Rapid resolution liquid chromatography (RRLC) analysis of amino acids using pre-column derivatization

A rapid resolution liquid chromatography (RRLC) method was developed for the simultaneous determination of 23 amino acids in rat serum after pre-column derivatization with 2,4-dinitrofluorobenzene (DNFB). The amino acid derivatives were separated on an Agilent Zorbax Eclipse Plus C(18) (4.6 mm×50 mm, 1.8 μm) column at 45°C. Ultraviolet (UV) detection was set at 360 nm. Good separation of 23 amino acids was achieved within 10 min with a ternary gradient elution of mobile phase at a flow rate of 1.5 mLmin(-1). Calibration curves were linear over the range from 1 to 500 μmolL(-1) with coefficients 0.9962 or better for each amino acid. The lower limits of quantification (LLOQ) of all 23 amino acids were 1μmolL(-1) with signal-to-noise (S/N) ratio ≥4. Intra- and Inter-day precisions, expressed as relative standard deviation (RSD) percentages, were ranged from 0.32% to 3.09% and 0.67% to 5.82%, respectively. Finally, it was successfully applied to the determination of amino acids in rat serum with recoveries ranged from 90.8% to 106.0% and RSD percentages ranged from 1.78% to 4.68%, respectively. The results showed that the proposed method provided a shorter elution time, better resolution and sharper peak shapes for all amino acids. Compared with the conventional high performance liquid chromatography (HPLC) methods, even some ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), the established RRLC method was superior performance.