FLUORESCENCE-BASED HPLC QUANTIFICATION OF NEOPTERIN AND TOTAL NEOPTERIN IN HUMAN PLASMA: PRACTICAL ANALYTICAL CONSIDERATIONS AND CLINICAL APPLICATION

Abstract

This work describes the use of fluorescence-based HPLC to quantify neopterin and total neopterin in human plasma. Blood collection, plasma preparation, acetonitrile protein precipitation, triiodide-induced selective oxidation of 7,8-dihydroneopterin to neopterin, isocratic chromatographic separation on an amino-bonded silica stationary phase, and fluorescence detection are the analytical steps. The clinical cohort data discussed herein were previously reported by Raajasekar et al. (2025) and are reinterpreted here exclusively from an analytical and preparative-biotechnology perspective. The original clinical study did not report formal analytical validation parameters such as recovery, precision, linearity, limit of detection, limit of quantification, and matrix effect assessment. Future validation work should address these parameters. The manuscript provides systematic steps to address analytical issues such as poor peak shape, variable triiodide oxidation, fluorescence quenching, and baseline instability using evidence-based methods. Plasma and total neopterin levels were significantly higher in stroke patients compared to age-matched controls in 61 carotid endarterectomy patients (Raajasekar et al., 2025) (11.3 vs. 7.7 nM, p = 0.0372 and 46.37 vs. 19.29 nM, p = 0.0001). This manuscript analyses the dataset from an analytical methodology perspective, focusing on sample preparation, chromatographic workflow, triiodide oxidation, and quantification of plasma and total neopterin.