Quantification of theobromine and caffeine in saliva, plasma and urine via liquid chromatography–tandem mass spectrometry: A single analytical protocol applicable to cocoa intervention studies

Targeted analyses of clinically relevant metabolites in human biofluids often require extensive sample preparation (e.g., desalting, protein removal and/or preconcentration) prior to quantitation. In this report, a single ultra-centrifugation based sample pretreatment combined with a designed liquid chromatography–tandem mass spectrometry (LC–MS/MS) protocol provides selective quantification of 3,7-dimethylxanthine (theobromine) and 1,3,7-trimethylxanthine (caffeine) in human saliva, plasma and urine samples. The optimized chromatography permitted elution of both analytes within 1.3 min of the applied gradient. Positive-mode electrospray ionization and a triple quadruple MS/MS instrument operated in multiple reaction mode were used for detection. 13C3 isotopically labeled caffeine was included as an internal standard to improve accuracy and precision. Implementing a 20-fold dilution of the isolated low MW biofluid fraction prior to injection effectively minimized the deleterious contributions of all three matrices to quantitation. The assay was linear over a 160-fold concentration range from 2.5 to 400 μmol L−1 for both theobromine (average R2 0.9968) and caffeine (average R2 0.9997) respectively. Analyte peak area variations for 2.5 μmol L−1 caffeine and theobromine in saliva, plasma and urine ranged from 5 and 10% (intra-day, N = 10) to 9 and 13% (inter-day, N = 25) respectively. The intra- and inter-day precision of theobromine and caffeine elution times were 3 and <1% for all biofluids and concentrations tested. Recoveries for caffeine and theobromine ranged from 114 to 118% and 99 to 105% at concentration levels of 10 and 300 μmol L−1. This validated protocol also permitted the relative saliva, plasma and urine distribution of both theobromine and caffeine to be quantified following a cocoa intervention.