MSACL 2019 EU Abstract

Reversed-phase UPLC-MS/MS Analysis of Plasma Catecholamines and Metanephrines for Clinical Research

Jonathan P. Danaceau (1), Li Qianqian (2), Songlin Yu (3), Lisa J Calton (4) and Gareth Hammond (4)
(1) Waters Corporation, Milford, MA, USA (2) Waters Corporation, Beijing, China (3) Department of Clinical Laboratory, Peking Union Medical College & Chinese Academy of Medical Sciences, China (4) Waters Corporation, Wilmslow, UK

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Gareth Hammond (Presenter) Waters Corporation

Relevant Financial Disclosures (within past 24 months) Stock/Bonds Waters Corporation Salary Waters Corporation Conflict mitigation NOT REQUIRED.

Background: Analysis of plasma catecholamines and metanephrines for clinical research is challenging due to their low concentrations (pg/mL) and the extreme polar nature of the analytes. HILIC LC-MS/MS has been used successfully but can be challenging to implement. To address this issue, we have developed a reversed-phase UPLC-MS/MS method that employs a previously developed mixed-mode SPE procedure and combines it with reversed-phase chromatography using the Waters UPLC™ HSS-PFP column.

Methods: Plasma samples (250 µL) were loaded onto Waters Oasis™ WCX µElution plates and diluted 1:1 with 50 mM NH4COOH containing 1 ng/mL internal standards. After loading, samples were washed with 200 µL of 50 mM NH4COOH and methanol (MeOH) and eluted with MeOH containing 2% formic acid (FA). After evaporation (5 min.) samples were reconstituted in 50 µL of 97:2:1 Water:ACN:FA, resulting in a 5X sample concentration. Separation was achieved using a Waters ACQUITY UPLC™ HSS-PFP column. All analytes were analyzed using quantifier and qualifier MRM transitions with deuterated internal standards on a Waters Xevo TQ-S micro mass spectrometer.

Results: The method enabled rapid separation in 1.5 minutes (3.5 minutes injection to injection) for norepinephrine (NE), epinephrine (EP), normetanephrine (NMT), dopamine (DA), metanephrine (MTN), and 3-methoxytyramine (3-MT). SPE recovery was consistent and removed phospholipids. Analyte recoveries from 5 plasma lots had %CVs <20% with matrix effects also <20% for all analytes. Calibration curves were linear from 2 – 20,000 pg/mL for all compounds with correlation coefficients (r2) >0.999 over five occasions. Accuracy was within 10% bias and precision was under 12% across all 4 QC levels (15, 30, 750, and 15,000 pg/mL). The method was analytically sensitive, with LLOQs of 10, 5, 5, 5, 2, and 1 pg/mL for NE, EP, NMT, DA, MTN, and 3-MT, respectively.

Conclusions: The challenge was met by using Oasis mixed-mode SPE combined with UPLC separation on the HSS-PFP column and detection by the Xevo TQ-S micro UPLC-MS/MS system. The method demonstrates excellent linearity, analytical sensitivity, precision and accuracy, while providing high sample throughput capabilities for the analysis of plasma catecholamines and metanephrines for clinical research purposes.

For Research Use Only, Not for use in diagnostic procedures.