{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T20:07:25Z","timestamp":1788898045398,"version":"build-2803163510"},"reference-count":26,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2024,8,12]],"date-time":"2024-08-12T00:00:00Z","timestamp":1723420800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Mass spectrometry (MS) is a widely used analytical technique including medical diagnostics, forensic toxicology, food and water analysis. The gold standard for quantifying compounds involves using stable isotope-labeled internal standards (SIL-IS). However, when these standards are not commercially available, are prohibitively expensive, or are extremely difficult to synthesize, alternative external quantification techniques are employed. We hereby present a novel, convenient and cheap quantification approach\u2014quantification via post column infusion (PCI). As a proof of concept, we demonstrated PCI quantification for the immunosuppressant tacrolimus in whole blood using liquid chromatography\u2013tandem mass spectrometry (LC-MS\/MS). The validation results met the criteria according to the guideline on bioanalytical method validation of the European Medicine Agency (EMA), achieving imprecisions and inaccuracies with coefficient of variation and relative bias below 15%. Anonymized and leftover whole blood samples from immunosuppressed patients receiving tacrolimus were used for method comparison (PCI quantification vs. conventional internal standard (IS) quantification). Both methods showed strong agreement with a Pearson correlation coefficient of r = 0.9532. This novel PCI quantification technique (using the target analyte itself) expands the quantification options available in MS, providing reliable results, particularly when internal standards are unavailable or unaffordable. With the current paper, we aim to demonstrate that our innovative PCI technique has great potential to overcome practical issues in quantification and to provide guidance on how to incorporate PCI in existing or new LC-MS methods. Moreover, this study demonstrated a more convenient method for correcting matrix effects in comparison to alternative PCI techniques.<\/jats:p>","DOI":"10.3390\/molecules29163829","type":"journal-article","created":{"date-parts":[[2024,8,12]],"date-time":"2024-08-12T10:34:59Z","timestamp":1723458899000},"page":"3829","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Standardization via Post Column Infusion\u2014A Novel and Convenient Quantification Approach for LC-MS\/MS"],"prefix":"10.3390","volume":"29","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5110-346X","authenticated-orcid":false,"given":"Katharina","family":"Habler","sequence":"first","affiliation":[{"name":"Institute of Laboratory Medicine, LMU University Hospital, LMU Munich, Marchioninistr. 15, 81377 Munich, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arber","family":"Rexhaj","sequence":"additional","affiliation":[{"name":"Institute of Laboratory Medicine, LMU University Hospital, LMU Munich, Marchioninistr. 15, 81377 Munich, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Felix L.","family":"Happich","sequence":"additional","affiliation":[{"name":"Institute of Laboratory Medicine, LMU University Hospital, LMU Munich, Marchioninistr. 15, 81377 Munich, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Vogeser","sequence":"additional","affiliation":[{"name":"Institute of Laboratory Medicine, LMU University Hospital, LMU Munich, Marchioninistr. 15, 81377 Munich, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,8,12]]},"reference":[{"key":"ref_1","first-page":"3","article-title":"Electrospray ionisation mass spectrometry: Principles and clinical applications","volume":"24","author":"Ho","year":"2003","journal-title":"Clin. Biochem. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1080\/0265203021000060887","article-title":"Analysis of acrylamide by LC-MS\/MS and GC-MS in processed Japanese foods","volume":"20","author":"Ono","year":"2003","journal-title":"Food Addit. Contam."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"7047","DOI":"10.1007\/s00216-015-8857-1","article-title":"Detecting a wide range of environmental contaminants in human blood samples--combining QuEChERS with LC-MS and GC-MS methods","volume":"407","author":"Plassmann","year":"2015","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_4","first-page":"19","article-title":"Principles and Applications of Liquid Chromatography-Mass Spectrometry in Clinical Biochemistry","volume":"30","author":"Pitt","year":"2009","journal-title":"Clin. Biochem. Rev."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"470","DOI":"10.1007\/s12302-009-0082-0","article-title":"Stabilisotopenverd\u00fcnnungsanalysen zur Quantifizierung organischer Spurenkomponenten in der Lebensmittelanalytik","volume":"21","author":"Rychlik","year":"2009","journal-title":"Environ. Sci. Eur."},{"key":"ref_6","first-page":"334","article-title":"Measuring Mass: From Positive Rays to Proteins by Michael A. Grayson","volume":"94","author":"Gove","year":"2003","journal-title":"Isis"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"5678","DOI":"10.1021\/ac8013065","article-title":"A Brief History of Mass Spectrometry","volume":"80","author":"Griffiths","year":"2008","journal-title":"Anal. Chem."},{"key":"ref_8","first-page":"1761","article-title":"Quantification of vancomycin in human serum by LC-MS\/MS","volume":"51","author":"Kobold","year":"2013","journal-title":"Clin. Chem. Lab. Med."