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...

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Published in:	Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 878; no. 3; pp. 409 - 416
Main Authors:	Ptolemy, Adam S., Tzioumis, Emma, Thomke, Arjun, Rifai, Sami, Kellogg, Mark
Format:	Journal Article
Language:	English
Published:	Amsterdam Elsevier B.V 01-02-2010 Elsevier
Subjects:	Analysis Analytical, structural and metabolic biochemistry Biological and medical sciences Cacao - chemistry Caffeine Caffeine - analysis Caffeine - blood Caffeine - chemistry Caffeine - urine Chromatography, Liquid - methods Cocoa Fundamental and applied biological sciences. Psychology General pharmacology LC–MS/MS Limit of Detection Medical sciences Pharmacology. Drug treatments Plasma Reference Standards Reproducibility of Results Saliva Saliva - chemistry Spectrometry, Mass, Electrospray Ionization Tandem Mass Spectrometry - methods Theobromine Theobromine - analysis Theobromine - blood Theobromine - chemistry Theobromine - urine Time Factors Urine Xanthines - pharmacokinetics Urine Plasma Theobromine Saliva Cocoa LC–MS/MS Caffeine Biological fluid Cardiotonic agent Purine derivatives CNS stimulant Psychotropic HPLC chromatography Blood plasma Respiratory analeptic Diuretic LC-MS/MS Alkaloid Mass spectrometry MS/MS Xanthine derivatives Quantitative analysis
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Summary:	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. 13C 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 μmol 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 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.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23
ISSN:	1570-0232 1873-376X
DOI:	10.1016/j.jchromb.2009.12.019