The validation of the analytical technique for the determination of polyamines in cerebral tissue using HPLC based on o-phthalaldehyde post-column derivatization is described. The polyamines were separated in a LiChrospher100 RP18 column. Elution gradient was formed with two mobile phases: A (sodium acetate 0.1 M + sodium octanesulphonate 0.01 M, pH = 4.5) and B (sodium acetate 0.2 M + sodium octanesulphonate 0.01 M)/acetonitrile (10:3), pH = 4.5) in a 1.2 ml/min flow rate. The derivative eluent was monitored by fluorescence (excitation, 345 nm; emission, 455 nm). Besides excellent linearity (putrescine, r = 0.9816; spermidine, r = 0.9920; spermine, r = 0.9901), the technique demonstrated intra and inter-day precision (< 20%) as well as recovery (spermidine = 92.56%; spermine = 84.47%). Quantification limits were 0.22 pM for putrescine, 76.44 pM for spermidine and 51.44 pM for spermine. The method demonstrated to be robust, simple and highly reproducible for polyamine determination in tissues.
Validation; Analytical technique; HPLC; Polyamines
