Separation and determination of amino acids, creatinine, bioactive amines and nucleic acid bases by dual-mode gradient ion-pair chromatography.

A simple and fast high-performance liquid chromatography method for the analysis of amino acids and biological bases such as creatinine was developed using a technique termed "dual-mode gradient ion-pair chromatography". A butyl-silica reversed-phase column and water-acetonitrile eluent containing sodium dodecyl sulfate (SDS) and perchloric acid were used for the separation. A concentration gradient of acetonitrile from 15 to 35% provided a good separation of such organic cations. Since change in concentration of acetonitrile causes change in distribution equilibrium of SDS between mobile and stationary phases, a complete regeneration of the column to the initial state is required for the reproducible separations. Completion of the reequilibrium was indicated by a system peak appearing in the UV chromatogram and by conductivity measurements. The formation mechanism of the system peak was revealed. A flow-rate gradient from 1 to 2 ml/min was introduced in addition to the concentration gradient to shorten the cycle time of the chromatography. More than twenty kinds of amino acids, creatine and creatinine were simultaneously separated within 50 min and the cycle time was 80 min including the reequilibration time. A post-column derivatization fluorescence detection system was usable as well as UV detection. This elution system was also useful for the separation of bioactive amines and nucleic acid bases. The developed method was applied to the simultaneous determination of urinary creatinine and diagnostic amino acids due to inherited metabolic disorders.