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UPLC-MS/MS Method Simultaneously Determines Plasma Catecholamines and Their Metabolites

By LabMedica International staff writers
Posted on 27 Jan 2021
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Image: ACQUITY I-Class UPLC coupled with Xevo TQ-S mass spectrometer (Photo courtesy of Waters Corporation).
Image: ACQUITY I-Class UPLC coupled with Xevo TQ-S mass spectrometer (Photo courtesy of Waters Corporation).
Phaeochromocytomas and paragangliomas (PPGLs) are rare tumors of adrenal chromaffin cells or extra-adrenal paraganglia. They are difficult to diagnose because of the non-specific clinical symptoms such as hypertension, palpitations, flushing and sweating.

Inappropriate catecholamines (CAs) production was normally used for the diagnosis of PPGLs in the clinical laboratory. New guideline recommends screening of fractionated urinary metanephrines (MNs, the metabolites of CAs) or plasma free MNs using high performance liquid chromatography with electrochemical detection (HPLC-ECD) or liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) as MNs are continuously secreted by the tumor.

Laboratory Medical Scientists at the Sichuan Provincial People’s Hospital (Sichuan, China) and their colleagues collected plasma samples in EDTA anticoagulant tubes by venipuncture in a seated position from patients. Plasma samples were pretreated with solid-phase extraction, followed by a 3-minute UPLC-MS/MS analysis to quantify epinephrine (E), norepinephrine (NE), dopamine (DA), metanephrine (MN), normetanephrine (NMN) and 3-methoxytyramine (3-MT), simultaneously.

A Waters ACQUITY I-Class UPLC coupled with Xevo TQ-S mass spectrometer (Waters Corporation, Milford, MA, USA) was used for analysis. Chromatographic separation was carried out using an Agilent Pursuit 3 PFP column (150 × 2.0 mm, 3.0 μm) (Agilent Technologies, Santa Clara, CA, USA) kept at 40 °C during analysis. The UPLC-MS/MS method was comprehensively verified and its diagnostic efficiency on PPGLs was tested using seven PPGLs and 408 non-PPGLs patient plasma samples.

After the UPLC-MS/MS method was verified, the seven PPGLs and 408 non-PPGLs patient plasma samples were used to assess its diagnostic efficiency. The median concentrations of the six analytes in the PPGLs patients were determined to be: E, 0.36 nmol/L; NE, 8.47 nmol/L; DA, 0.09 nmol/L; MN, 0.26 nmol/L; NMN, 6.26 nmol/L; and 3-MT, 5.35 pg/mL. The results showed that the median concentration of E of PPGLs group was significantly higher than that of cerebral hemorrhage group, but showed no significant difference to the other six non-PPGLs groups. The median concentration of NE of PPGLs group was significantly higher than almost all the non-PPGLs groups, except for cardiac diseases group. The median concentration of NMN of PPGLs group was significant higher than all non-PPGLs groups.

The authors concluded their study presented a UPLC-MS/MS method with high sensitivity and selectivity, good extraction recoveries and low matrix interferences for determining the concentrations of catecholamines and metabolites in human plasma. ROC curve analysis showed that the developed method provided excellent diagnostic performance in patients tested for PPGLs, which was validated to be an effective tool for screening or ruling out of PPGLs in clinical practice. The study was published on in the January 2021 issue of the journal Clinical Biochemistry.

Related Links:
Sichuan Provincial People’s Hospital
Waters Corporation
Agilent Technologies

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