MSACL 2024 Abstract

Validation of a Simple Dilute-and-Shoot LCMSMS Method for Urinary Metanephrines: Time to Break Free?

Gerard Fernando(1), Jason L Robinson (1,2,3)
1 Health PEI, Charlottetown, Prince Edward Island, Canada 2 University of Prince Edward Island, Charlottetown, Prince Edward Island, Canada 3 Dalhousie University, Halifax, NS, Canada

Jason Robinson, PhD (Presenter) Health PEI Provincial Laboratory Services

Presenter Bio: I am a Clinical Biochemist practicing on Prince Edward Island, Canada. I am interested in novel testing approaches and interpretation within the clinical laboratory. I'm presently focused on implementing and developing mass spec methodologies for the first time on the island using LCMSMS and PSMSMS. I did my Clinical Biochemistry fellowship training at the University of Calgary following an academic post-doc at the Baylor College of Medicine in Houston, Texas. My graduate training was in the area of amino acid metabolism of the newborn at Memorial University of Newfoundland. I am passionate about mentorship in science and am an avid outdoorsmen. I live in Charlottetown with my wife and growing family.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Introduction: Pheochromocytoma is a tumor of the adrenomedullary chromaffin cells. These tumors commonly produce excessive amounts of the catecholamine metabolites metanephrine, normetanephrine, and 3-methoxytyramine, which are collectively referred to as metanephrines and are generally specific for pheochromocytomas. Indeed, quantitation of fractionated urine or plasma free metanephrines are the first line biochemical test for the investigation of pheochromocytoma. Often, the fractionated urine metanephrines are measured in an acidified 24-hour collection, followed by in vitro acid hydrolysis to deconjugate urine metanephrines prior to measurement by LCMSMS or HPLC. However, the amount of free, non-conjugated urine metanephrines are more clinically relevant versus the total deconjugated amount; and moreover, urine free metanephrines are reported to be highly stable in refrigerated urine without acid preservative. We hypothesized that by measuring the free fraction of metanephrines in urine we could establish a streamlined and concise laboratory workflow in addition to more convenient collection requirements for patients.

Objectives: To develop an accurate and robust LCMSMS method to detect urinary metanephrines using a UHPLC coupled with a Thermo TSQ Quantis tandem mass spectrometer.

Methods: The mass spectrometer was tuned using pure calibrator standards for metanephrine, normetanephrine and 3-methoxytyramine. Deuterated internal standards were used for each compound. Final sample preparation involved diluting urine samples with 4-volumes of mobile phase A (10 mM ammonium formate; pH 2.8), and 2-volumes of internal standard in mobile phase B (100% methanol). Samples were injected with UHPLC through a pentafluorophenyl column (150 mm x 2.1 mm; 1.9 µm) and a 13-minute gradient method eluted all three compounds of interest with column reconditioning. The concentration of each target was determined using commercial calibrators, and within-run precision and repeatability studies were performed using commercial QC material with CLSI based protocols. The method was extensively compared to an accredited referral laboratory using non-linear regression analysis and via an ISO15189 proficiency testing program for urine free metanephrines by LCMSMS. We also evaluated sample stability and various preanalytical preparations such as solid-phase extraction.

Results: Within-run precision of all targets was 0.9 to 2.5%; and repeatability was 2.7 to 3.1%. The limit of quantitation was between 1.3 to 1.5 nmol/L and the method was linear to ~10000 nmol/L with R^2>0.99. Our method was compared to a referral lab using 105 clinical samples and we report an acceptable negative bias (-1.2 to -14 nmol/L) and comparison across the 3 metanephrine targets (R2 = 0.82 to 0.92). Importantly, samples above the clinical cut-offs were 100% concordant with the referral laboratory. Method accuracy was considered acceptable based on the performance of 3 proficiency testing events with an LCMS peer group of 11. Solid-phase extraction did not measurably improve our analytical sensitivity based on signal-to-noise and the instrument performed excellent with only the minimum amount of vendor recommended weekly maintenance. We established reference intervals in nmol/d using non-acidified 24-hour urines submitted to our laboratory, and transferred intervals from a referral lab as well as the literature. The samples were stable refrigerated or frozen for 4 weeks.

Conclusions: We validated a simple and robust methodology for detecting urine free metanephrines, which allowed us to repatriate a large volume send-out test. The collection requirement allows for more convenient patient collection protocols and the ability to perform more tests on 24-hour collections.