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Their quantification in biological samples is analytically demanding due to their high polarity, chemical instability, and the presence of multiple oxidation states. This review presents an integrated overview of pterin occurrence, structure, and physicochemical properties, followed by a detailed discussion of sample preparation strategies designed to ensure compound stability and analytical accuracy. Methods such as chemical oxidation, photochemical derivatization, and antioxidant stabilization are evaluated in the context of various biological matrices. We further examine state\u2010of\u2010the\u2010art analytical techniques that combine separation with detection, including capillary electrophoresis, gas and liquid chromatography coupled with fluorescence, UV, electrochemical, or mass spectrometric detection. Particular attention is given to recent advances in LC\u2013MS techniques, including both tandem mass spectrometry (LC\u2013MS\/MS) and high\u2010resolution approaches (e.g., HPLC\u2013Q\/TOF\u2010MS), which have greatly improved the sensitivity, selectivity, and throughput of pterin analysis, especially in combination with HILIC separation mode. These developments support the growing use of pterins as biomarkers in clinical diagnostics and physiological research, and underscore the importance of robust, matrix\u2010appropriate analytical protocols tailored to the specific challenges posed by this compound class.<\/jats:p>","DOI":"10.1002\/jssc.70483","type":"journal-article","created":{"date-parts":[[2026,7,6]],"date-time":"2026-07-06T07:05:09Z","timestamp":1783321509000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["An Overview of Pterin Analysis in Biological Samples: From Occurrence and Properties to Sample Pretreatment Combined With Hyphenated Separation Techniques"],"prefix":"10.1002","volume":"49","author":[{"given":"Jind\u0159ich","family":"Brejcha","sequence":"first","affiliation":[{"name":"Department of Philosophy and History of Science Faculty of Science Charles University  Prague Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8483-5739","authenticated-orcid":false,"given":"Zuzana","family":"Bosakova","sequence":"additional","affiliation":[{"name":"Department of Analytical Chemistry Faculty of Science Charles University  Prague Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,7,6]]},"reference":[{"key":"e_1_2_10_2_1","volume-title":"The Biochemistry of Natural Pigments","author":"Britton G.","year":"1983"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00216\u2010019\u201001574\u2010y"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jnatprod.9b00351"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.1039\/9781849734714-00158"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/B978-044453346-3.50351-6"},{"key":"e_1_2_10_7_1","volume-title":"Comprehensive Heterocyclic Chemistry: The Structure, Reactions, Synthesis, and Uses of Heterocyclic Compounds","author":"Katritzky A. 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