Exploratory Development Of High Performance Capillary Electrophoresis To The Diagnosis Of Pheochromocytoma And Neuroblastoma

ObjectiveTo establish a method for the detection of vanillylmandelic acid (VMA),homovanillic acid(HVA) and creatinine(Cr) in random urine by high performance capillary electrophoresis(HPCE),which can be applied to the diagnosis of pheochromocytoma and neuroblastoma.Methods1.Separation conditions,including running buffer,separation mode, wavelength,separation voltage and specification of capillary tube,were optimized in order to achieve baseline resolution of VMA,HVA and Cr in both standards and urine samples.2.The novel method provided here was validated systematically, including linearity,reproducibility,recovery,interference and comparison tests.3.Random urine samples from 100 healthy adults and 100 healthy infants were collected and detected for establishing the reference ranges of VMA/Cr and HVA/Cr,respectively.Additionally,7 pathological urine samples from 5 patients with pheochromocytoma and 2 patients with neuroblastoma were quantified by this method. 4.From a healthy volunteer,a patient with pheochromocytoma and an infant with neuroblastoma,5 random urine samples were taken consecutively and analyzed by our method in order to observe the dynamic process of VMA and HVA secretion in human urine.Results1.Separations were carried out with 120 mmol/L phosphate buffer (pH 6.80) in an uncoated fused-silica capillary tube(47 cm×75μm I.D.) by capillary zone electrophoresis(CZE).Diluted urine samples were directly injected by pressure(1 p.s.i.,4s),separated at the voltage of 20 kV and monitored by a diode array detector(DAD) at 200 nm.Under these conditions,separations of Cr,VMA and HVA could be completed in 13 min with high resolution.2.The linearity ranges were 0～500,0～500 and 0～4000μmol/L for VMA,HVA and Cr,respectively,with correlation coefficients(r) between 0.9972 and 0.9991(P＜0.001).The limits of detection(S/N=3) for VMA,HVA and Cr were 1.0,1.0 and 50.0μmol/L,respectively.3.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.4.This CZE method presented high accuracy.The average recoveries were 98.36%for VMA,93.56%for HVA and 98.85%for Cr. Such compounds possibly appearing in human urine as catecholamines, 5-hydroxytryptamine and albumen didn't interfere with the detection of VMA,HVA and Cr.5.This CZE method correlated well with HPLC method and correlation coefficients(r) of VMA and HVA were 0.9549(P＜0.001) and 0.9451(P＜0.001),respectively.6.Skewness distributions were presented for VMA/Cr and HVA/Cr in random urine(n=100) 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.7.After observing the secreting regularity of VMA and HVA in random urine,we found that the absolute concentrations of VMA and HVA changed a lot during 24 hours,while the ratios of VMA/Cr and HVA/Cr had a smaller changing extent.The latter proved to be more stable and scientific to be taken as the analysis index.Conclusions The CZE method devised here for direct measurement of urinary VMA,HVA and Cr is simple,fast and precise with high reproducibility, which may meet the need for routine detection and mass screening of pheochromocytoma and neuroblastoma.