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. (13)C(3) 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 micromol L(-1) for both theobromine (average R(2) 0.9968) and caffeine (average R(2) 0.9997) respectively. Analyte peak area variations for 2.5 micromol 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 micromol 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.
Ptolemy
Adam S
AS
Department of Laboratory Medicine, Children's Hospital Boston, 300 Longwood Avenue, Boston, MA 02115, USA. adam.ptolemy@childrens.harvard.edu
Tzioumis
Emma
E
Thomke
Arjun
A
Rifai
Sami
S
Kellogg
Mark
M
D002099:Cacao / Q000737:chemistry*; D002110:Caffeine / Q000032:analysis* / Q000097:blood / Q000737:chemistry / Q000652:urine; D002853:Chromatography, Liquid / Q000379:methods*; D057230:Limit of Detection; D012015:Reference Standards; D015203:Reproducibility of Results; D012463:Saliva / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013805:Theobromine / Q000032:analysis* / Q000097:blood / Q000737:chemistry / Q000652:urine; D013997:Time Factors; D014970:Xanthines / Q000493:pharmacokinetics
D014970:Xanthines; C008514:methylxanthine; D002110:Caffeine; D013805:Theobromine