title: Quantification of caffeine in human saliva by Nuclear Magnetic Resonance as an alternative method for cytochrome CYP1A2 phenotyping.
abstract: The first step in caffeine metabolism is mediated for over 95% by the CYP1A2 isoform of cytochrome P450. Therefore, CYP1A2 activity is most conveniently measured through the determination of caffeine clearance. The HPLC quantification of caffeine is fully validated and is the most widely used method. It can be performed on saliva, which is gaining importance as a diagnostic biofluid and permits easy and low invasive sampling. Here, we present a quantitative (1)H nuclear magnetic resonance (NMR) method to determine caffeine in human saliva. The procedure is simple because it involves only an ultra-filtration step and a direct extraction in a deuterated solvent, yielding a matrix that is then analyzed. The reliability of this NMR method was demonstrated in terms of linearity, accuracy, recovery, and limits of detection (LoD). Good precision (relative standard deviation, RSD <4%), a recovery of >95% and LoD of 6.8·10(-7) mol L(-1) were obtained. The method was applied to samples collected from different volunteers over 24h following a single oral dose of about 100mg of caffeine administered with either coffee beverage or a capsule.
authors.affiliation: Department of Chemical Sciences, University of Padova, via Marzolo 1, 35131 Padova, Italy. Electronic address: elisabetta.schievano@unipd.it.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Schievano
authors.affiliation: Department of Chemical Sciences, University of Padova, via Marzolo 1, 35131 Padova, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Finotello
authors.affiliation: illycaffè S.p.A., via Flavia 110, 34147 Trieste, Italy.
authors.forename: Luciano
authors.initials: L
authors.lastname: Navarini
authors.affiliation: Department of Chemical Sciences, University of Padova, via Marzolo 1, 35131 Padova, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Mammi
mesh_terms: D002110:Caffeine / Q000032:analysis* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism*; D006801:Humans; D057230:Limit of Detection; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D012463:Saliva / Q000737:chemistry* / Q000378:metabolism
chemical_list: D002110:Caffeine; C504573:CYP1A2 protein, human; D019388:Cytochrome P-450 CYP1A2
keywords: Caffeine; Cytochrome P450 1A2; Phenotyping; Quantitative Nuclear Magnetic Resonance; Saliva