Simultaneous Quantification of Antioxidants Paraxanthine and Caffeine in Human Saliva by Electrochemical Sensing for CYP1A2 Phenotyping

• Main Authors: Rozalia-Maria Anastasiadi, Federico Berti, Silvia Colomban, Claudio Tavagnacco, Luciano Navarini, Marina Resmini
• Format: Article
• Language: English
• Published: MDPI AG 2021-12-01
• Series: Antioxidants
• Subjects: paraxanthine; caffeine; CYP1A2 phenotyping; antioxidants; human saliva; differential pulse voltammetry
• Online Access: https://www.mdpi.com/2076-3921/10/1/10

The enzyme CYP1A2 is responsible for the metabolism of numerous antioxidants in the body, including caffeine, which is transformed into paraxanthine, its main primary metabolite. Both molecules are known for their antioxidant and pro-oxidant characteristics, and the paraxanthine-to-caffeine molar ratio is a widely accepted metric for CYP1A2 phenotyping, to optimize dose–response effects in individual patients. We developed a simple, cheap and fast electrochemical based method for the simultaneous quantification of paraxanthine and caffeine in human saliva, by differential pulse voltammetry, using an anodically pretreated glassy carbon electrode. Cyclic voltammetry experiments revealed for the first time that the oxidation of paraxanthine is diffusion controlled with an irreversible peak at ca. +1.24 V (vs. Ag/AgCl) in a 0.1 M H₂SO₄ solution, and that the mechanism occurs via the transfer of two electrons and two protons. The simultaneous quantification of paraxanthine and caffeine was demonstrated in 0.1 M H₂SO₄ and spiked human saliva samples. In the latter case, limits of detection of 2.89 μM for paraxanthine and 5.80 μM for caffeine were obtained, respectively. The sensor is reliable, providing a relative standard deviation within 7% (<i>n</i> = 6). Potential applicability of the sensing platform was demonstrated by running a small scale trial on five healthy volunteers, with simultaneous quantification by differential pulse voltammetry (DPV) of paraxanthine and caffeine in saliva samples collected at 1, 3 and 6 h postdose administration. The results were validated by ultra-high pressure liquid chromatography and shown to have a high correlation factor (r = 0.994).