{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,25]],"date-time":"2026-03-25T11:35:12Z","timestamp":1774438512001,"version":"3.50.1"},"reference-count":42,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2019,8,7]],"date-time":"2019-08-07T00:00:00Z","timestamp":1565136000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia","award":["PTDC\/NEU-NMC\/0205\/2012, POCI-01-0145-FEDER-007440 (ref.; UID\/NEU\/04539\/2019), POCI-01-0145-FEDER-016428 (ref.: SAICTPAC\/0010\/2015), POCI-01-0145-FEDER-016795 (ref.: PTDC\/NEU-SCC\/7051\/2014), POCI-01-0145-FEDER-029311 (ref.: PTDC\/BTM-TEC\/29311\/2017), POCI-0"],"award-info":[{"award-number":["PTDC\/NEU-NMC\/0205\/2012, POCI-01-0145-FEDER-007440 (ref.; UID\/NEU\/04539\/2019), POCI-01-0145-FEDER-016428 (ref.: SAICTPAC\/0010\/2015), POCI-01-0145-FEDER-016795 (ref.: PTDC\/NEU-SCC\/7051\/2014), POCI-01-0145-FEDER-029311 (ref.: PTDC\/BTM-TEC\/29311\/2017), POCI-0"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Caffeine is one of the most widely consumed psycho-stimulants. The study of the beneficial effects of caffeine consumption to decrease the risk of developing several neuropsychiatric pathologies is receiving increasing attention. Thus, accurate and sensitive methods have been developed, mainly by LC-MS\/MS, in order to quantify caffeine and its metabolites. These quantifications of caffeine and its metabolites by LC-MS\/MS require a considerable effort to select or find a surrogate matrix, without the compounds of interest, to be used in the calibration curves. Thus, we evaluated the possibility of using calibration curves prepared in solvent instead of calibration curves prepared in human plasma. Results show that the calibration curves prepared in solvent and in human plasma were similar by comparing their slopes and interceptions, and the accuracy and precision were within the limits of acceptance for both calibration curves. This work demonstrates that, by using internal standards, it is possible to use a calibration curve in solvent instead of a calibration curve in plasma to perform an accurate and precise quantification of caffeine and theobromine.<\/jats:p>","DOI":"10.3390\/molecules24162863","type":"journal-article","created":{"date-parts":[[2019,8,7]],"date-time":"2019-08-07T10:56:38Z","timestamp":1565175398000},"page":"2863","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":39,"title":["Validation of an LC-MS\/MS Method for the Quantification of Caffeine and Theobromine Using Non-Matched Matrix Calibration Curve"],"prefix":"10.3390","volume":"24","author":[{"given":"Vera M.","family":"Mendes","sequence":"first","affiliation":[{"name":"CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0392-1118","authenticated-orcid":false,"given":"Margarida","family":"Coelho","sequence":"additional","affiliation":[{"name":"CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal"},{"name":"Chemistry Department, Faculty of Sciences and Technology, University of Coimbra, 3004-535 Coimbra, Portugal"}]},{"given":"Angelo R.","family":"Tom\u00e9","sequence":"additional","affiliation":[{"name":"CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal"},{"name":"Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra, 3000-456 Coimbra, Portugal"}]},{"given":"Rodrigo A.","family":"Cunha","sequence":"additional","affiliation":[{"name":"CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal"},{"name":"Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2087-4042","authenticated-orcid":false,"given":"Bruno","family":"Manadas","sequence":"additional","affiliation":[{"name":"CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2019,8,7]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"294","DOI":"10.1016\/j.neubiorev.2016.09.001","article-title":"A review of caffeine\u2019s effects on cognitive, physical and occupational performance","volume":"71","author":"McLellan","year":"2016","journal-title":"Neurosci. Biobehav. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"384","DOI":"10.1124\/pr.117.014407","article-title":"Interindividual Differences in Caffeine Metabolism and Factors Driving Caffeine Consumption","volume":"70","author":"Nehlig","year":"2018","journal-title":"Pharmacol. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1016\/j.fct.2013.10.042","article-title":"Beverage caffeine intakes in the U.S.","volume":"63","author":"Mitchell","year":"2014","journal-title":"Food Chem. Toxicol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1080\/0265203021000007840","article-title":"Effects of caffeine on human health","volume":"20","author":"Nawrot","year":"2003","journal-title":"Food Addit. Contam."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"EFSA NDA Panel (EFSA Panel on Dietetic Products, Nutrition and Allergies) (2015). Scientific Opinion on the safety of caffeine. Efsa J., 13, 4102.","DOI":"10.2903\/j.efsa.2015.4102"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1250","DOI":"10.1080\/10408398.2016.1247252","article-title":"Caffeine intake and its sources: A review of national representative studies","volume":"58","author":"Verster","year":"2018","journal-title":"Crit. Rev. Food Sci. Nutr."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"563","DOI":"10.1016\/S0149-7634(98)00050-5","article-title":"Are we dependent upon coffee and caffeine? A review on human and animal data","volume":"23","author":"Nehlig","year":"1999","journal-title":"Neurosci. Biobehav. Rev."