ANISOIN: A USEFUL PRE-CHROMATOGRAPHIC DERIVATIZATION FLUOROGENIC REAGENT FOR HPLC ANALYSIS OF GUANIDINO COMPOUNDS

The analysis of several aliphatic guanidino compounds is of considerable importance in pharmaceutical and biological field, but it is quite difficult due to their poor detectability owing to the absence of a strong chromophore and fluorophore. Pre-column chemical derivatization in combination with fluorescence detection constitutes an effective approach to overcome the problem. A great variety of reagents has been used for the derivatization of amines, thiols and carboxylic acids, while few probes (i.e. benzoin, 9,10-phenanthrenequinone) have been described for the determination of guanidino compounds. In the present communication 4,4’-dimethoxybenzoin (anisoin) (I) is proposed as new useful fluorescent labelling reagent for HPLC determination of guanidino compounds such as arginine. Arginine is an amino acid that is best known as a growth hormone releaser. It is also used in certain conditions accompanied by hyperammonaemia and as acidifying agent. Anisoin was found to react rapidly and selectively with the guanidino function to give stable and highly fluorescent adducts. The RP-HPLC separations were performed at 33±2°C on a Phenomenex Prodigy 5ODS (250 x 3.2 mm i.d.) with gradient elution using a mobile phase consisting of a mixture A/B where A=0.05 M TEA phosphate buffer (pH 4 or pH 5) and B=acetonitrile, at a flow rate of 0.4 ml/min. The fluorescence intensity at l=435 nm (excitation at l=325 nm) was monitored. The proposed method was applied with satisfactory results to the determination of arginine in commercial pharmaceutical formulations and guanidino compounds in human urine.