摘要
以6-氨基喹啉基-N-羟基琥珀酰-亚氨基甲酸酯为柱前衍生化试剂,结合HPLC技术,首次建立了定量分析CHO细胞培养上清中氨基酸浓度的方法。选用Agilent 1260型高效液相色谱仪,AccQ-TagTM柱,以流动相A和流动相B[乙腈-水(60∶40,体积比)]进行梯度洗脱,荧光激发波长为250nm,发射波长为395nm,柱温为42.5℃,进样量为5μL。结果表明:21种氨基酸的线性回归系数均大于0.99,连续6次测定培养上清中氨基酸组分峰面积RSD均小于5%;21种氨基酸9次测定的平均加标回收率为96.8%,RSD为4.93%。该方法快速、简便、重复性好、结果准确,符合动物细胞培养上清中氨基酸分析的要求。
A HPLC method was first developed for the determination of amino acids from CHO cell culture supernatant using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate as pro-column derivatization reagent. The analysis of amino acids was performed on Agilent 1260 HPI.C using AccQ-TagTM column. Mobile phase consis- ted of concentrated liquid-water（1 ： 10） （A） and acetonitrile-water（60 ： 40） （B） with gradient elution. The ex- citation wavelength was 250 nm and the emission wavelength was 395 nm. The column temperature was 42.5 ℃ and the injection volume was 5 μL. The results showed that the linear regression coefficients were grea- ter than 0.99 for 21 kinds of amino acids. The precisions of peak areas were all less than 5% with 6 consecutive injections. The average recovery（n=9） was 96.8% with RSD of 4.93% for 21 kinds of amino acids. In summa- ry,this method is rapid,simple,reproducible,accurate,and it is suitable for determination of amino acids from animal cell culture supernatant.
出处
《化学与生物工程》
CAS
2014年第2期68-71,78,共5页
Chemistry & Bioengineering
基金
"重大新药创制"科技重大专项资助项目(2012ZX09105301)
