Modified Method for Serum Paraxanthine/Caffeine Ratio: An Index of CYP1A2 Activity

Abstract

The ratio of paraxanthine/caffeine is generally used to be an index of CYP1A2 activity.

The assay of serum paraxanthine/caffeine ratio was modified from the method of Koch J.P.et al. The validation of a reverse phase high performance liquid chromatography (HPLC) method with UV detection for both paraxanthine and caffeine in serum was described. The optimum time of blood sampling after caffeine intake was detected in a pilot study. Each subject took a 180 mg single oral dose of caffeine solution. Blood samples were collected before and 1, 2, 3, 4, 5, 6 and 8 hours after caffeine intake and analyzed further by HPLC. The assay validation was shown as these parameters. The lower limit of detection of the assay was 0.125 μg/ml and 0.25 μg/ml, for paraxanthine and caffeine, respectively. Accuracy expressed as% recovery, those range were 97.73 - 105.49 % and 95.84 - 100.63 %, for paraxanthine and caffeine, respectively. The precision expressed as relative standard deviation, the results were 2.88% and 5.25% for intraday and interday assay of paraxanthine, and 3.07% and 5.78% for intraday and interday assay of caffeine. Linearity of calibration curve of both were covered 0 - 8 μg/ml (R2=0.9999). Serum samples were stable when stored at -70°C for 24 weeks. The best sampling time of serum paraxanthine/caffeine ratio was 5 hours after caffeine intake. This method is simplified and reliable for serum paraxanthine/caffeine ratio determination as an index of CYP1A2 activity.