Zlata D. Clarka, M. Laura Parnasb and Elizabeth L. Frankb Determination of Urinary Metanephrine and Normetanephrine by LC-MS/MS Following Solid Phase Extraction Zlata D. Clarka, M. Laura Parnasb and Elizabeth L. Frankb aARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108 bDepartment of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT 84112 Introduction Materials and Methods Results The determination of metanephrine (M) and normetanephrine (NM) (Figure 1) urine concentrations is used in clinical diagnosis of pheochromocytoma, a rare but potentially fatal tumor arising from the chromaffin cells of the adrenal medulla, as well as related neurogenic tumors. 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 MS 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. Assay Overview M and NM were subjected to acid hydrolysis at 90 °C for 15 min to release the analytes from their glucuronide and sulfate forms. The target compounds were then isolated from the hydrolyzed urine using solid phase extraction (SPE) on PWCX mixed mode cation exchange sorbent (SPEware Corp.) in 96-well format. The SPE eluates were injected directly without a drying step and separated using a Restek Ultra II PFP column (2.1 x 50 mm, 3 µm) on an Agilent 1200 HPLC system. The mobile phase was 0.2% formic acid in water/methanol. Gradient elution produced well-resolved peaks within 2 minutes. Representative chromatograms of normal and abnormal patient specimens are shown in Figure 2. Detection was accomplished using an Applied Biosystems API 3200 triple quadrupole mass spectrometer with electrospray ionization in positive mode. Isotopically labeled internal standards (IS) and a five point calibration curve were used for quantitation in the new LC-MS/MS assay. Data was acquired in multiple-reaction monitoring (MRM) mode with two transitions (quantifier and qualifier) for each analyte and internal standard. LC-MS/MS System Applied Biosystems API 3200 triple quadrupole mass spectrometer CTC Analytics HTC PAL autosampler Agilent 1200 Series LC system Restek Ultra II PFP column (2.1 x 50 mm, 3 µm) Conditions Mobile phase A 0.2% formic acid in water LC Gradient: Mobile phase B 0.2% formic acid in 100% methanol Flow rate 0.5 mL/min Injection volume 15 µL Column temperature 30 °C Linearity Standards prepared at 25, 100, 500, 2000, and 7000 nmol/L.. The method is linear to 7000 nmol/L (Figure 3). Analytical Sensitivity LOQ (nmol/L) LOD (nmol/L) Metanephrine 10 2.5 Normetanephrine 5 2.5 Precision Three concentrations of controls run in duplicate over 20 days. Mean %CV Metanephrine (nmol/L) within run between-day total Low 124 1.6 0.9 1.9 Level I 946 1.0 2.5 2.7 Level II 1818 1.6 1.3 2.0 Normetanephrine (nmol/L) within run between-day total Low 432 1.9 1.1 2.2 Level I 3149 1.5 2.1 2.6 Level II 4725 1.2 2.2 2.5 Accuracy/Method Comparison Patient samples (n = 275) were analyzed by both the newly developed method and the method currently in use (Figure 4). Deming regression: Metanephrine y= 1.050x – 45.44; Sy/x= 104.98; R= 0.9891 Normetanephrine y= 1.079x – 191.44; Sy/x= 396.56; R= 0.9635 Reference Interval Literature reference intervals (below) were verified using 127 patient urines submitted for Cu or As routine testing. Metanephrine 152 - 1775 nmol/d Normetanephrine 273 - 3548 nmol/d MRM transitions Quantifier Qualifier Metanephrine m/z 180  165 m/z 180  148 Normetanephrine m/z 166  134 m/z 166  106 Metanephrine (1,2-13C2, 15N) m/z 183  168 m/z 183  151 d3-Normetanephrine (α-d2, β-d1) m/z 169  137 m/z 169  109 Time (min) Mobile phase B (%) 0.0 5 1.5 2.5 50 2.8 3.0 4.5 Results FIGURE 1: Chemical structures of metanephrines FIGURE 2: Example chromatograms FIGURE 3: Linearity experiments graphical data Metanephrine Normetanephrine Normetanephrine Metanephrine A. Normal urine specimen B. Abnormal urine specimen Intensity (cps) Time (min) M - IS Conclusions NM - IS We have developed and validated a robust 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. M NM FIGURE 4: Method Comparison References Metanephrine Normetanephrine M 1. Wu AHB, ed. Tietz Clinical Guide to Laboratory Tests, 4th ed. St. Louis: Saunders, 2006: 1798 pp. 2. Crockett DK, Frank EL, Roberts WL. Rapid analysis of metanephrine and normetanephrine in urine by gas chromatography-mass spectrometry. Clin Chem 2002; 48: 332-337. 3. Whiting MJ. Simultaneous measurement of urinary metanephrines and catecholamines by liquid chromatography with tandem mass spectrometric detection. Ann Clin Biochem 2009; 46: 129-136. 4. Taylor RL, Singh RJ. Validation of liquid chromatography-tandem mass spectrometry method for analysis of urinary conjugated metanephrine and normetanephrine for screening of pheochromocytoma. Clin Chem 2002; 48: 533-539. NM M - IS NM - IS