| Objective:1.To establish a simple,cost-effective direct electrochemical method for the determination of free metanephrines(MNs)in spot urine.2.To investigate the diagnostic efficacy of free MNs in pheochromocytoma(PHEO).The concentration of spot urinary free MNs from the patients with PHEO and controls were detected by the established method.The plasma free MN and NMN of all the subjects were also measured by HPLC-ED method.Methods:1.Cyclic voltammetry(CV)and differential pulse voltammetry(DPV)were used to qualitatively and quantitatively analyze MNs.The electrode is a screen printed carbon electrode(SPCE): a work electrode and a counter electrode are printed by carbon thick liquid;reference electrode is printed by Ag/Ag Cl thick liquid.2.Spot urine sample after solid phase extraction(SPE)was added into PBS(100.0 m M,p H 8.0)and mixed completely.Then,50 μL of the mixed solution was added to the working electrode on the SPCE,which was connected to the electrochemical workstation.Subsequently,the electrochemical signal was detected by DPV technique.3.The concentration of spot urinary free MNs of PHEO patient samples obtained by the established method was compared with the HPLC-ED method.4.The established method was carried out to detect spot urine free MNs,including controls(n=63)and PHEO patients(n=22).The concentration of plasma free MN and NMN in all samples was also determined by HPLC-ED method.ROC curve was employed to assess optimal diagnostic efficacy.Results:1.The linearity of free MNs in spot urinary was in the range of 0.25~12.5mg/L.The regression equation was Y = 0.916 X-0.127,with the correlation coefficient of 0.994.The lowest limit of quantification for MNs was 0.20 mg/L and the limit of detection was 0.05 mg/L.The relative standard deviation(RSD)for intra-day precisions were ranged from 5.9% to 9.7% and 6.8% to 9.9% for inter-day.The recoveries for different concentrations of MNs were ranged from 82.0% to 101.1% for intra-day,and 81.0% to 96.5% for inter-day,respectively.2.The concentration of free MNs for PHEO urine samples obtained by the established method were significantly correlated with MNs measured by the HPLC-ED method(r=0.941,p=0.000).3.In this study,the established method of detection of spot urinary free MNs and HPLC-ED method detection of plasma free MN and NMN concentrations of PHEO were significantly higher than the control group(p<0.001).The area under the ROC curve of MNs was 0.981 with the sensitivity of 95.5% and the specificity of 92.4% at the cut-off value of 0.404 mg/L in spot urinary samples.When the cut-off value of NMN and MN were 0.540 mg/L and 0.128 mg/L,the area under the ROC curve of NMN was 0.975 with the sensitivity of 90.9% and the specificity of 95.2%,the area under the ROC curve of MN was 0.916 with the sensitivity of 86.4% and the specificity of 85.7%.Conclusions:1.A simple and cost-effective electrochemical detection method was developed using unmodified SPCE for the quantitation of free MNs in spot urinary samples.Furthermore,the use of SPCE renders the electrochemical detection technique several additional advantages such as easy and cost-effective electrode fabrication and capablility of mass production,which makes it attractive for clinical diagnostic applications.2.Compared with plasma free MN and NMN,spot urinary free MNs showed a superior sensitivity for diagnosis of PHEO.Based on the convenienience and non-invasive,spot urinary free MNs was suitable for early diagnosis and prognosis of PHEO. |
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