Elsevier

Journal of Chromatography B

Volume 879, Issue 31, 1 December 2011, Pages 3673-3680
Journal of Chromatography B

Urinary metanephrines by liquid chromatography tandem mass spectrometry: Using multiple quantification methods to minimize interferences in a high throughput method

https://doi.org/10.1016/j.jchromb.2011.10.005Get rights and content

Abstract

Determination of urinary metanephrines is requested frequently for the differential diagnosis and monitoring of pheochromocytoma. Although numerous methods have been developed, interferences are common and hinder most available assays. This study describes the development, validation and implementation of a reliable high-throughput LC–MS/MS method for the measurement of metanephrine and normetanephrine in urine. Metanephrine and normetanephrine were isolated from urine samples subjected to acid hydrolysis using solid phase extraction on a mixed mode cation exchange sorbent in 96-well format. The extracts were injected directly onto a Restek perfluorophenyl column and separated isocratically in 0.2% formic acid in 5% methanol with a gradient cleanout step to 50% methanol. Detection was accomplished using an API 3200 triple quadrupole mass spectrometer with electrospray ionization in positive mode. Data were acquired in multiple reaction monitoring mode. Three transitions were monitored for metanephrine and its deuterated internal standard; two transitions were monitored for normetanephrine and its deuterated internal standard. Two quantification methods were used to address metanephrine interferences without reducing throughput. The method was linear to 15,000 nmol/L. The limits of detection and quantification were 2.5 and 10 nmol/L, respectively. Within run, between-day and total imprecision values were at or below 1.9%, 2.5% and 2.7% for both analytes. The method correlated well with our previously used GC–MS method. Injection-to-injection time was 6 min. The validated LC–MS/MS method for measurement of metanephrine and normetanephrine in urine specimens was placed into service in August 2010 and has performed exceptionally well.

Highlights

► We developed and validated a robust LC–MS/MS method for urinary metanephrines. ► Specificity was enhanced and interferences resolved using multiple mass transitions. ► This sensitive, high-throughput LC–MS/MS method is suitable for clinical use.

Introduction

Pheochromocytoma, a neuroendocrine tumor arising from the chromaffin cells of the adrenal medulla, is a rare and often overlooked cause of secondary hypertension [1]. Although most pheochromocytomas are benign, tumor production of excess catecholamines has potentially fatal consequences due to the potent impact of these compounds on the cardiovascular system [2]. Therefore, correct diagnosis and timely treatment of these tumors are crucial.

Over the last several decades, various biochemical tests for catecholamines and their metabolites have been used for differential diagnosis [3]. The measurement of plasma free and/or urinary fractionated metanephrines was recommended as appropriate testing for initial assessment by the First International Symposium on Pheochromocytoma [4]. While plasma metanephrines have been reported to have a higher clinical diagnostic value than urinary metanephrines [5], [6], [7], both tests have significant advantages over tests for the parent catecholamines and other metabolites [4]. Assessment of urine metanephrine excretion has been proposed as the appropriate test for screening a population in which the pre-test probability of disease is low [8]. Some of the advantages of testing for urinary as opposed to plasma metanephrines are higher analyte concentrations, which makes analysis less challenging, and a non-invasive sample collection that minimizes expenditure of time and effort by the medical staff [4].

In our laboratory, urinary metanephrines is a high-volume test frequently requested by medical providers. Previously, this test was performed using a GC–MS method [9]. Although a significant improvement over lower throughput HPLC assays with high incidence of interferences, the GC–MS methods suffer from longer run times and more cumbersome sample preparation than required for an LC–MS/MS platform. The increasing availability of this powerful technique in clinical laboratories is evidenced by the growing number of publications describing LC–MS/MS methods for urinary [10], [11], [12] and plasma metanephrines [13], [14], [15]. This study describes the development and validation of a simple, reliable, high throughput LC–MS/MS method for the analysis of metanephrine and normetanephrine in urine. Potential interferences are mitigated by the use of multiple quantification methods.

Section snippets

Chemicals and reagents

HPLC-grade methanol was purchased from JT-Baker; formic acid (99%) and hydrochloric acid (HCl, 37%) from VWR (West Chester, PA); and sodium hydroxide (NaOH), sodium hydrogen phosphate and sodium dihydrogen phosphate from Sigma–Aldrich (St. Louis, MO). Racemic metanephrine·HCl was purchased from Toronto Research Chemicals (North York, Ontario, CAN); rac-normetanephrine·HCl, dopamine·HCl, 3-methoxytyramine·HCl, rac-norepinephrine (+)-bitartrate, and rac-epinephrine base from Sigma–Aldrich; rac

Sample preparation and LC–MS/MS conditions

The acid hydrolysis procedure used in our previous GC–MS method [9] was re-optimized. Several combinations of final HCl concentration (0.3, 0.6, and 3 M), temperature (20, 60 and 90 °C), and duration (15 and 30 min) were evaluated using standard solutions (50 and 4000 nmol/L), three quality control materials as well as low (90 nmol/L for MN and 250 nmol/L for NM) and high (5300 nmol/L for both MN and NMN) urine pools. Measurements were carried out in duplicate. Analyte concentration yields were

Discussion

In current practice urinary and/or plasma metanephrines are the biochemical tests of choice for the diagnosis and assessment of pheochromocytoma. Some argue that while the clinical sensitivity of both tests is similar, plasma metanephrines supersede urinary metanephrines in clinical specificity [5], [6]. However, the concentrations of metanephrines in plasma are 2–3 orders of magnitude lower than those in urine, presenting a greater analytical challenge. Consequently, urinary metanephrine

Conclusions

In conclusion, we have developed, thoroughly characterized and validated a robust, high-throughput method for the determination of urinary metanephrines. This method utilizes a simple LC–MS/MS system and a 96-well format SPE amenable to automation. Occasional interferences occurring on the two MN transitions chosen initially were resolved by addition of a third transition and the use of two quantification methods. The assay was placed into service in August 2010 and has performed exceptionally

Acknowledgements

We would like to thank Dr. Mark Kushnir, Mr. Seyed Sadjadi and the staff of the Mass Spectrometry 2 Laboratory for their assistance during this project. This study was supported by the ARUP Institute for Clinical and Experimental Pathology®.

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Parts of these data were presented at the MSACL conference in San Diego, CA, February 2010, and at the annual meeting of the American Association for Clinical Chemistry in Anaheim, CA, July 2010.

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