC-MS assay for quercetin and isorhamnetin in mouse and human plasma: application to
Purnajai Srivijay Saravanan Vanaja1, Subodh Mishra2, John Douglas Cleary2
1Department of Pharmaceutics, Virginia Commonwealth University School of Pharmacy, Richmond, VA, USA.
Background:
Quercetin, a dietary flavonoid with emerging therapeutic relevance in myotonic dystrophy type 1 (DM1), has low solubility and poor oral bioavailability. Enzymatically modified isoquercitrin (EMIQ), a water-soluble prodrug, raises systemic quercetin exposure. Pharmacokinetic studies require a sensitive assay that uses minimal sample volume.
Research Design And Methods:
We developed a single-quadrupole liquid chromatography-mass spectrometry (LC-MS) assay for free quercetin, total quercetin (after enzymatic hydrolysis of glucuronide and sulfate conjugates), and the methylated metabolite isorhamnetin in mouse and human plasma. The method used protein precipitation, 10 µL of plasma, reversed-phase C18 separation, and single-ion recording of [M+H]+ adducts. Validation followed a fit-for-purpose approach consistent with M10 guidelines, and the assay was applied to plasma from EMIQ-treated DM1 and wild-type mice (15 g/L for 6 and 12 weeks).
Results:
Calibration curves showed r2 > 0.99, with an LLOQ of 0.070 µM for quercetin in both matrices. The assay was successfully validated for quercetin in mouse and human plasma. Total quercetin and isorhamnetin were quantifiable in all treated mice. Exploratory analysis suggested glucuronidation as the major conjugation pathway.
Conclusions:
This simple, cost-effective microsampling assay suits preclinical and translational studies of EMIQ in DM1, though the conjugation findings remain exploratory.
