摘要
目的建立高效毛细管电泳技术快速检测随机尿中香草扁桃酸（vanillylmandelic acid，VMA）、高香草酸（homovaniilic acid，HVA）和肌酐（creatinine，Cr）的方法。方法在毛细管区带电泳模式下，以120mmol／L NaH2PO4-Na2HPO4（pH值6．80）为缓冲液，在非涂层石英毛细管（47cm×75μm内径）中进行电泳。样品离心后直接稀释压力进样4s，20kV电压分离后通过二极管阵列检测器（DAD）检测（λ=200nm）。对该法进行系统的方法学评价后，测定健康成人及儿童随机尿标本各100份，建立成人与儿童尿VMA／Cr、HVA／Cr参考值。结果尿中VMA、HVA和Cr在13min内出峰。VMA、HVA和Cr分别在0～500、0—500、0—4000μmol／L范围内呈良好的线性，相关系数（r）在0．9972—0．9991之间（P〈0．01），检出限（S／N=3）分别为1．0、1．0和50．0μmol／L。尿中VMA、HVA和Cr迁移时间的平均批内（n=10）变异系数（CV）分别为0．58％、0．56％和0．25％，平均批间（n=10）CV分别为0．95％、1．00％和0．48％；峰面积的平均批内（n=10）CV分别为3．78％、3．97％和2．76％，平均批间（n=10）CV分别为4．60％、4．08％和4．42％。VMA、HVA和Cr平均回收率分别为98．36％、93．56％和98．85％。儿茶酚胺、5-羟色胺和清蛋白等对测定结果无干扰。该法与HPLC法有较好的相关性，VMA、HVA浓度的相关系数（r）分别为0．9549（P〈0．01）和0．9451（P〈0．01）。健康成人与儿童随机尿标本的VMA／Cr、HVA／Cr比值均呈偏态分布（n=100），以百分位数法建立其95％参考值，成人VMA／Cr、HVA／Cr分别为0—4．26和0～1．69（μmol／mm01），儿童VMA／Cr、HVA／Cr分别为0～10．39和0～4．31（μmol／mmol）。结论该法可同时检测随机尿中VMA、HVA和Cr，样品用量少，且稀释后直接进样，操作简便、快速，准确度和精密度高，易于自动化，是尿液VMA、HVA常规检测及相关疾病筛查的理想方法。
Objective To establish a method for detecting urinary vanillylmandelic acid （VMA）, homovanillic acid （HVA） and creatinine （Cr） simuhaneously by high performance capillary electrophoresis （HPCE）. Methods The separations were carried out using a 120 mmol/L phosphate buffer （pH 6. 80） in a fused-silica capillary tube of 47 cm×75 μm I.D. by capillary zone electrophoresis （CZE）. Injections were made by using the pressure mode for 4 s at 1 p. s.i. after samples were centrifuged and diluted. The detections were monitored by a diode-array detector （DAD） at 200 nm after samples were separated at a voltage of 20 kV. The method developed was validated systematically and applied to urine samples from healthy adults （n = 100） and children （n = 100） for establishing the reference ranges of VMA/Cr and HVA/Cr, respectively. Results Under these conditions, the separations of VMA, HVA and Cr could be completed within 13 min. The linearity ranges of VMA, HVA and Cr were 0-500, 0-500 and 0-4 000 μmol/ L, respectively, with the correlation coefficients （r） between 0. 997 2 and 0. 999 1 （ P 〈 0. 01 ）. The detection limits （S/N = 3 ） were 1.0 μmol/L for VMA, 1.0μmol/L for HVA and 50. 0 μmol/L for Cr. The mean within-run （n = 10） CVs of migration time for VMA, HVA and Cr in urine were 0. 58% , 0. 56% and 0. 25% respectively, while the mean between-run （n = 10） CVs of migration time were 0. 95%, 1.00% and 0. 48% respectively. The mean within-run （n = 10） CVs of peak area for VMA, HVA and Cr were 3.78% ,3. 97% and 2. 76% respectively, while the mean between-run （ n = 10 ） CVs of peak area were 4. 60% , 4.08% and 4.42% respectively. The average recoveries were 98.36% for VMA, 93.56% for HVA and 98.85% for Cr. Other compounds in human urine such as catecholamines, 5-hydroxytryptamine and albumen didn＇t interfere with the assay. The correlation between CE method and HPLC method was good. And the correlation coefficients （r） of VMA and HVA were 0. 954 9（P 〈0. 01 ） and 0. 945 1 （P 〈0. 01 ）, respectively. Skewness distributions were presented for VMA/Cr and HVA/Cr in random urine from both adults and children, and the 95% reference ranges were established by the percentile method. For adults, the reference ranges of VMA/Cr and HVA/Cr were 0-4.26 and 0-1.69 （ μmol/mmol）, respectively. For children, the reference ranges of VMA/Cr and HVA/Cr were 0-10. 39 and 0-4. 31 （μmol/mmol）, respectively. Conclusions The CE method devised here for direct measurement of urinary VMA, HVA and Cr is simple, fast,precise and automatic with good repeatability. It is an ideal method for routine detection and mass screening of pheochromocytoma and neuroblastoma.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2009年第10期1148-1154,共7页
Chinese Journal of Laboratory Medicine
关键词
电泳
毛细管
香草扁桃酸
高香草酸
肌酸
尿
Electrophoresis,capillary
Vanilmandelic acid
Homovanillic acid
Creatinine
Urine
