Caffeine and paraxanthine HPLC assay for CYP1A2 phenotype assessment using saliva and plasma

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<jats:title>Abstract</jats:title><jats:p>Caffeine has been extensively used as a probe to measure CYP1A2 activity in humans with caffeine clearance or the paraxanthine (major metabolite of caffeine) to caffeine concentration ratio being regarded as the preferred metric. A simple reverse‐phased C<jats:sub>18</jats:sub>HPLC assay using ethyl acetate liquid–liquid extraction was developed to quantitate caffeine and paraxanthine concentrations in saliva and plasma. The mobile phase consisted of acetonitrile–acetic acid–H<jats:sub>2</jats:sub>O (100:1:899) and analytes were quantitated with UV detection at 280 nm. The extraction recovery for paraxanthine and caffeine was approximately 70% in both saliva and plasma. The assay was linear over the concentration ranges 0.05–2.50 and 0.05–5.00 µg/mL, for paraxanthine and caffeine, respectively, in saliva. In plasma the assay was linear over the ranges 0.025–2.50 and 0.025–5.00 µg/mL for paraxanthine and caffeine, respectively. Intra‐ and inter‐assay precision and accuracy were less than 15%. Detection limits were 0.015 µg/mL for paraxanthine and caffeine in saliva, while it was 0.005 µg/mL for paraxanthine and caffeine in plasma. Utility was established in samples collected from two healthy volunteers who abstained from caffeine for 24 h and received a single 100 mg oral dose of caffeine. The assay developed is a robust, simple and precise technique to measure caffeine and paraxanthine in saliva and plasma of healthy volunteers after a single oral dose of caffeine. Copyright © 2010 John Wiley & Sons, Ltd.</jats:p>

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