邻苯二甲醛柱前衍生反相高效液相色谱法测定CHO细胞内氨基酸浓度

Determination of Amino Acids in CHO Cells by Reversed-phase High Performance Liquid Chromatography with o-Phthaldialdelhyde Pre-column Derivatization

用邻苯二甲醛进行柱前衍生,HypersilAA-ODS柱分离,梯度洗脱,紫外检测,在17min内分离测定了中国仓鼠卵巢(CHO)细胞内19种氨基酸的浓度。紫外检测器的检出限为0.10～0.23μmol/L;12次连续测定25μmol/L氨基酸标准溶液的保留时间,精密度小于0.3%,面积精密度小于3%。在一定浓度范围内,氨基酸组分和内标物峰面积的比值与氨基酸浓度呈良好的线性关系,相关系数为0.9979～0.9999。用内标工作曲线法对胞内氨基酸浓度进行了定量分析,氨基酸回收率为87%～106%。

Nineteen amino acids in Chinese hamster ovary (CHO) cells were determinated by reversed-phase high performance liquid chromatography with o-phthaldialdelhyde pre-column derivatization. The separation of amino acid derivatives was carried out on a Hypersil AA-ODS column by gradient elution, and was detected with an ultraviolet detector. The detection limits of the ultraviolet detector ranged from (0.10) to (0.23 μmol/L); the retention time precision was below (0.3%) and area precision was below 3% over 12 consecutive runs for a (25μmol/L) amino acid standard solution. A good linear relationship exists between the peak area ratio of amino acids and internal standard in certain range of concentrations, and the correlation coefficients ranged from (0.997 9) to (0.999 9). Concentrations of intracellular amino acids were quantitatively analyzed with standarded curves method based on internal standardization, and the recovery of amino acids ranged from 87% to 106%.