【作者】 茅惠明； 魏玮； 熊伍军； 陈炳官；

【Author】 MAO Hui-ming~(*1),WEI Wei~2,XIONG Wu-jun~3 and CHEN Bing-guan~1(1.Department of Central Laboratory,Shanghai East Hospital,Tongji University,Shanghai 200120;2.Department of tumour Shanghai East Hospital,Tongji University, Shanghai 200120;3.Department of digestion,Shanghai East Hospital,Tongji University,Shanghai 200120)

【机构】 上海同济大学附属东方医院中心实验室； 上海同济大学附属东方医院肿瘤科； 上海同济大学附属东方医院消化内科；

【摘要】 建立一种用反相高效液相色谱(RP-HPLC)同时测定血浆L-精氨酸和L-瓜氨酸浓度的方法并应用于临床。血浆样本用三氯乙酸及在75～80℃温育15 min除蛋白,离心10000 g,15 min后,上清液用苯基异硫氰酸酯(PITC)溶液衍生化,氮气吹干,流动相A定容。梯度洗脱,流动相A:500 mL含4.77 g NaAc,12.5 mL乙腈,125μL10 mmol/L EDTA;流动相B:由75 mL甲醇,225 mL乙腈及200 mL超纯水混合组成。采用PICO-TAG色谱柱[300×3.9 mm,5μm]分离样本,柱温46℃,流速1.0mL/min,进样量20μL,紫外检测器检测,波长254 nm。L-精氨酸和L-瓜氨酸浓度线性范围0～1000μmol/L,线性回归方程分别为Y=892.1697+4278.031 X(Syx=6767.05,Sa=2757.27,Sb=7.167)及Y=15531.02+4272.131X(Syx=30160,Sa=13345,Sb=33.99),两者r~2均大于0.9999;精氨酸和瓜氨酸的方法检测限分别为0.0476μmol/L和0.0201μmol/L,定量下限(LOQ)分别为0.448μmol/L和0.240μmol/L。日内变异系数CV(%)分别为3.2%和3.4%,日间变异系数CV(%)分别为4.9%和7.2%。平均回收率分别在86.69%～93.06%及86.22%～118.9%之间。用本法测定的39例健康人(男23例,女21例,23～73岁)血浆L-精氨酸和L-瓜氨酸浓度分别为58.19±16.43μmol/L及60.77±9.18μmol/L。采用三氯乙酸沉淀血浆蛋白并加温,减少了热变性蛋白的干扰并有利于延长色谱柱的使用寿命;所建方法灵敏、重现性好、结果可靠,可应用于临床和科研。更多还原

【Abstract】 Citrulline and arginine play an important role in the course of physiology and pathology in human.We developed a new method for simultaneously determining L-citrulline and L-arginine in plasma by using RP-HPLC with ultraviolet detection.The plasma samples were deproteinized by adding trichloroacetic acid and heating up suitably.After centrifugating,the supemate was derivatized by phenyl-isothiocyanate(PITC) solution.We used nitrogen gas to evaporate the mixture to dryness,and the sample was reconstituted in mobile phase A.A PICO-TAG column was selected as separation column at 46℃.A gradient elution was carried out with a flow rate at 1.0 mL/min.The linearities were over the range from 0 to at least 1000μmol/L for L-citrulline and L-arginine.R~2 were all above 0.9999 for the both.LOD were 0.0201μmol/L and 0.0476μmol/L,respectively.LOQ were 0.240μmol/L and 0.448μmol/L, respectively.Intra-and inter-day CVs were<3.40%and were<7.2%,respectively.The average recovery rates were 86.22%～118.9%.The concentrations of healthy human were 60.77±9.18μmol/L and 58.19±16.43μmol/L,respectively.This approach is a reliable and efficient analytical platform for simultaneous determination of citrulline and arginine in plasma.更多还原