• Application Note

Simultaneous, Sensitive LC-MS Analysis of Plasma Metanephrines

Simultaneous, Sensitive LC-MS Analysis of Plasma Metanephrines
  • Joanne Adaway
  • Brian Keevil
  • University Hospital of South Manchester
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For research use only. Not for use in diagnostic procedures.

This is an Application Brief and does not contain a detailed Experimental section.

Abstract

This Application brief demonstrates the simultaneous and high sensitivity analysis of plasma metanephrines by LC-MS for clinical research.

Benefits

Measurement of several important plasma biogenic amines

Introduction

Development of LC-MS methods for simultaneous measurement of plasma metanephrines has been challenging. Efficient sample pre-treatment strategies have yet to be defined and measurement of these molecules can be complicated by the difficult chromatographic separation of metanephrines such as 3-methoxytyramine (3-MT), from other metanephrines. Development of a good separation has proven problematic and can lead to overestimation of metanephrine levels. Many laboratories also do not measure 3-MT as the assays they use may not have the analytical sensitivity to measure this low-level analyte. To effectively study these important biogenic amines, a research method to measure metanephrine, normetanephrine and 3-MT simultaneously is needed.

Structures of biogenic amines
Figure 1: Structures of biogenic amines.

Experimental

Method details

LC System:

ACQUITY UPLC with ACQUITY UPLC Online SPE Manager

Mass Spectrometer:

Xevo TQ-S

Column:

Atlantis HILIC, 2.1 x 50 mm, 3 μm

Sample Preparation:

ACQUITY UPLC Online SPE Manager (OSM)

SPE:

MassTrak WCX OSM Cartridge

Sample preparation method

Plasma samples were diluted 1:1 with deuterated internal standard and then centrifuged through a 10 k MW cutoff centrifugation spin filter device to remove proteins from the sample. 

After filtration, an aliquot of sample was injected into the online SPE equipped LC-MS system. SPE was performed by the online SPE system as follows:

After SPE, samples were analyzed by LC-MS using the following gradient conditions:

gradient

Results and Discussion

LC-MS Analysis of metanephrine and 3-methoxytyramine
Figure 2: LC-MS Analysis of metanephrine and 3-methoxytyramine. The 3-MT elutes before the metanephrine to help avoid potential interference from ionic crosstalk and isobaric interfaces from metanephrine that could lead to the overestimation of 3-MT in the analysis of these molecules.
Recovery determination of online SPE method for biogenic amines
Figure 3: Recovery determination of online SPE method for biogenic amines. The breakthrough, recovery, and and adsorption were determined for each analyte. The online SPE method demonstrated no breakthrough or adsorption and excellent recovery for all three analytes.
Plasma metanephrine analysis using LC-MS with online SPE.
Figure 4: Plasma metanephrine analysis using LC-MS with online SPE.
Plasma normetanephrine analysis using LC-MS with online SPE.
Figure 5: Plasma normetanephrine analysis using LC-MS with online SPE.
Plasma 3-methoxytyramine analysis using LC-MS with online SPE
Figure 6: Plasma 3-methoxytyramine analysis using LC-MS with online SPE.

Conclusion

In this study, a clinical research method has been developed for the simultaneous measurement of three plasma biogenic amines. The method utilizes LC-MS and an online SPE system. This combination of SPE sample preparation coupled with the analytical power of LC-MS is able to deliver an efficient assay for these important plasma metanephrines.

The method developed here provides:

  • Simultaneous analysis of metanephrine, normetanephrine, and 3-methoxytyramine from plasma
  • Effective separation of 3-MT from metanephrines to avoid interferences and overestimation
  • Highly efficient SPE sample preparation integrated with LC-MS
  • Very good analytical sensitivity (LLOQs for all analytes in the low picomolar ranges) 

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