Determination of caffeine and its metabolites in human skin homogenate by high-performance liquid chromatography.
Zang-LY; DeHaven-JI; Soderholm-SC
Proceedings of the International Conference on Occupational and Environmental Exposures of Skin to Chemicals: Science & Policy, Arlington, VA, September 8-11, 2002. Morgantown, WV: National Institute for Occupational Safety and Health, 2002 Sep; :1
A method has been developed for the separation and quantification of caffeine and its metabolites in human skin homogenate. The method development was based on high-performance liquid chromatography (HPLC) equipped with UV-visible diode array detector and a Symmetry Shield RP8 column (3 mm, 2.1 mm x 150 mm). Beta-hydroxyethltheophylline (ISTD) was used as the internal standard for quantitation. The analytes in human skin homogenate were extracted with a Waters Oasis HLB solid phase extraction plate or by liquid-liquid extraction using methanol-methylene chloride (1:2, v/v). The mobile phase consisted of 20 mM ammonium acetate and acetonitrile at the gradient ratios from 100:0 for 5.5 min to 90:10 for 10 min at a constant flow rate of 0.3 mL/min. The compounds were monitored at 275 nm and a reference wavelength 360 nm with 100 nm widths. The elution order of these species was found to be 3-methyluric acid, 7-methyluric acid, 1-methyluric acid, 37-methyluric acid, 17-dimethyluric acid, 13-dimethyluric acid, 7-methylxanthine, 3-methylxanthine, 1-methylxanthine, 37-dimethylxanthine, 17-dimethylxanthine, 13-dimethylxanthine, ISTD, and 137-methylxanthine. The limits of quantification (LOQ) for these compounds were 1.0 - 2.5 ng per total injection. Reproducibility of the sample handling and HPLC assay had a relative standard deviation of +/- 10%. The average recoveries were better than 90% for liquid-liquid extraction and 82% for solid phase extraction.
Metabolites; Chromatographic-analysis; Analytical-methods; Skin-tests; Skin; Quantitative-analysis; Analytical-processes; Analytical-chemistry
Proceedings of the International Conference on Occupational and Environmental Exposures of Skin to Chemicals: Science & Policy, Arlington, VA, September 8-11, 2002