反相高效液相色谱结合柱前衍生化法测定人血清中L-门冬酰胺

Determination of L-asparagine in human serum on o-phthalaldehyde derivatives by RP-HPLC with pre-column derivatization

目的:建立反相高效液相色谱法(RP-HPLC)快速准确测定人血清中L-门冬酰胺(L-ASN)。方法:用已耗竭L-ASN的人血清为空白生物基质建立标准曲线,邻苯二甲醛在线柱前衍生样品,ZORBAX Eclipse AAA柱(4.6 mm×150 mm,5μm)分离衍生化产物。采用内标法根据荧光响应值定量分析。结果:L-ASN与内标高丝氨酸的衍生物均在16 min内出峰,在2～100μmol.L-1的范围内方法线性关系良好(r>0.999),最低定量限为2μmol.L-1,日内与日间精密度(RSD)均小于12.9%,回收率在96.9%～108.3%之间,平均提取回收率为58.2%,血清中L-ASN-80℃冻存四周保持稳定。结论:建立了RP-HPLC结合柱前衍生化测定人血清中L-ASN的方法,并应用于临床血清样品中L-ASN的定量测定。

Objective：To develop a rapid and accurate reversed phase high performance liquid chromatography（RP-HPLC） method for determination of L-asparagine（L-ASN） in human serum.Method：The calibration curves were established with a specific L-ASN-exhausted serum,which had been treated by spiked asparaginase.Amino acids derivatization with o-phthalaldehyde was performed automatically by the auto-sampler.After separated on a ZORBAX Eclipse AAA column,the o-phthalaldehyde derivatives were quantitated by internal standard method depending on the responding of fluorescence.Results：Both of the analyte L-ASN and the internal standard homoserine were eluted within 16 min.The calibration curves were linear over the concentration range of 2-100 μmol·L-1（r〉0.999）and the lower limit of quantification（LLOQ） was 2 μmol·L-1.The inter-day and intra-day precisions（RSD） were ≤12.9%,and the accuracy ranged from 96.9% to 108.3%.The average extraction recovery was 58.2%.L-ASN was proved to be stable storded at-80 ℃ for 4 weeks in human serum.Conclusion：An RP-HPLC combined pre-column derivatization method was successfully established,and was applied to quantitate L-ASN in clinical serum samples.