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 84108bDepartment of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT 84112

Materials and Methods Results

Conclusions

Introduction

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.

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

Mean %CV

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 ComparisonPatient 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

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.

References

FIGURE 3: Linearity experiments graphical data

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.

FIGURE 1: Chemical structures of metanephrines

Assay OverviewM 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 SystemApplied Biosystems API 3200 triple quadrupole mass spectrometerCTC Analytics HTC PAL autosamplerAgilent 1200 Series LC systemRestek Ultra II PFP column (2.1 x 50 mm, 3 µm)

ConditionsMobile phase A 0.2% formic acid in water LC Gradient:Mobile phase B 0.2% formic acid in 100% methanolFlow rate 0.5 mL/minInjection volume 15 µLColumn temperature 30 °C

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 5 2.5 50 2.8 50 3.0 54.5 5

FIGURE 4: Method Comparison

Metanephrine Normetanephrine

Metanephrine Normetanephrine

Results

FIGURE 2: Example chromatograms

A. Normal urine specimen

B. Abnormal urine specimen

Inte

nsity

(cps

)In

tens

ity (c

ps)

Time (min)

Time (min)

M

NM

M - IS

NM - IS

NM - IS

NM

M

M - IS

Normetanephrine

Metanephrine

HO

O

H3C

HN

CH3

OH

HO

O

H3C

OH

NH2