title: Caffeine and paraxanthine HPLC assay for CYP1A2 phenotype assessment using saliva and plasma.
abstract: 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(18) 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(2)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.
authors.affiliation: Faculty of Pharmacy, The University of Sydney, NSW 2006, Australia.
authors.forename: Vidya
authors.initials: V
authors.lastname: Perera
authors.forename: Annette S
authors.initials: AS
authors.lastname: Gross
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: McLachlan
mesh_terms: D019540:Area Under Curve; D002110:Caffeine / Q000008:administration & dosage / Q000032:analysis / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism*; D004355:Drug Stability; D006801:Humans; D016018:Least-Squares Analysis; D008297:Male; D010641:Phenotype; D015203:Reproducibility of Results; D012463:Saliva / Q000737:chemistry* / Q000201:enzymology; D012680:Sensitivity and Specificity; D013806:Theophylline / Q000032:analysis / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics*; D055815:Young Adult
chemical_list: D002110:Caffeine; D013806:Theophylline; C504573:CYP1A2 protein, human; D019388:Cytochrome P-450 CYP1A2; C021183:1,7-dimethylxanthine