Analytical and Bioanalytical Chemistry

, Volume 406, Issue 3, pp 771–784 | Cite as

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

  • Michael E. Rybak
  • Ching-I Pao
  • Christine M. Pfeiffer
Research Paper


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.


The determination of caffeine and its metabolites by LC-MS/MS. Both positive and negative ion mode electrospray ionization were used to maximize measurement sensitivity and selectivity, allowing the development of a robust method suitable for estimating caffeine exposure and metabolic phenotyping in population studies


Caffeine Urine Biomarkers Dietary intake Phenotyping Mass spectrometry NHANES 



The authors would like to thank Teresa D. Douglas of the Nutritional Health Sciences Program, Graduate Division of Biological and Biomedical Sciences, Emory University (Atlanta, GA), for her contributions in standard and sample preparation, and Patrick W. Simon of the Centers for Disease Control and Prevention, National Center for Environmental Health, for his contributions in sample preparation and analyses related to this work.


The findings and conclusions in this report are those of the authors and do not necessarily represent the official views or positions of the Centers for Disease Control and Prevention/Agency for Toxic Substances and Disease Registry or the Department of Health and Human Services.


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Copyright information

© Springer-Verlag Berlin Heidelberg (outside the USA) 2013

Authors and Affiliations

  • Michael E. Rybak
    • 1
  • Ching-I Pao
    • 1
  • Christine M. Pfeiffer
    • 1
  1. 1.U.S. Centers for Disease Control and PreventionNational Center for Environmental HealthAtlantaUSA

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