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A New O-phthaldialdeyde (OPA) Solution for Fluorescence HPLC Amine Group Detection without Boric Acid Preparation

Gilberto Joao Padovan, Izabel Arruda Leme, Priscila Giacomo Fassini, Nelson Iucif Junior and Julio Sergio Marchini

Traditionally o-phthaldialdeyde (OPA) is prepared with borate buffer for pre- and postcolumn reactions. The objective of this study was to show that the preparation of OPA for HPLC amine group detection using alkalinized water (pH 10.4), instead of boric acid is methodologically feasible, yielding results similar to those obtained with boric acid. This procedure can eliminates eventual line system precipitation, such as at the cuvette detector and at in the tubing line after the "T" connection. Another important feature of this procedure is that it eliminates the potential risk of borax poisoning among users. The sensitivity of the detection signal did not change at all. Standard polyamine and biogenic amine was used for both OPA preparations and HPLC analysis. The correlation coefficient (r) was 0.97 and the recovery ~ 90% for both water and borate buffer preparations. The inter-assay coefficient of variability (CV) for both the water and borate procedures ranged from 7.9 to 15.2% and the intra-assay CV ranged from 1.8 to 5.9%. Consequently, water can replace boric acid to advantage in the OPA process. Therefore, using water instead of boric acid in OPA preparation results in less environmental residue contamination by the HPLC detritus, as well eliminating future risk of boric acid poisoning.