Quantification of caffeine in human saliva by Nuclear Magnetic Resonance as an alternative method for cytochrome CYP1A2 phenotyping

• We present a quantitative 1H NMR method to determine caffeine in human saliva.
• This NMR method was applied to follow caffeine clearance for CYP1A2 phenotyping.
• Caffeine clearance was determined for the first time by quantitative NMR in saliva.

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 1H 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 24 h following a single oral dose of about 100 mg of caffeine administered with either coffee beverage or a capsule.

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