Analysis of polyamines in biological samples by HPLC involving pre-column derivatization with o-phthalaldehyde and N-acetyl-L-cysteine.
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abstract
Polyamines (putrescine, spermine and spermidine) play a crucial role in the regulation of cell growth, differentiation, death and function. Accurate measurement of these substances is essential for studying their metabolism in cells. This protocol describes detailed procedures for sample preparation and HPLC analysis of polyamines and related molecules (e.g., agmatine and cadaverine) in biological samples. The method is optimized for the deproteinization of samples, including biological fluids (e.g., 10 l), plant and animal tissues (e.g., 50 mg), and isolated/cultured cells (e.g., 1 10(6) cells). The in-line reaction of polyamines with o-phthalaldehyde and N-acetyl-L-cysteine yields fluorescent derivatives which are separated on a reversed-phase C18 column and detected by a fluorometer at an excitation wavelength of 340 nm and an emission wavelength of 450 nm. The total running time for each sample (including column regeneration on the automated system) is 30 min. The detection limit is 0.5 nmol/ml or 0.1 nmol/mg tissue in biological samples. The assays are linear between 1 and 50 M for each of the polyamines. The accuracy (the nearness of an experimental value to the true value) and precision (agreement between replicate measurement) of the HPLC method are 2.5-4.2 % and 0.5-1.4 %, respectively, for biological samples, depending on polyamine concentrations and sample type. Our HPLC method is highly sensitive, specific, accurate, easily automated, and capable for the analysis of samples with different characteristics and small volume/amount, and provides a useful research tool for studying the biochemistry, physiology, and pharmacology of polyamines and related substances.
