Liquid chromatography Mass-spectrometry (LC-MS) is a widely used method for the quantitation of plasma metanephrines.
In 2% of our patients, 6 different uncharacterised co-eluting endogenous substances were found to interfere with the LC-MS/MS assay leading to inability to report results. We investigated the use of MRM3 detection to improve the selectivity of this important diagnostic assay.
Metanephrine and Normetanephrine were isolated from plasma samples using weak cation exchange solid phase extraction prior to analysis. Prepared samples were injected onto a Shimadzu Prominence HPLC system and chromatographic separation was achieved with hydrophilic interaction liquid chromatography. Mass spectrometric detection was performed using an ABSciex QTRAP 5500 mass spectrometer operating in positive ion electrospray mode.
Normetanephrine and Metanephrine were quantitated by two different processes. MRM transitions were created for each fragment produced by collision induced dissociation of the target analytes. By use of linear ion trap technology, each fragment was then fragmented again to produce MRM3 transitions. All MRM and MRM3 transitions were then assessed for analytical specificity.
Over a 6 month period, 21 patient samples were identified as being affected by one or more interferences. Retrospective review of drug history could not yet identify a cause for these interferences.
No MRM transition fragments were capable of removing all the interferences. Each fragment was fragmented again and MRM3 transitions were then assessed. For metanephrine 180/149/121 and for normetanephrine 166/134/79, all interferences were removed.
Using these new MRM3 transitions a sample comparison was completed against the existing MRM method (Metanephrine 180/148, Normetanephrine 166/134) showing good agreement with slopes of 1.04 and 1.01 (Deming Regression) and bias of -3.2% and 0.2% for Metanephrine and Normetanephrine, respectively.
This is the first demonstration of MRM3 technology for the interference-free quantitation of plasma metanephrines contributing to improved quality of patient results.