Multiplexed Dilute-and-Shoot Liquid Chromatography-Multiple-Reaction Monitoring Mass Spectrometry Clinical Assay for Metanephrines and Catecholamines in Human Urine

Abstract

Background: Quantifying urinary catecholamines and metanephrines is essential for the clinical screening and diagnosis of neuroendocrine tumours. HPLC with electrochemical detection (HPLC-ECD) is commonly used for this type of analysis but requires extensive sample cleanup. Simple and rapid dilute-and-shoot LC-multiple-reaction monitoring (MRM)-MS assays have been developed for quantitating these analytes in urine but have not yet been validated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Methods: A simple dilute-and-shoot sample preparation without derivatization was used. C18 RP-UPLC-MRM-MS and positive-ion ESI were used, usually with two transitions per analyte being monitored. Certified deuterated internal standards were used for each analyte. Results: This assay was validated according to the CLSI C62-A guidelines, including accuracy/trueness, imprecision, sensitivity, specificity, carryover, stability, and linearity. The final MRM-MS method was compared to the established HPLC-ECD clinical chemistry reference method. The run time was reduced from 25 min to 5 min. Conclusions: A simple, robust, rapid, and cost-effective LC-MRM-MS assay for measuring urinary catecholamines and metanephrines was developed and validated according to the CLSI guidelines. This validated method requires minimal sample manipulation before analysis and provides sensitivity, specificity, and improved precision. The implementation of this assay in clinical laboratories will facilitate early and accurate diagnosis.

Keywords: adrenal gland; catecholamines; dilute-and-shoot; liquid chromatography–multiple-reaction monitoring mass spectrometry (LC-MRM-MS); metanephrines; paraganglioma; pheochromocytoma; urine.

Figure 1

Extracted ion chromatograms (XICs) for MN with and without ACN. XICs for metanephrine’s two transitions (quantifier and qualifier) showing a poor peak shape obtained when ACN was used in the dilution step. (A) An XIC with ACN in the dilution step. (B) An XIC without ACN, using only 0.1% FA in the dilution step.

Figure 2

Extracted ion chromatograms (XICs). XICs were obtained for the multiplexed detection of norepinephrine (NE), epinephrine (EP), normetanephrine (NMN), dopamine (DA), metanephrine (MN), and 3-methoxytyramine (3MT), using a reversed-phase C18 column, in a spiked catecholamine-free urine sample (250 µg/L each). The response of the analytes is expressed as intensity (y-axis) vs. time (x-axis).