HPLC method for urinary theobromine determination: Effect of consumption of cocoa products on theobromine urinary excretion in children

Clin Biochem. 2015 Nov;48(16-17):1138-43. doi: 10.1016/j.clinbiochem.2015.06.022. Epub 2015 Jul 2.


Objective: To validate a simple method of urinary theobromine determination, to assess urinary theobromine levels in 80 healthy children and to relate these levels to consumption of cocoa products.

Design and methods: Urine samples were diluted, directly injected into an HPLC system, separated by gradient elution on a C18 column, and detected by UV spectrometry. The method was validated for linearity, limits of detection and quantification, imprecision, accuracy, recovery and interferences. The proposed method was used to assess 12-h day and 12-h night urinary theobromine excretion by 80 healthy children, divided into four groups based on consumption of cocoa products. In addition, urinary excretion of magnesium and oxalate, also present in cocoa, was measured in these four groups.

Results: The method was linear to a theobromine concentration of 278μmol/L (50mg/L). LOD and LOQ for urine samples, diluted 1:5 (vol/vol) with water, were 1.1 and 3.6μmol/L respectively. Within-run and between-run imprecisions (CV) were each <2%. Average recovery was 99%, and analysis of a certified reference sample showed an error <2.5%. Theobromine excretion levels were significantly higher in healthy children with higher consumption of cocoa products (p<0.001), but oxalate (p=0.098) and magnesium (p=0.068) excretion levels did not differ significantly.

Conclusion: This validated method resulted in urinary theobromine determination with 100% recovery, without sample pretreatment. Urinary theobromine levels in healthy children were directly related to their consumption of cocoa products.

Keywords: Cocoa consumption in children; HPLC; Theobromine determination; Uric acid nephrolithiasis; Urinary theobromine levels.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Cacao / metabolism*
  • Child
  • Child, Preschool
  • Chromatography, High Pressure Liquid / methods
  • Female
  • Humans
  • Magnesium / urine
  • Male
  • Oxalates / urine
  • Theobromine / urine*


  • Oxalates
  • Magnesium
  • Theobromine