MSACL 2019 EU Abstract

LC-MS/MS Analysis of Plasma Epinephrine and Norepinephrine

Marianne Lerbæk Bergmann and Anne Vibeke Schmedes
Lillebaelt Hospital, Department of Biochemistry and Immunologyl, Vejle

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Marianne Bergmann (Presenter) Lillebaelt Hospital >> POSTER (PDF)

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

Background:
The catecholamines, epinephrine (E) and norepinephrine (NE) are important both as neurotransmitters and hormones, affecting body functions such as blood pressure, heart rate, lipolysis, glycogenolysis, fatty acid mobilization and body temperature regulation. Measurement of E and NE in plasma is therefore of great interest in medical research. However, because of the low concentrations of E and NE in plasma (reference levels of 0.03 - 0.98 nmol/L for E and 0.63 – 4.5 for NE), and the instability of the catechol group, this is not an easy task. Furthermore, E and NE are very small and polar molecules, causing additional challenges to LC-MS/MS method development, as they are poorly retained on reverse-phase C18 columns, and elute in solvent with very low organic content (maximum 5%), which leads to poor ionization in the mass spectrometer.

Methods:
Plasma E and NE was extracted using solid phase extraction on Waters Sep-Pak Alumina B, 100 mg 96 well extraction plates. After elution the samples were diluted with the ion-pairing reagent, 1-Heptane Sulfonic Acid (HSA) and analyzed on a Waters Acquity UPLC with Xevo TQ-S tandem mass spectrometer operated in electrospray positive mode (ES+). The chromatographic separation was achieved with a Phenomenex Kinetex Biphenyl (100 x 2.1 mm, 2.6 µm) column and gradient elution with mobile phases consisting of 0.1% formic acid in water (mobile phase A) and methanol (mobile phase B).

Results:
The method is calibrated using seven in-house prepared calibrators. The calibration curve is linear up to 10 nmol/L. Intermediate precision for control samples is <7% and the limit of quantification (LOQ) is 0.20 and 0.02 nmol/L for NE and E, respectively.

Conclusions:
We have developed an LC-MS/MS method for simultaneous measurement of E and NE in plasma. The assay has good performance characteristics and is sensitive enough to measure plasma levels in the entire normal range. Furthermore, this method has been used in research projects to demonstrate changes in plasma E and NE levels in hypoglycemic diabetics.