Keson Trakunram, Benjamas Janchawee, Pritsana Raungrut, and Sathaporn Prutipanlai

DOI: 10.14456/sjst-psu.2018.116

pp. 759 - 766

Abstract

A solid-phase extraction (SPE) method for use with high performance liquid chromatography to measure caffeine (CF)
and its metabolites in rat urine was developed and validated. Oasis HLB cartridges were used and washed with water, 1% acetic
acid: 3% methanol-water (0.5:99.5 v/v), and 1% ammonium hydroxide: 3% methanol-water (0.5:99.5 v/v), respectively, and
eluted with methanol:acetronitrile (80:20 v/v). Method validation showed good linearity (r>0.9990) over all calibration ranges
for CF and its metabolites. The method was precise (1.0–17.6%RSD) and accurate (–13.8–(+) 16.0%DEV). The SPE method
resulted in a high recovery for all analytes (72.9–98.9%). The lower limits of quantification were 0.1 µg/ml (PX), 0.25 µg/ml (CF
and theophylline), and 0.5 µg/ml (theobromine and 1,3,7-trimethyluric acid). This method was successfully applied to determine
the urinary profiles of CF and its metabolites, and the caffeine metabolic ratios in rats receiving a single oral dose of CF.