Clinical Chemistry
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Clinical Chemistry 54: 1729-1732, 2008. First published August 7, 2008; 10.1373/clinchem.2008.104083
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(Clinical Chemistry. 2008;54:1729-1732.)
© 2008 American Association for Clinical Chemistry, Inc.

Brief Communications

Isopropanol Protein Precipitation for the Analysis of Plasma Free Metanephrines by Liquid Chromatography–Tandem Mass Spectrometry

Luke C. Marney, Thomas J. Laha, Geoffrey S. Baird, Petrie M. Rainey and Andrew N. Hoofnaglea

Department of Laboratory Medicine, University of Washington, Seattle, WA;

aaddress correspondence to this author at: Department of Laboratory Medicine, Campus Box 357110, 1959 NE Pacific St, Room NW120, University of Washington Medical Center, Seattle, WA 98115-7110. Fax (206) 598-6189; e-mail ahoof{at}u.washington.edu.


Abstract

Background: High-performance liquid chromatography–tandem mass spectrometric (LC-MS/MS)1 analysis of plasma free metanephrines is the most diagnostically sensitive and specific screening test for the diagnosis of pheochromocytoma. We sought to develop an in-house method for this expensive test

Methods: We used off-line isopropanol protein precipitation of plasma to remove interfering substances before LC-MS/MS analysis. We compared the extraction efficiency and limits of quantification of protein precipitation to those of previously reported solid-phase techniques.

Results: The new method had limits of quantification of 0.09 nmol/L and 0.17 nmol/L for metanephrine and normetanephrine, respectively. Method comparison with a previously described solid-phase extraction method revealed Deming regression slopes of 0.904 and 0.994, intercepts of 0.007 and 0.023, and SEs of the residuals (Sy|x) of 0.071 and 0.284 for metanephrine and normetanephrine, respectively. Extraction efficiency of isopropanol protein precipitation was 66% for metanephrine and 35% for normetanephrine, results that were superior to the efficiencies of 4% and 1% for our adapted solid-phase extraction method. No ion suppression was observed at the retention times for metanephrine and normetanephrine.

Conclusions: Isopropanol protein precipitation is a novel and effective off-line sample preparation method for metanephrines that offers a less expensive alternative to on-line solid-phase extraction for low-volume testing and requires a sample volume of only 200 µL. The mass spectrometric analysis time is equivalent to that of solid-phase techniques.






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Copyright © 2008 by the American Association for Clinical Chemistry.