Plasma And Urinary Free Metanephrines Measured By HPLC-ED For Diagnose Of Pheochromocytoma

Objective：1. To establish a method for determination of free metanephrines(MNs), including normetanephrine (NMN) and metanephrine (MN) inplasma and urine by high performance liquid chromatography withelectrochemical detection (HPLC-ED).2. To investigate the plasma free MNs in the diagnosis and differentialdiagnosis of pheochromocytoma and prognosis. The developed methodwas applied to determine the concentrations of plasma free MNs innormal volunteers, patients with primary hypertension, patients withadrenal mass (who were excluded pheochromocytoma) and patients withpheochromocytoma.3. To investigate urinary free MNs and the ratio of metanephrines tocreatinine (MNs/Cr) in diagnosis of pheochromocytoma. The developedmethod was applied to determine the concentrations of urinary free MNsin control group (aforementioned patients with primary hypertension andpatients with adrenal mass) and patients with pheochromocytoma, thenMNs/Cr was measured. Methods:1. The free metanephrines in plasma and urine were extracted usingthe cation exchange solid phase extraction. A ZORBAX SB-C18column(3.0×100mm,3.5μm) was used for analyzing at a temperature of30℃.The separation was carried out with the mobile phase consisting of0.1mol/L sodium dihydrogen phosphate,0.4mmol/L EDTA-Na2and0.5mmol/L sodium octane sulfonate (pH3.2): acetonitrile(95.5:4.5) at a flowrate of0.6ml/min. The electrochemical detector was set at potential of+0.85V.50μl samples was injected, and the detection time was20minute.2. The method was carried out to detect plasma free MNs, includingnormal volunteers (n=26), patients with primary hypertension (n=23),patients with adrenal mass (n=24) and patients with pheochromocytoma(n=21).3. The method was carried out to detect urinary free MNs, includingcontrol group (n=18) and patients with pheochromocytoma (n=7), thenmeasure the MNs/Cr.Results:1. The retention time of NMN and MN were4.3and6.7min,respectively. In plasma, the assay was linear from25to2000ng/L for bothNMN and MN. The limit of detection was11ng/L for NMN and12ng/Lfor MN. The intra-day CVs were less than4.4%, and the inter-day CVswere less than6.3%. The average recoveries for NMN and MN from plasma were in the range of86.6~117.0%.In urine, the the assay was linear from25to2500μg/L for both NMNand MN. The limit of detection was5.9μg/L for NMN and11.3μg/L forMN. The intra-day CVs were less than5.9%, and the inter-day CVs wereless than6.0%. The average recoveries for NMN and MN from urine werein the range of92.8~103.4%.2. The concentrations of plasma free MNs in patients withpheochromocytoma were significantly increased compared with those ofother groups (P<0.05); The sensitivies and specificies of plasma free MNsfor diagnosis of pheochromocytoma were100%and89%respectively;Plasma concentrations of free MNs showed significant positiverelationships with tumors size in patients with pheochromocytomas (NMN:r=0.952, P<0.001. MN: r=0.510, P=0.018). Plasma concentrations of freeMNs in patients with pheochromocytoma dropped rapidly after surgery.3. The concentrations of urinary free MNs and MNs/Cr in patientswith pheochromocytoma were significantly increased compared withcontrol group (P<0.05); The concentrations of urinary free MNs andMNs/Cr showed significant positive relationships with the concentrationsof plasma free MNs (NMN: r=0.889, P <0.001；NMN/Cr：r=0.799, P<0.001；MN: r=0.828, P=0.018；MN/Cr：r=0.703, P <0.001). Thesensitivities of urinary free MNs and MNs/Cr for diagnosis ofpheochromocytoma were both85.9%, but the specificities of urinary free MNs (NMN44.4%, MN22.2%) were significantly lower than MNs/Cr(NMN/Cr88.9%, MN/Cr88.9%).Conclusions:1. High performance liquid chromatography with electrochemicaldetection was developed to determine free MNs in plasma and urine. Thismethod is cheap, sensitive, accurate and is well suitable for research andclinical routine measurement.2. Plasma free MNs are good markers for the diagnosis anddifferential diagnosis of pheochromocytoma and prognosis.3.24h urinary fractionated MNs can be replace by the test of urinaryfree MNs/Cr for the diagnosis of pheochromocytoma.