Validation of a high-performance liquid chromatography assay for quantification of caffeine and paraxanthine in human serum in the context of CYP1A2 phenotyping.
In this study the validation of a reversed-phase high-performance liquid chromatography (HPLC) method, with UV-detection, for both caffeine and paraxanthine in human serum is described. This method is feasible for cytochrome P450 1A2 (CYP1A2) phenotyping, according to the results of a pilot study. With this HPLC method caffeine and paraxanthine can be determined selectively and specifically. In the expected concentration range, caffeine recoveries were 98-108% (within-run variation 4.0-6.4%, between-run variation 6.4-8.8%), paraxanthine recoveries were 96.6-97.5% (within-run variation 5.0-7.2%, between-run variation 7.2-10.8%). The limits of detection for caffeine and paraxanthine using this HPLC system were 0.3 and 0.1 mg/L, respectively. Linear calibration curves for both caffeine and paraxanthine were obtained in the concentration range 0.5-30 mg/L (r > 0.9999. Serum samples were stable for a week, when stored at -20 and +4 degrees C.
University of Utrecht, Faculty of Pharmacy, Department of Analysis and Toxicology, The Netherlands.
J P
JP
Koch
G W
GW
ten Tusscher
J G
JG
Koppe
H J
HJ
Guchelaar
D002110:Caffeine / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism*; D006801:Humans; D010641:Phenotype; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet; D013806:Theophylline / Q000097:blood*
D002110:Caffeine; D013806:Theophylline; D019388:Cytochrome P-450 CYP1A2; C021183:1,7-dimethylxanthine