Development and validation of a derivatized liquid chromatography–tandem mass spectrometry method for polyamine analysis and its application to rat tissues with a comparative non-derivatized approach

Polyamines, including spermidine (SPD), spermine (SPM), putrescine (PUT), cadaverine (CAD), and their acetylated derivatives, play essential roles in cellular proliferation, differentiation and apoptosis. Accurate quantification in biological matrices is critical for understanding tissue and subcellular polyamine metabolism in health and disease. In this study, we developed and validated a derivatized LC–MS/MS method in serum as a matrix to quantify seven polyamines, using two isotope-labelled analogues as internal standards. Method validation included precision, accuracy, limits of detection (LOD) and quantification (LOQ), linear range, recovery and matrix effects. The technique was applied to measure polyamine concentrations in rat tissues and subcellular fractions (S9, cytosol and microsomes). In addition, a non-derivatized LC–MS/MS approach was compared for the quantification of high-abundance polyamines in heart tissue. The derivatized method demonstrated robust analytical performance, with acceptable precision (RSD < 20%), low LoDs (0.022–0.049 μM), and excellent linearity (0.3125–20 μM; R² > 0.995). Across rat tissues, SPD and SPM were the predominant polyamines (e.g., spleen S9: SPD 809.12 nmol/g, SPM 496.33 nmol/g), whereas acetylated polyamines were detected at substantially lower levels and were largely absent in microsomal fractions. PUT and CAD were reliably detected only after derivatization. Comparative analysis showed that the non-derivatized LC–MS approach provided quantitatively consistent measurements of SPD and SPM in heart tissue fractions but exhibited limited sensitivity to diamines. Overall, this study establishes a reliable LC–MS/MS approach for polyamine profiling in tissues and subcellular fractions and clarifies the appropriate use of derivatized and non-derivatized strategies.