Measurement of Plasma Free Metanephrine and Normetanephrine by Liquid Chromatography-Tandem Mass Spectrometry for Diagnosis of Pheochromocytoma

Susan A. Lagerstedt, Dennis J. O'Kane, Ravinder Jit Singh

Research output: Contribution to journalArticle

92 Citations (Scopus)

Abstract

Background: Quantification of plasma free metanephrines is usually accomplished by HPLC with electrochemical detection, but sample preparation is labor-intensive and time-consuming, run times are long, and interfering substances sometimes obscure the relevant peaks. The aim of this study was to develop a sensitive and specific LC-MS/MS method for plasma free metanephrines. Methods: After solid-phase extraction, chromatographic separation of normetanephrine (NMN) and metanephrine (MN) was accomplished by use of a cyano analytical column. NMN, MN, d3-NMN, and d3-MN positive ions were detected in the multiple-reaction monitoring mode using the specific transitions m/z 166→134, 180→148, 169→137, and 183→151, respectively, with an atmospheric pressure chemical ionization source. Results: Multiple calibration curves exhibited consistent linearity and reproducibility. Interassay imprecision values (CV; n = 20) for NMN at 0.64, 1.9, and 2.7 nmol/L were 6.6%, 7.8%, and 13%, respectively. Interassay CV for MN at 0.60, 1.2, and 2.1 nmol/L (n = 20) were 9.2%, 6.8%, and 9.8%, respectively. The mean recoveries of NMN and MN relative to the internal standard were 100% and 96%, respectively. The assays were linear between 0.20 and 10.0 nmol/L. Deming regression of HPLC and LC-MS/MS results yielded slopes of 0.93 (95% confidence interval, 0.89-0.98) and 0.89 (0.85-0.93) and y-intercepts of -0.16 and 0.03 nmol/L for NMN (n = 132) and MN (n = 92), respectively. Conclusions: This novel LC-MS/MS approach provides a precise, rapid, and specific alternative method to HPLC for the quantification of the low nanomolar concentrations of free metanephrines in plasma.

Original languageEnglish (US)
Pages (from-to)603-611
Number of pages9
JournalClinical Chemistry
Volume50
Issue number3
DOIs
StatePublished - Mar 2004

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Normetanephrine
Metanephrine
Liquid chromatography
Pheochromocytoma
Tandem Mass Spectrometry
Liquid Chromatography
Mass spectrometry
Plasmas
High Pressure Liquid Chromatography
Atmospheric Pressure
Solid Phase Extraction
Ion sources
Calibration
Atmospheric pressure
Assays
Positive ions
Personnel
Confidence Intervals
Ions

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Measurement of Plasma Free Metanephrine and Normetanephrine by Liquid Chromatography-Tandem Mass Spectrometry for Diagnosis of Pheochromocytoma. / Lagerstedt, Susan A.; O'Kane, Dennis J.; Singh, Ravinder Jit.

In: Clinical Chemistry, Vol. 50, No. 3, 03.2004, p. 603-611.

Research output: Contribution to journalArticle

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title = "Measurement of Plasma Free Metanephrine and Normetanephrine by Liquid Chromatography-Tandem Mass Spectrometry for Diagnosis of Pheochromocytoma",
abstract = "Background: Quantification of plasma free metanephrines is usually accomplished by HPLC with electrochemical detection, but sample preparation is labor-intensive and time-consuming, run times are long, and interfering substances sometimes obscure the relevant peaks. The aim of this study was to develop a sensitive and specific LC-MS/MS method for plasma free metanephrines. Methods: After solid-phase extraction, chromatographic separation of normetanephrine (NMN) and metanephrine (MN) was accomplished by use of a cyano analytical column. NMN, MN, d3-NMN, and d3-MN positive ions were detected in the multiple-reaction monitoring mode using the specific transitions m/z 166→134, 180→148, 169→137, and 183→151, respectively, with an atmospheric pressure chemical ionization source. Results: Multiple calibration curves exhibited consistent linearity and reproducibility. Interassay imprecision values (CV; n = 20) for NMN at 0.64, 1.9, and 2.7 nmol/L were 6.6{\%}, 7.8{\%}, and 13{\%}, respectively. Interassay CV for MN at 0.60, 1.2, and 2.1 nmol/L (n = 20) were 9.2{\%}, 6.8{\%}, and 9.8{\%}, respectively. The mean recoveries of NMN and MN relative to the internal standard were 100{\%} and 96{\%}, respectively. The assays were linear between 0.20 and 10.0 nmol/L. Deming regression of HPLC and LC-MS/MS results yielded slopes of 0.93 (95{\%} confidence interval, 0.89-0.98) and 0.89 (0.85-0.93) and y-intercepts of -0.16 and 0.03 nmol/L for NMN (n = 132) and MN (n = 92), respectively. Conclusions: This novel LC-MS/MS approach provides a precise, rapid, and specific alternative method to HPLC for the quantification of the low nanomolar concentrations of free metanephrines in plasma.",
author = "Lagerstedt, {Susan A.} and O'Kane, {Dennis J.} and Singh, {Ravinder Jit}",
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T1 - Measurement of Plasma Free Metanephrine and Normetanephrine by Liquid Chromatography-Tandem Mass Spectrometry for Diagnosis of Pheochromocytoma

