柱前衍生反相高效液相色谱法同时测定家兔关节微透析液中的精氨酸和瓜氨酸

目的 建立一种稳定的同时测定家兔关节微透析液中精氨酸和瓜氨酸的方法.方法 利用微透析技术从抗原诱导性关节炎家兔的关节腔中取样,荧光检测器反相高效液相色谱法检测样品中精氨酸和瓜氨酸浓度.衍生剂为邻苯二甲醛(OPA),色谱柱为Boston Green C18柱(4.6 mm×250 mm,5μm),流动相A为甲醇,B为50mmol·L-1乙酸钠(用冰乙酸调pH=4.5),采用二元梯度洗脱,λEx=342 nm,λEm=446 nm,流速1 mL· min-1,柱温25℃.结果 两种氨基酸在40 min内得到了很好的分离,精氨酸和瓜氨酸分别在0.054～6.480 μg· mL-1(r=0.9993),0.0525～6.3000 μg·mL-1 (r=0.999)内呈良好的线性关系,检测限分别为12.58 ng·mL-1和9.08 ng·mL-1,精密度RSD分别为0.86％和1.04％.结论 该方法简便、灵敏、准确,适用于关节微透析液中精氨酸和瓜氨酸的同时测定.

Simultaneous Determination of Arginine and Citruline in Rabbit Joint Microdialysis Fluid by Reverse-phase High Performance Liquid Chromatography with Pre-column Derivatization

Objective To establish a stable method for the simultaneous determination of arginine and citruline in rabbit joint microdialysis fluid.Methods The samples were collected by microdialysis from the joint cavity of rabbits,which suffered antigen-induced arthritis.The concentrations of arginine and citruline in the samples were determined by high performance liquid chromatography-fluorescence detector(HPLC-FLD).The samples were derived with o-phthalaldehyde(OPA),and the separation was carried out on a Boston Green C18 column(4.6 mm × 250 mm,5 μm).The mobile phase consisted of A(methanol) and B(50 mmol·L-1 sodium acetate,pH=4.5,adjusted with acetic acid),and binary gradient elution was performed.The detection wavelength was λEx=342 nm,λEm=446 nm,the flow rate was 1 mL·min-1 and the column temperature was maintained at 25 ℃.Results Two kinds of amino acids can be separated well within 40 min,the arginine and citruline obtained good linearity in the range of 0.054~6.480 μg·mL-1(r=0.9993)and 0.0525~6.3000 μg·mL-1(r=0.999),the limit of detection was 12.58 ng·mL-1 and 9.08 ng·mL-1,and the precision RSD(%) was 0.86 % and 1.04 %,respectively.Conclusion The method is convenient,sensitive and reliable,and is suitable for the simultaneous determination of arginine and citruline in joint microdialysis fluid.