title: Development and validation of a reversed-phase HPLC method for CYP1A2 phenotyping by use of a caffeine metabolite ratio in saliva.
abstract: CYP1A2 is important for metabolizing various clinically used drugs. Phenotyping of CYP1A2 may prove helpful for drug individualization therapy. Several HPLC methods have been developed for quantification of caffeine metabolites in plasma and urine. Aim of the present study was to develop a valid and simple HPLC method for evaluating CYP1A2 activity during exposure in xenobiotics by the use of human saliva. Caffeine and paraxanthine were isolated from saliva by liquid-liquid extraction (chlorophorm/isopropanol 85/15v/v). Extracts were analyzed by reversed-phase HPLC on a C18 column with mobile phase 0.1% acetic acid/methanol/acetonitrile (80/20/2 v/v) and detected at 273nm. Caffeine and paraxanthine elution times were <13min with no interferences from impurities or caffeine metabolites. Detector response was linear (0.10-8.00µg/ml, R(2) >0.99), recovery was >93% and bias <4.47%. Intra- and inter-day precision was <5.14% (n=6). The limit of quantitation was 0.10µg/ml and the limit of detection was 0.018±0.002µg/mL for paraxanthine and 0.032±0.002µg/ml for caffeine. Paraxanthine/caffeine ratio of 34 healthy volunteers was significantly higher in smokers (p<0.001). Saliva paraxanthine/caffeine ratios and urine metabolite ratios were highly correlated (r=0.85, p<0.001). The method can be used for the monitoring of CYP1A2 activity in clinical practice and in studies relevant to exposure to environmental and pharmacological xenobiotics.
authors.affiliation: Laboratory of Pharmacology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Greece.
authors.forename: Elias
authors.initials: E
authors.lastname: Begas
authors.affiliation: Laboratory of Pharmacology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Greece.
authors.forename: Evangelos
authors.initials: E
authors.lastname: Kouvaras
authors.affiliation: Laboratory of Chemistry, Faculty of Medicine, School of Health Sciences, University of Thessaly, Greece.
authors.forename: Andreas K
authors.initials: AK
authors.lastname: Tsakalof
authors.affiliation: Laboratory of Pharmacology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Greece.
authors.forename: Maria
authors.initials: M
authors.lastname: Bounitsi
authors.affiliation: Laboratory of Pharmacology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Greece.
authors.forename: Eftihia Konstadinos
authors.initials: EK
authors.lastname: Asprodini
mesh_terms: D002110:Caffeine / Q000378:metabolism*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism*; D006801:Humans; D057230:Limit of Detection; D010641:Phenotype; D012015:Reference Standards; D015203:Reproducibility of Results; D012463:Saliva / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
chemical_list: D002110:Caffeine; D019388:Cytochrome P-450 CYP1A2
keywords: CANCER; CYP1A2; DRUG METABOLISM; PHENOTYPE; SALIVA