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AU - O'Kane, Dennis J.

AU - Singh, Ravinder Jit

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N2 - Background: Quantification of plasma free metanephrines is usually accomplished by HPLC with electrochemical detection, but sample preparation is labor-intensive and time-consuming, run times are long, and interfering substances sometimes obscure the relevant peaks. The aim of this study was to develop a sensitive and specific LC-MS/MS method for plasma free metanephrines. Methods: After solid-phase extraction, chromatographic separation of normetanephrine (NMN) and metanephrine (MN) was accomplished by use of a cyano analytical column. NMN, MN, d3-NMN, and d3-MN positive ions were detected in the multiple-reaction monitoring mode using the specific transitions m/z 166→134, 180→148, 169→137, and 183→151, respectively, with an atmospheric pressure chemical ionization source. Results: Multiple calibration curves exhibited consistent linearity and reproducibility. Interassay imprecision values (CV; n = 20) for NMN at 0.64, 1.9, and 2.7 nmol/L were 6.6%, 7.8%, and 13%, respectively. Interassay CV for MN at 0.60, 1.2, and 2.1 nmol/L (n = 20) were 9.2%, 6.8%, and 9.8%, respectively. The mean recoveries of NMN and MN relative to the internal standard were 100% and 96%, respectively. The assays were linear between 0.20 and 10.0 nmol/L. Deming regression of HPLC and LC-MS/MS results yielded slopes of 0.93 (95% confidence interval, 0.89-0.98) and 0.89 (0.85-0.93) and y-intercepts of -0.16 and 0.03 nmol/L for NMN (n = 132) and MN (n = 92), respectively. Conclusions: This novel LC-MS/MS approach provides a precise, rapid, and specific alternative method to HPLC for the quantification of the low nanomolar concentrations of free metanephrines in plasma.

AB - Background: Quantification of plasma free metanephrines is usually accomplished by HPLC with electrochemical detection, but sample preparation is labor-intensive and time-consuming, run times are long, and interfering substances sometimes obscure the relevant peaks. The aim of this study was to develop a sensitive and specific LC-MS/MS method for plasma free metanephrines. Methods: After solid-phase extraction, chromatographic separation of normetanephrine (NMN) and metanephrine (MN) was accomplished by use of a cyano analytical column. NMN, MN, d3-NMN, and d3-MN positive ions were detected in the multiple-reaction monitoring mode using the specific transitions m/z 166→134, 180→148, 169→137, and 183→151, respectively, with an atmospheric pressure chemical ionization source. Results: Multiple calibration curves exhibited consistent linearity and reproducibility. Interassay imprecision values (CV; n = 20) for NMN at 0.64, 1.9, and 2.7 nmol/L were 6.6%, 7.8%, and 13%, respectively. Interassay CV for MN at 0.60, 1.2, and 2.1 nmol/L (n = 20) were 9.2%, 6.8%, and 9.8%, respectively. The mean recoveries of NMN and MN relative to the internal standard were 100% and 96%, respectively. The assays were linear between 0.20 and 10.0 nmol/L. Deming regression of HPLC and LC-MS/MS results yielded slopes of 0.93 (95% confidence interval, 0.89-0.98) and 0.89 (0.85-0.93) and y-intercepts of -0.16 and 0.03 nmol/L for NMN (n = 132) and MN (n = 92), respectively. Conclusions: This novel LC-MS/MS approach provides a precise, rapid, and specific alternative method to HPLC for the quantification of the low nanomolar concentrations of free metanephrines in plasma.

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