An LC-MRM assay for the quantification of metanephrines from dried blood spots for the diagnosis of pheochromocytomas and paragangliomas

Richard, Vincent R.; Zahedi, René P.; Eintracht, Shaun; Borchers, Christoph H.

The quantitation of metanephrine (MN), normetanephrine (NMN), and 3-methoxytyramine (3MT) – referred to as metanephrines -- by LC-MS/MS is the gold-standard for screening for pheochromocytoma and paragangliomas (PPGLs), tumors of the adrenal gland and the peripheral nervous system. An assay for metanephrines from dried blood spots (DBSs) would be of high clinical utility as it simplifies sample collection, enables remote sampling, and could increase compliance with the clinical recommendation for supine sampling. Moreover, DBS sampling facilitates the measurement of blood-derived metanephrines in pediatric patients – where DBSs are well-established – in order to diagnose neuroblastomas. Here, we adapted an established derivatization-based LC-MRM-MS assay for plasma catecholamines, and optimized the sample extraction, LC, and MS parameters to produce a fast, sensitive, and robust method for the measurement of metanephrines from DBSs, including 3-methoxytyramine. The DBS samples were excised, derivatized with phenyl isothiocyanate (PITC) on-spot, extracted, and measured by LC-MRM-MS. To validate assay suitability and performance, we assessed the linearity, precision, accuracy, recovery, and matrix effects of the method, and determined the stability of metanephrines in DBSs under different storage conditions. Assay performance for NMN, MN, and 3MT was sufficient for quantitation from a single DBS within a linear range from 40 – 2000 pg/mL MN and NMN were stable in DBSs for 2 weeks, whereas 3MT was stable for one week regardless of storage temperature. Altogether, this work represents the first quantitative LC-MS/MS method for metanephrines from DBSs and provides a novel opportunity for the diagnosis of PPGLs and neuroblastomas in the future.