Comprehensive qualitative and quantitative analysis of three main polyamines in Pyropia spp. (Rhodophyta): method establishment, optimization and validation

Polyamines are essential for the growth and development of plants and algae. Although polyamines have been studied in various macroalgae and chromatographic methods have been applied for their quantification, fully validated HPLC protocols specifically adapted to the complex matrix of commercially important red seaweeds such as Pyropia spp. remain limited. In this study, key procedures, including sample pretreatment, derivatization, and chromatographic separation, were optimized using two pre-column derivatization reagents, FMOC-Cl and DNS-Cl, and two reliable HPLC methods were established for the quantitative analysis of total polyamines (including free and bound forms) in Pyropia spp. FMOC-Cl enabled rapid derivatization with minimal chromatographic background, but showed relatively lower response for spermidine. In contrast, DNS-Cl required a longer reaction time and produced higher background signals, but provided better sensitivity and stability, particularly for low-abundance polyamines such as spermidine. Both derivatization methods showed excellent linearity across the tested ranges, with correlation coefficients (R 2 ) ≥ 0.993. The linear ranges were 0.1–8.8 µg mL −1 for FMOC-Cl derivatives and 0.2–10.8 µg mL −1 for DNS-Cl derivatives. For DNS-Cl derivatives, the relative standard deviations (RSDs) of retention time and peak area were ≤ 0.58% and ≤ 2.18%, respectively, and the limit of detection (LOD) was 0.05 µg mL −1 . For FMOC-Cl derivatives, the corresponding RSDs were ≤ 0.74% and ≤ 2.42%, and the LOD was 0.01 µg mL −1 . Application of these methods to Pyropia spp. revealed that putrescine was the predominant polyamine, accounting for more than 50% of the total polyamine content, and that leafy thalli contained higher total polyamine levels than filamentous thalli. This study provides fundamental data on polyamines in Pyropia and a reliable analytical framework for other commercially important macroalgae.