An Overview of Pterin Analysis in Biological Samples: From Occurrence and Properties to Sample Pretreatment Combined With Hyphenated Separation Techniques.

Pterins are a structurally diverse group of biologically active compounds within the pteridine family, with key roles in pigmentation, redox metabolism, light sensing, and cellular signaling across a wide range of organisms. 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-of-the-art 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-MS techniques, including both tandem mass spectrometry (LC-MS/MS) and high-resolution approaches (e.g., HPLC-Q/TOF-MS), 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-appropriate analytical protocols tailored to the specific challenges posed by this compound class.