|Zlata D. Clark(a), M. Laura Parnas(b), Elizabeth L. Frank(b)|
(a) ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108
(b) Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT 84112
The determination of urinary metanephrine (M) and normetanephrine (NM) concentrations is used in clinical diagnosis of pheochromocytoma, a rare but potentially fatal tumor arising from the chromaffin cells of the adrenal medulla. In our laboratory, a GC-MS method is used to measure M and NM concentrations. Separation and detection are accomplished in 13 minutes. The assay is robust, although double derivatization is required to provide unique fragments for both analytes. Increased reagent cost and limited availability prompted our investigation of an alternate method.
The goal of this study was to develop and validate a high-throughput LC-MS/MS method for the measurement of urinary M and NM to replace the current GC-MS based assay.
M and NM were isolated from urine using mixed mode cation exchange SPE and separated using a Restek Ultra II PFP column (2.1 x 100 mm, 3 µm) on an Agilent 1200 HPLC system. The mobile phase was 50 mM formic acid in water/methanol. Gradient elution produced well-resolved peaks within 5 minutes. Detection was accomplished with an Applied Biosystems API 3200 triple quadrupole mass spectrometer with electrospray ionization in positive mode. Deuterated internal standards were used for quantification. A five point calibration curve was used in the new LC-MS/MS assay; the GC-MS method used a single calibrator. Data was acquired in the multiple-reaction monitoring (MRM) mode. The MRM transitions were: m/z 180 → 165 for M, m/z 166 → 134 for NM, m/z 183 → 168, for d3-M, and m/z 169 → 137 for d3-NM.
A subset of specimens received by the laboratory for urine metanephrines testing was analyzed using both the GC-MS and the LC-MS/MS methods. Concentrations ranged from 17-1856 µg/L for M and from 40-8073 µg/L for NM. The method comparison parameters using Deming regression analysis were as follows: For M, the number of samples (n) was 25, slope 1.002, intercept -0.9 µg/L, standard error of the estimate (Sy/x) 42.4 µg/L, and correlation coefficient (R) 0.996; for NM, n was 24, slope 0.969, intercept 122.3 µg/L, Sy/x 276.5 µg/L, and R 0.989.
We have developed an LC-MS/MS method for the measurement of metanephrine and normetanephrine in urine specimens. This method has higher analytical sensitivity and increased throughput compared to the GC-MS method in current use and allows elimination of a cumbersome and costly sample derivatization step.