2,7-Dimethyl-3,8-dinitrodipyrazolo[1,5-a:1′,5′-d]pyrazine-4,9-dione: A new labelling reagent for liquid chromatographic analysis of amino acids Original Research Article

The use of 2,7-dimethyl-3,8-dinitrodipyrazolo[1,5-a:1′,5′-d]pyrazine-4,9-dione as pre-column reagent for LC analysis of amino acids is proposed. The compound reacts (30 min at 68 °C in presence of 0.04 M triethylamine in a dimethylsulfoxide–water mixture) with primary and secondary amino function and the stable resulting adducts can be chromatographed under reversed-phase conditions and detected at λ = 280 nm. The derivatization conditions were optimized by a series of experiments. The effect of temperature, triethylamine concentration and reagent on the reaction was investigated. The yield of the glycine derivative was found to be quantitative at a reagent amino acid molar ratio of about 6 by comparison with an authentic specimen of synthesized glycine adduct. Application of the method to quality control of commercially available oral polyaminoacid formulations is described.