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1053\/beem.2000.0055","article-title":"Central nervous system stimulants","volume":"14","author":"George","year":"2000","journal-title":"Baillieres Best Pract. Res. Clin. Endocrinol. Metab."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"71","DOI":"10.2174\/1570159X13666141210215655","article-title":"Caffeine: Cognitive and physical performance enhancer or psychoactive drug?","volume":"13","author":"Cappelletti","year":"2015","journal-title":"Curr. Neuropharmacol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1046\/j.1468-1331.2002.00421.x","article-title":"Does caffeine intake protect from Alzheimer\u2019s disease?","volume":"9","author":"Maia","year":"2002","journal-title":"Eur. J. Neurol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"S117","DOI":"10.3233\/JAD-2010-091249","article-title":"Caffeine and coffee as therapeutics against Alzheimer\u2019s disease","volume":"20","author":"Arendash","year":"2010","journal-title":"J. Alzheimers Dis."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1002\/ana.1052","article-title":"Prospective study of caffeine consumption and risk of Parkinson\u2019s disease in men and women","volume":"50","author":"Ascherio","year":"2001","journal-title":"Ann. Neurol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1016\/j.mam.2016.11.011","article-title":"Low, but not high, dose caffeine is a readily available probe for adenosine actions","volume":"55","author":"Fredholm","year":"2017","journal-title":"Mol. Aspects Med."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2153","DOI":"10.1007\/s00018-007-7051-9","article-title":"Caffeine analogs: Biomedical impact","volume":"64","author":"Daly","year":"2007","journal-title":"Cell Mol. Life Sci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"S25","DOI":"10.3233\/JAD-2010-091384","article-title":"Caffeine, adenosine receptors, and synaptic plasticity","volume":"20","author":"Costenla","year":"2010","journal-title":"J. Alzheimers Dis."},{"key":"ref_16","first-page":"201","article-title":"The role of adenosine receptors in the central action of caffeine","volume":"7","author":"Daly","year":"1994","journal-title":"Pharmacopsychoecologia"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"S3","DOI":"10.3233\/JAD-2010-1379","article-title":"Caffeine and adenosine","volume":"20","author":"Ribeiro","year":"2010","journal-title":"J. Alzheimers Dis."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e3900","DOI":"10.1002\/bmc.3900","article-title":"Measurement of caffeine and its three primary metabolites in human plasma by HPLC-ESI-MS\/MS and clinical application","volume":"31","author":"Chen","year":"2017","journal-title":"Biomed. Chromatogr."},{"key":"ref_19","first-page":"180","article-title":"Disposition of caffeine and its metabolites in man","volume":"224","author":"Williams","year":"1983","journal-title":"J. Pharmacol. Exp. Ther."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"476","DOI":"10.1016\/j.neuropharm.2012.11.029","article-title":"Psychostimulant pharmacological profile of paraxanthine, the main metabolite of caffeine in humans","volume":"67","author":"Orru","year":"2013","journal-title":"Neuropharmacology"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/0304-3940(82)90290-7","article-title":"Effects of caffeine and theophylline on adenosine and benzodiazepine receptors in human brain","volume":"30","author":"Boulenger","year":"1982","journal-title":"Neurosci. Lett."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1016\/j.yrtph.2017.07.025","article-title":"Caffeine and cardiovascular health","volume":"89","author":"Turnbull","year":"2017","journal-title":"Regul. Toxicol. Pharmacol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"284","DOI":"10.1016\/j.ijcard.2013.12.237","article-title":"Cardiovascular health and caffeine consumption. A population-based study in rural Ecuador","volume":"172","author":"Mera","year":"2014","journal-title":"Int. J. Cardiol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1016\/0742-8413(94)00048-F","article-title":"Caffeine and coffee: Effects on health and cardiovascular disease","volume":"109","author":"Chou","year":"1994","journal-title":"Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"S1","DOI":"10.3233\/JAD-2010-01420","article-title":"Therapeutic opportunities for caffeine in Alzheimer\u2019s disease and other neurodegenerative disorders","volume":"20","author":"Cunha","year":"2010","journal-title":"J. Alzheimers Dis."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"S221","DOI":"10.3233\/JAD-2010-091525","article-title":"Caffeine exposure and the risk of Parkinson\u2019s disease: A systematic review and meta-analysis of observational studies","volume":"20","author":"Costa","year":"2010","journal-title":"J. Alzheimers Dis."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"553","DOI":"10.1080\/19390211.2016.1275916","article-title":"Neuroprotective and Therapeutic Effect of Caffeine on the Rat Model of Parkinson\u2019s Disease Induced by Rotenone","volume":"14","author":"Khadrawy","year":"2017","journal-title":"J. Diet Suppl."