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. 2020 Dec 24;10(1):10.
doi: 10.3390/antiox10010010.

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

Rozalia-Maria Anastasiadi  1 , Federico Berti  2 , Silvia Colomban  3 , Claudio Tavagnacco  2 , Luciano Navarini  3 , Marina Resmini  1
Affiliations

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

Rozalia-Maria Anastasiadi et al. Antioxidants (Basel). .

Abstract

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% (n = 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).

Keywords: CYP1A2 phenotyping; antioxidants; caffeine; differential pulse voltammetry; human saliva; paraxanthine.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Chemical structures of caffeine and its primary metabolites.
Figure 2
Figure 2
(a) Cyclic voltammograms of an equimolar mixture (200 μΜ) of paraxanthine (PX) and caffeine (CAF) in the optimal concentration for the various supporting electrolytes on the glassy carbon (GC) electrode with a scan rate of 100 mV s−1; (b) cyclic voltammograms of blank 0.1 M H2SO4 (black dashed line), 500 μM CAF (solid blue line) and 500 μM PX (solid blue line) in 0.1 M H2SO4 on the GC electrode with a scan rate of 100 mV s−1. The chemical structures of the paraxanthine (blue) and caffeine (black) are also given in the figure; (c) cyclic voltammograms of 500 μM PX at various pH values of H2SO4 on the GC electrode with a scan rate of 100 mV s−1. The lower lines are the reverse scan of the corresponding upper lines of the same color; (d) the effect of pH on the peak potential (E) and peak current (I) of PX.
Scheme 1
Scheme 1
Overall proposed oxidation mechanism of paraxanthine in 0.1 M H2SO4 on the GC electrode.
Figure 3
Figure 3
(a) Cyclic voltammograms of 400 μM PX at various scan rates (from a to k): 5, 10, 20, 40, 60, 80, 100, 200, 400, 600 and 800 mV s−1 in 0.1 M H2SO4 on the GC electrode; (b) logI against logv; (c) I against v1/2.
Figure 4
Figure 4
(a) DP voltammograms of equimolar mixtures of paraxanthine (PX, EPX = +1.15 V) and caffeine (CAF, ECAF = +1.36 V): blank, 1, 2, 5, 8, 20, 40, 60, 80, 120, 150, 180 and 200 μM in 0.1 M H2SO4 on the GC electrode at a scan rate of 5 mV s−1; corresponding calibration curves by (b) differential pulse voltammetry (DPV) and (c) UHPLC-UV.
Figure 5
Figure 5
(a) DP voltammograms of paraxanthine (PX, EPX = +1.25 V) and caffeine (CAF, ECAF = +1.45 V) extracted from spiked saliva on the GC electrode at a scan rate of 5 mV s−1; PX and CAF concentrations assuming quantitative extraction: blank saliva (black dashed line), 25 μM and 5 μM (red), 50 μM and 12.5 μM (green), 62.5 μM and 20 μM (blue), 75 μM and 25 μM (light blue), 87.5 μM and 32.5 μM (pink), 100 μM and 37.5 μM (yellow), 112.5 μM and 45 μM (dark yellow), 125 μM and 50 μM (blue); corresponding calibration curves by (b) DPV and (c) UHPLC-UV.
Figure 6
Figure 6
(a) DP voltammograms containing paraxanthine (PX, EPX = +1.24 V) and caffeine (CAF, ECAF = +1.44 V), isolated from saliva, in 0.1 M H2SO4 on the GC electrode, with a scan rate of 5 mV s−1: predose as blank saliva (B saliva), 1, 3 and 6 h upon administration of a single 200 mg CAF dose from a male volunteer; (b) CAF and PX concentration-time profiles by DPV and UHPLC for a male volunteer in three different days upon administration of a single 200 mg CAF dose with SD of the three measurements as error bars; (c) PX/CAF molar ratios vs. time (h) by DPV (filled circles and solid lines) and UHPLC-UV (empty circles and dashed lines) for all five subjects (F1 and F2 for females and M1, M2 and M3 for males); (d) Correlation plot of PX/CAF molar ratios obtained by DPV vs. UHPLC.
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