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1016\/j.cca.2016.11.029","article-title":"An LC-MS\/MS based candidate reference method for the quantification of total gentamicin in human serum and plasma using NMR characterized calibrator material","volume":"464","author":"Lucha","year":"2017","journal-title":"Clin. Chim. Acta"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1406","DOI":"10.1373\/clinchem.2008.105643","article-title":"Instrument-specific matrix effects of calibration materials in the LC-MS\/MS analysis of tacrolimus","volume":"54","author":"Vogeser","year":"2008","journal-title":"Clin. Chem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"4065","DOI":"10.1002\/rcm.3311","article-title":"Using calibration approaches to compensate for remaining matrix effects in quantitative liquid chromatography\/electrospray ionization multistage mass spectrometric analysis of phytoestrogens in aqueous environmental samples","volume":"21","author":"Kang","year":"2007","journal-title":"Rapid Commun. Mass Spectrom."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"910","DOI":"10.1007\/s00216-003-2442-8","article-title":"Evaluation of three calibration methods to compensate matrix effects in environmental analysis with LC-ESI-MS","volume":"378","author":"Reemtsma","year":"2004","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1016\/S0021-9673(01)01429-7","article-title":"Matrix effect and correction by standard addition in quantitative liquid chromatographic-mass spectrometric analysis of diarrhetic shellfish poisoning toxins","volume":"943","author":"Ito","year":"2002","journal-title":"J. Chromatogr. A"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1093\/jaoac\/93.2.355","article-title":"Multi-Analyst, Multi-Matrix Performance of the QuEChERS Approach for Pesticide Residues in Foods and Feeds Using HPLC\/MS\/MS Analysis with Different Calibration Techniques","volume":"93","author":"Lehotay","year":"2010","journal-title":"J. AOAC Int."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1172","DOI":"10.1021\/jf803040g","article-title":"LC-MS\/MS quantitation of hop-derived bitter compounds in beer using the ECHO technique","volume":"57","author":"Intelmann","year":"2009","journal-title":"J. Agric. Food Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1016\/S0021-9673(02)01196-2","article-title":"Alternative calibration approaches to compensate the effect of co-extracted matrix components in liquid chromatography-electrospray ionisation tandem mass spectrometry analysis of pesticide residues in plant materials","volume":"973","author":"Poustka","year":"2002","journal-title":"J. Chromatogr. A"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"4107","DOI":"10.1021\/ac990312o","article-title":"Postcolumn Introduction of an Internal Standard for Quantitative LC\u2212MS Analysis","volume":"71","author":"Choi","year":"1999","journal-title":"Anal. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"80","DOI":"10.1016\/j.chroma.2018.01.001","article-title":"Post-column infusion of internal standard quantification for liquid chromatography-electrospray ionization-tandem mass spectrometry analysis\u2014Pharmaceuticals in urine as example approach","volume":"1535","author":"Rossmann","year":"2018","journal-title":"J. Chromatogr. A"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1016\/j.chroma.2013.12.066","article-title":"Using a postcolumn-infused internal standard for correcting the matrix effects of urine specimens in liquid chromatography-electrospray ionization mass spectrometry","volume":"1327","author":"Liao","year":"2014","journal-title":"J. Chromatogr. A"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1097","DOI":"10.1021\/acs.jproteome.6b01011","article-title":"Development of a Postcolumn Infused-Internal Standard Liquid Chromatography Mass Spectrometry Method for Quantitative Metabolomics Studies","volume":"16","author":"Liao","year":"2017","journal-title":"J. Proteome Res."},{"key":"ref_21","unstructured":"Geyer, R., Halg, W., and Vogeser, M. (2009). Functional Check and Variance Compensation in Mass Spectrometry. (13\/379,180), U.S. Patent."},{"key":"ref_22","unstructured":"European Medicines Agency (2011). Guideline on Bioanalytical Method Validation."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Dubbelman, A.-C., van Wieringen, B., Roman Arias, L., van Vliet, M., Vermeulen, R., Harms, A., and Hankemeier, T. (2024). Strategies for using post-column infusion of standards to correct for matrix effect in LC-MS-based quantitative metabolomics. ChemRxiv Anal. Chem., 1\u20139.","DOI":"10.26434\/chemrxiv-2024-w524w"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Li, W., Zhang, J., and Tse, F.L.S. (2013). Use of internal standards in LC-MS bioanalysis. Handbook of LC-MS Bioanalysis: Best Practices, Experimental Protocols, and Regulations, John Wiley & Sons, Inc.","DOI":"10.1002\/9781118671276"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"412","DOI":"10.1093\/jaoac\/86.2.412","article-title":"Fast and Easy Multiresidue Method Employing Acetonitrile Extraction\/Partitioning and \u201cDispersive Solid-Phase Extraction\u201d for the Determination of Pesticide Residues in Produce","volume":"86","author":"Anastassiades","year":"2003","journal-title":"J. AOAC Int."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Regal, P., Lamas, A., Franco, C.M., and Cepeda, A. (2019). Veterinary Drugs: Progress in Multiresidue Technique. Encyclopedia of Food Chemistry, Elsevier.","DOI":"10.1016\/B978-0-08-100596-5.21809-X"}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/29\/16\/3829\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T15:35:33Z","timestamp":1760110533000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/29\/16\/3829"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,8,12]]},"references-count":26,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2024,8]]}},"alternative-id":["molecules29163829"],"URL":"https:\/\/doi.org\/10.3390\/molecules29163829","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,8,12]]}}}