Validation and sample bioanalysis of protoporphyrin IX in rat plasma by the surrogate matrix approach

Protoporphyrin IX (PPIX) plays a pivotal role in the heme biosynthesis pathway and serves as both a valuable biomarker in clinical diagnostics and a photosensitizer in photodynamic applications. Despite its physiological importance, accurate quantification of endogenous PPIX in biological matrices remains challenging due to the lack of an analyte-free authentic control matrix and inherent baseline variability. This study describes the development and validation of a high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) for PPIX quantification in rat plasma, applying a surrogate matrix strategy in full compliance with the International Council for Harmonisation M10 guideline. Endogenous PPIX was removed from rat plasma by visible light-induced analyte stripping, enabling the in-house preparation of a surrogate matrix, for accurate calibration sample generation. The method showed appropriate selectivity, excellent linearity over the range of 10 and 700 ng/mL (r ≥ 0.995), satisfactory precision and accuracy across all validation levels. The lower limit of quantification was established at 10 ng/mL. The stability of both the analyte and internal standard was confirmed under various conditions, including 3 freeze-thaw cycles, short- and long-term storage, and autosampler residence. The method was successfully applied in an in vivo study in which male rats were treated with aminolevulinic acid to induce PPIX formation, thereby confirming its suitability for study sample analysis. This fluorescence-based HPLC method offers a practical and cost-effective solution for monitoring PPIX in plasma samples. The bioanalytical method validation using a surrogate matrix approach for endogenous PPIX quantification fully aligned with current international regulatory standards may set a precedent for future method development in this field.