Extraction of caffeine and its metabolite paraxanthine from urine and measuring both of them by high-performance liquid chromatography.

Abstract

Background & Aim: Caffeine is an alkaloid found in common drinks such as coffee and tea and carbonated drinks. Since it has a stimulant effect on the nervous system، its consumption is monitored in sporting events. Caffeine is metabolized by the cytochrome CYP1A2 enzyme to its primary metabolite، paraxanthine. The aim of this research is to study the validity of the method of extracting and analyzing caffeine and paraxanthine together in urine by high performance liquid chromatography (HPLC)، as it is an available device.

Materials and methods: Caffeine and paraxanthine were extracted from urine by solid phase extraction، then analyzed by HPLC with the presence of antipyrine as an internal standard.

Results: The results showed that the method was specific for caffeine and paraxanthine، as clear peaks appeared for them without interference from endogenous substances in urine. The method was linear between concentrations of 10-160 µg/ml for caffeine and 10-80 µg/ml for paraxanthine. The extraction yield was more than 96% for both substances. The capacity factor، the number of theoretical layers، the appendix factor، the accuracy and the accuracy were calculated and showed the suitability of the system. The limit of detection (LOD) was 0.25 µg/ml for caffeine، and 0.27 µg/ml for paraxanthine، while the limit of quantification (LOQ) was 0.77 µg/ml for caffeine and 0.84 µg/ml for paraxanthine.

Conclusions: This method can be adopted to measure caffeine and paraxanthine in urine with a cost-effective and available device، whether in sports organizations that are interested in determining caffeine levels in urine or in studying the activity of the enzyme CYP1A2.