Determination of urine caffeine and its metabolites by use of high-performance liquid chromatography-tandem mass spectrometry: estimating dietary caffeine exposure and metabolic phenotyping in population studies.

We have developed and validated a high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for determining urine caffeine and 14 caffeine metabolites suitable for estimating caffeine exposure and metabolic phenotyping in population studies. Sample preparation consisted solely of a series of simple reagent treatments at room temperature. Stable isotope-labeled analogs were used as internal standards for all analytes. We developed rapid LC-MS/MS separations for both positive and negative ion mode electrospray ionizations to maximize measurement sensitivity. Limits of detection were 0.05-0.1 μmol/L depending on the analytes. Method imprecision, based on total coefficients of variation, was generally <7 % when analyte concentration was >1 μmol/L. Analyte recoveries were typically within 10 % of being quantitative (100 %), and good agreement was observed among analytes measured across different MS/MS transitions. We applied this method to the analysis of a convenience set of human urine samples (n = 115) and were able to detect a majority of the analytes in ≥99 % of samples as well as calculate caffeine metabolite phenotyping ratios for cytochrome P450 1A2 and N-acetyltransferase 2. Whereas existing LC-MS/MS methods are limited in number of caffeine metabolites for which they are validated, or are designed for studies in which purposely elevated caffeine levels are expected, our method is the first of its kind designed specifically for the rapid, sensitive, accurate, and precise measurement of urine caffeine and caffeine metabolites at concentrations relevant to population studies.