Determination of Polyamines as Their Benzoylated Derivatives by HPLC

Abstract

Almost all of the methods available to analytical biochemists have been applied to polyamine analysis at some time. The diversity of the methods used is an indication of the difficulties experienced in attempts to measure the polyamine content of biological samples. The quantitation of polyamines by high-pressure liquid chromatography (HPLC) of then benzoyl derivatives was described by Redmond and Tseng (1) in 1979 and has been modified at intervals ever since (2–12). The advantages of this technique are its relative simplicity and the need for only very basic HPLC equipment—pump, column, injection valve, and UV-detector. The main disadvantage, as noted by Seiler (13,14), is that benzoyl chloride, in addition to undergoing condensation with primary and secondary amines (Fig. 1), may also react with imidazole nitrogens and phenolic hydroxyl groups. However, benzoylation results in fewer side reactions than are seen with dansyl chloride and hence extensive clean-up procedures may not be necessary. The main contaminants in the chromatogram are benzoyl chloride, benzoic acid, benzoic anhydride, and methylbenzoate, the first two being quantitatively the most important. Contrary to the accepted dogma, some benzoyl chloride may survive the derivatization procedure and be extracted and applied to the column; its presence can be detected by a peak in the chromatogram (Fig. 2) and by its odor if the top of the column is unscrewed; use of a guard column (or cartridge) will reduce the deleterious effects benzoyl chloride on the analytical column. Benzoic anhydride may comigrate with spermine, benzoic acid can interfere with the separation of N-acetylputrescine and N-(3-aminopropyl)pyrrolidin-2-one (7), and methyl benzoate can overlap N ¹-acetylspermidine, depending on the solvent mixture used. Clean-up steps have been recommended by some authors (12,15) and do result in the reduction/removal of some contaminating peaks. However, if these do not interfere then thus step can be omitted. Methanol-water is the most widely used solvent system and most reported separations have been performed isocratically (1–5,7,8,10–12), obviating the need for a gradient maker or two pump system, although use of a gradient may enable the simultaneous detection of other compounds (6,9). Acetonitrile-water mixtures, also used isocratically, can improve sensitivity (11). The characteristics of the assay have been extensively investigated (4,7,12).