用高效液相色谱(HPLC)法测定血清维生素C含量

目的 :　建立更简便、快速、准确、灵敏的测定血清维生素 C(VC)含量的方法 ,并对血清 VC稳定性进行研究。方法 :　血清样品经 1 0 %偏磷酸溶液提取 ,旋涡混匀 3 0 s,离心 1 0 min(4 0 0 0 r/min,4℃ )后 ,用带二极管阵列检测 (DAD)的高效液相色谱 (HPLC)仪对 VC进行定性定量分析。流动相为 1 0 0 mmol/L KH2 PO4 ,p H值为 3 .0 ,检测波长为 2 43 nm,流速为 1 1 ml/min。结果 :　在上述色谱条件下 ,血清中 VC能得到良好分离 ,该法的日内变异系数和日间变异系数分别为 0 .42 %和 2 .1 1 % ;回收率 82 %～ 88.5 % ;VC的最低检测限为 0 .0 5 μg/ml。在对血清 VC稳定性的研究中 ,发现血清 VC在放置过程中很容易被氧化降解 ,其偏磷酸提取液相对稳定 ,但在 5 d后含量也开始明显下降 ,到第 44d后血清和提取液中 VC含量均几乎完全消失。结论 :　建立了高速、高效、高灵敏度的测定血清 VC含量的 HPLC法。血清 VC应立即测定 ,或将样品用偏磷酸提取 ,保存在 -70℃ ,5 d以内分析完毕。

DETERMINATION OF VITAMIN C IN SERUM BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Objective: To assay the content and stability of vitamin C(VC) in serum by a simple, specific method. Methods: Serum samples were extracted by metaphosphoric acid, and analyzed on a column C 18 of HPLC with DAD detector at 234 nm. Mobile phase was 100 mmol/L KH 2PO 4, pH3.00. The flow rate was 1 ml/min. Results: Under this condition, the retention time of VC was 5.76 min. The within run and between run reproducibilities tests were 0.42% and 2.11% respectively, and the recovery rate ranged from 82% to 88.5%. Stored at -70 ℃ and compared to that of its metaphosphoric acid extract, serum VC content was stable only for two days, and then decreased rapidly, whereas it did not decrease in metaphosphoric acid extract until the fifth day. Conclusion: A simple, sensitive and reliable method of HPLC with DAD detector is established for measuring VC in either serum or fruit, such as orange juice. VC should be determined immediately or should be extracted with metaphosphoric acid as soon as possible, then the extracts are kept at -70 ℃, and analysed within 5 d.