Simultaneous determination of vanillylmandelic acid, homovanillic acid and creatinine in random urine by high performance capillary electrophoresis / 中华检验医学杂志

ABSTRACT

Objective To establish a method for detecting urinary vanillylmandelic acid (VMA), homovanillic acid (HVA) and creatinine (Cr) simultaneously 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, 1.0 μmol/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-rim (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 neuroblastoms.