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"S205","DOI":"10.3233\/JAD-2010-091459","article-title":"Effects of caffeine in Parkinson\u2019s disease: From neuroprotection to the management of motor and non-motor symptoms","volume":"20","author":"Prediger","year":"2010","journal-title":"J. Alzheimers Dis."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"S631","DOI":"10.1016\/j.dsx.2017.04.017","article-title":"The effect of acute caffeine intake on insulin sensitivity and glycemic control in people with diabetes","volume":"11","author":"Dewar","year":"2017","journal-title":"Diabetes Metab. Syndr."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1177\/0269881116668595","article-title":"A case-control field study on the relationships among type 2 diabetes, sleepiness and habitual caffeine intake","volume":"31","author":"Urry","year":"2017","journal-title":"J. Psychopharmacol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"182","DOI":"10.1007\/s13668-019-0274-1","article-title":"Coffee Drinking and Reduced Risk of Liver Cancer: Update on Epidemiological Findings and Potential Mechanisms","volume":"8","author":"Inoue","year":"2019","journal-title":"Curr. Nutr. Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"634","DOI":"10.1158\/1055-9965.EPI-15-0924","article-title":"Coffee Consumption and the Risk of Colorectal Cancer","volume":"25","author":"Schmit","year":"2016","journal-title":"Cancer Epidemiol. Biomarkers Prev."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2735","DOI":"10.1016\/j.foodchem.2013.05.069","article-title":"Simultaneous quantification of caffeine and its three primary metabolites in rat plasma by liquid chromatography-tandem mass spectrometry","volume":"141","author":"Choi","year":"2013","journal-title":"Food Chem."},{"key":"ref_34","first-page":"2085059","article-title":"HPLC Method for Quantification of Caffeine and Its Three Major Metabolites in Human Plasma Using Fetal Bovine Serum Matrix to Evaluate Prenatal Drug Exposure","volume":"2018","author":"Hernandez","year":"2018","journal-title":"J. Anal. Methods Chem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1016\/j.chroma.2014.05.047","article-title":"Derivation of uncertainty functions from validation studies in biological fluids: Application to the analysis of caffeine and its major metabolites in human plasma samples","volume":"1353","author":"Gassner","year":"2014","journal-title":"J. Chromatogr. A"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2198","DOI":"10.1016\/j.jchromb.2009.01.003","article-title":"Validation of bioanalytical LC-MS\/MS assays: Evaluation of matrix effects","volume":"877","author":"Lanckmans","year":"2009","journal-title":"J. Chromatogr. B Analyt. Technol. Biomed. Life Sci."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"10957","DOI":"10.1021\/acs.analchem.6b02596","article-title":"Relevance in the Use of Appropriate Internal Standards for Accurate Quantification Using LC-MS\/MS: Tauro-Conjugated Bile Acids as an Example","volume":"88","author":"Krautbauer","year":"2016","journal-title":"Anal. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1349","DOI":"10.4155\/bio-2018-0105","article-title":"Proposed selection strategy of surrogate matrix to quantify endogenous substances by Japan Bioanalysis Forum DG2015-15","volume":"10","author":"Wakamatsu","year":"2018","journal-title":"Bioanalysis"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"292","DOI":"10.1093\/chrsci\/49.4.292","article-title":"Validated HPLC method for determination of caffeine level in human plasma using synthetic plasma: Application to bioavailability studies","volume":"49","author":"Alvi","year":"2011","journal-title":"J. Chromatogr. Sci."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"8959","DOI":"10.1021\/ac5018265","article-title":"Selecting the correct weighting factors for linear and quadratic calibration curves with least-squares regression algorithm in bioanalytical LC-MS\/MS assays and impacts of using incorrect weighting factors on curve stability, data quality, and assay performance","volume":"86","author":"Gu","year":"2014","journal-title":"Anal. Chem."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1016\/S1570-0232(02)00244-1","article-title":"Linear regression for calibration lines revisited: Weighting schemes for bioanalytical methods","volume":"774","author":"Almeida","year":"2002","journal-title":"J. Chromatogr. B Analyt. Technol. Biomed Life Sci."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"419","DOI":"10.2217\/bmm.12.46","article-title":"Recommendations to standardize preanalytical confounding factors in Alzheimer\u2019s and Parkinson\u2019s disease cerebrospinal fluid biomarkers: An update","volume":"6","author":"Mollenhauer","year":"2012","journal-title":"Biomark. Med."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/24\/16\/2863\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T13:09:13Z","timestamp":1760188153000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/24\/16\/2863"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,8,7]]},"references-count":42,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2019,8]]}},"alternative-id":["molecules24162863"],"URL":"https:\/\/doi.org\/10.3390\/molecules24162863","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,8,7]]}}}