College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Oct 1;906:91-5. doi: 10.1016/j.jchromb.2012.08.030. Epub 2012 Sep 1.
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.
建立了一种快速分辨液相色谱（RRLC）方法，用于在柱前衍生化 2,4-二硝基氟苯（DNFB）后同时测定大鼠血清中的 23 种氨基酸。氨基酸衍生物在 Agilent Zorbax Eclipse Plus C（18）（4.6mm×50mm，1.8μm）柱上于 45°C 下分离。紫外（UV）检测设置在 360nm。在 1.5mLmin（-1）的流速下，用三元梯度洗脱实现了 23 种氨基酸在 10min 内的良好分离。校准曲线在 1 至 500μmolL（-1）范围内呈线性，每个氨基酸的系数均为 0.9962 或更好。所有 23 种氨基酸的定量下限（LLOQ）均为 1μmolL（-1），信噪比（S/N）≥4。日内和日间精密度分别以相对标准偏差（RSD）的百分比表示，范围为 0.32%至 3.09%和 0.67%至 5.82%。最后，该方法成功应用于大鼠血清中氨基酸的测定，回收率范围为 90.8%至 106.0%，RSD 百分比范围为 1.78%至 4.68%。结果表明，与传统高效液相色谱（HPLC）方法甚至一些超高效液相色谱串联质谱（UPLC-MS/MS）相比，该方法具有更短的洗脱时间、更好的分辨率和更尖锐的峰形。
