高效液相色谱法检测罗非鱼血浆中L-肉碱的方法研究

Application of High Performance Liquid Chromatography (HPLC) in Detection of L-carnitine Levels of Plasma in Tilapia

以罗非鱼为对象测定血浆中的肉碱含量,就流动相、柱温对色谱峰的影响等方面对样品处理方法及衍生化反应的条件进行研究,建立相应的高效液相色谱法,Kromasil Sil色谱柱(250mm×4.6mm,5μm),流动相为乙腈—柠檬酸缓冲液(90∶10,体积比)。每1000mL流动相中加入0.25mL三乙胺,流速1.2mL/min,柱温35℃,检测波长260nm,进样量为20μL。选取10%的对溴苯乙酰基溴为衍生试剂,70℃水浴90min。方法学上考察结果表明,该方法专属性高,其线性回归方程和相关系数分别为y=0.08376x+0.09663(n=6,r^2=0.9995),最低检测限为1μmol/L,日内和日间变异系数分别为1.79%~5.56%、9.83%~11.81%,平均回收率为95.83%,12h峰面积变化相对标准偏差为6.58%,可将其作为检测L-肉碱的分析方法。

The effects of mobile phase,column temperature to chromatographic peak,method of sample handling and derivation reaction conditions on L-carnitine levels of plasma in GIFT tilapia Oreochromis niloticus with body weight of（82.71±1.28）g,and the corresponding HPLC method was established under conditions of Kromasil Sil chromatographic column（250 mm×4.6 mm,5 microns）,and mobile phase consisting of acetonitrile-citric acid buffer solution（90∶10,V/V）,0.25 mL per 1 Lliquid phase joining triethylamine,flow rate of 1.2 mL/min,35 ℃ of column temperature,detection wavelength of 260 nm and 20μL of sample.The 10% of bromobenzene acetyl bromide was used as derivative reagent at 70 ℃water bath for 90 minutes.Methodological investigation revealed that the corresponding HPLC method has high specificity,with the equation of linear regression and the correlation coefficient of y=0.08376 x＋0.09663（n=6,r^2=0.9995）,the minimum detection limit of 1μmol/L,the intra-day and inter-day variation coefficients of 1.79%—5.56% and 9.83%—11.81%,respectively,the average recovery of 95.83%,and the 12 hours peak area relative standard deviation of 6.58%.The findings indicate that the established HPLC method can be used as an assay for detection of L-carnitine in fish.