Received on ,
Accepted on ,

Brief Communications

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

¹ Department of Laboratory Medicine, University of Washington, Seattle, WA

^* To whom correspondence should be addressed. E-mail: ahoof{at}u.washington.edu.

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 (S_y|m.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 methods. 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.