Multiple Reaction Monitoring with Multistage Fragmentation (MRM3) Detection Enhances Selectivity for LC-MS/MS Analysis of Plasma Free Metanephrines

BACKGROUND: LC-MS/MS with multiple reaction monitoring (MRM) is a powerful tool for quantifying target analytes in complex matrices. However, the technique lacks selectivity when plasma free metanephrines are measured. We propose the use of multistage fragmentation (MRM3) to improve the analytical selectivity of plasma free metanephrine measurement. METHODS: Metanephrines were extracted from plasma with weak cation exchange solid-phase extraction before separation by hydrophilic interaction liquid chromatography. We quantified normetanephrine and metanephrine by either MRM or MRM3 transitions m/z 1663134379 and m/z 18031493121, respectively. RESULTS: Over a 6-month period, approximately 1% (n = 21) of patient samples showed uncharacterized coeluting substances that interfered with the routine assay, resulting in an inability to report results. Quantification with MRM3 removed these interferences and enabled measurement of the target compounds. For patient samples unaffected by interferences, Deming regression analysis demonstrated a correlation between MRM3 and MRM methods of y = 1.00x = 0.00 nmol/L for normetanephrine and y = 0.99x + 0.03 nmol/L for metanephrine. Between the MRM3 method and the median of all LC-MS/MS laboratories enrolled in a quality assurance program, the correlations were y = 0.97x + 0.03 nmol/L for normetanephrine and y = 1.03x - 0.04 nmol/L for metanephrine. Imprecision for the MRM3 method was 6.2%-7.0% for normetanephrine and 6.1%-9.9% for metanephrine (n = 10). The lower limits of quantification for the MRM3 method were 0.20 nmol/L for normetanephrine and 0.16 nmol/L for metanephrine. CONCLUSIONS: The use of MRM3 technology improves the analytical selectivity of plasma free metanephrine quantification by LC-MS/MS while demonstrating sufficient analytical sensitivity and imprecision. (C) 2014 American Association for Clinical Chemistry