Isopropanol protein precipitation for the analysis of plasma free metanephrines by liquid chromatography-tandem mass spectrometry.
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/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 microL. The mass spectrometric analysis time is equivalent to that of solid-phase techniques.
Marney
Luke C
LC
Department of Laboratory Medicine, University of Washington, Seattle, WA, USA.
Laha
Thomas J
TJ
Baird
Geoffrey S
GS
Rainey
Petrie M
PM
Hoofnagle
Andrew N
AN
D019840:2-Propanol / Q000737:chemistry*; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D008676:Metanephrine / Q000097:blood*; D011506:Proteins / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D011506:Proteins; D008676:Metanephrine; D019840:2-Propanol