Joanne Adaway and Brian Keevil, University Hospital of South Manchester, UK

GOA L

To demonstrate the simultaneous and high sensitivity analysis of plasma metanephrines by LC/MS for clinical research.

BAC KG ROU N D

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.

Simultaneous, Sensitive LC/MS Analysis of Plasma Metanephrines

Measurement of several important

plasma biogenic amines.

Figure 1: Structures of biogenic amines.

2

HO

HN

OH

Metanephrine

O

HO

NHOH

Normetanephrine

O

2

HO

NH

3-methoxytyramine

O

T H E SO LU T IO N

In this study, a method for simultaneously measuring three biogenic amines (metanephrine, normetanephrine, 3-methoxytryamine) from plasma has been developed. This method takes advantage of the capabilities of an online SPE system (ACQUITY UPLC® Online SPE Manager) to enable the accurate measurement of these molecules.

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:

Step Solvent Volume (µL)

Cartridge conditioning 0.1% formic acid (v/v) in acetonitrile 200

Cartridge conditioning 2 0.1% formic acid (v/v) in acetonitrile 200

Cartridge conditioning 3 20% 10 mmol/L ammonium formate pH 3.2:80% acetonitrile 250

Cartridge conditioning 4 95% acetonitrile 250

Cartridge equilibration H2O 250

Sample load H2O 250

Cartridge wash H2O 200

Cartridge wash 2 95% acetonitrile 200

Clamp flush 95% acetonitrile 250

Mobile phase A: 100 mmol/L ammonium formate pH 3.2Mobile phase B: Acetonitrile

0

100

80

60

% M

obile

Pha

se B

40

20

01 2 3 4

Minutes

Plasma metanephrines gradient

5 6 7 8

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

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.

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.

MRM of 6 Channels ES+180.2 > 148.2 (metanephrine)

8.03e6

MRM of 6 Channels ES+155.2 > 123.2 (d4 3MT)

1.61e6

MRM of 6 Channels ES+151.2 > 119.2 (3MT)

4.09e4

2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 Min.

2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 Min.

2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 Min.

MRM of 6 Channels ES+180.2 > 148.2 (metanephrine)

7.74e7

MRM of 6 Channels ES+151.2 > 119.2 (3MT)

4.32e7

MRM of 6 Channels ES+166.2 > 134.2 (normetanephrine)

2.32e7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Min.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Min.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Min.

Tube adsorptionRecoveryBreakthrough

R E SU LT S

Metanephrine

Metanephrine

Metanephrine

Normetanephrine

3-MT

3-MT

Deuterated 3-MT

Figure 4: Plasma metanephrine analysis using LC/MS with online SPE.

Figure 5: Plasma normetanephrine analysis using LC/MS with online SPE.

Figure 6: Plasma 3-methoxytyramine analysis using LC/MS with online SPE.

6.56.05.55.04.54.03.53.02.52.01.51.00.5 7 Min.

6.5

4.50

4.00

3.50

3.00

2.50

Resp

onse

2.00

1.50

1.00

0.50

-0.00-0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

nmol/L

6.05.55.04.54.03.53.02.52.01.51.00.5 7 Min.

MRM of 6 Channels ES+180.2 > 148.2 (metanephrine)

2.06e6

Compound name: MetanephrineCorrelation coefficient: r=0.999933, r^2=0.999865Calibration curve: 0.479747* x + 0.00347414Response type: Internal Std (Ref 2), Height*(IS Conc./IS Height)Curve type: Linear, Origin: Exclude, Weighing: 1/x, Axis trans: None

MRM of 6 Channels ES+183.3 > 151.2 (d3 metanephrine)

3.56e6

6.5

Resp

onse

-0.00

0.40

0.80

1.20

1.60

2.00

2.40

-0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0nmol/L

6.05.55.04.54.03.53.02.52.01.51.00.5 7 Min.

6.56.05.55.04.54.03.53.02.52.01.51.00.5 7 Min.

MRM of 6 Channels ES+166.2 > 134.2 (normetanephrine)

3.86e5

Compound name: NormetanephrineCorrelation coefficient: r=0.999803, r^2=0.999606Calibration curve: 0.269543* x + 5.64015e-005Response type: Internal Std (Ref 4), Height*(IS Conc./IS Height)Curve type: Linear, Origin: Exclude, Weighing: 1/x, Axis trans: None

MRM of 6 Channels ES+169.2 > 137.2 (d3 normetanephrine)

1.20e6

6.56.05.55.04.54.03.53.02.52.01.51.00.5 7 Min.

6.56.05.55.04.54.03.53.02.52.01.51.00.5 7 Min.

Resp

onse

0.20

0.60

1.00

1.40

1.80

2.20

-0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0nmol/L

MRM of 6 Channels ES+151.2 > 119.2 (3MT)

1.01e6

Compound name: 3MTCorrelation coefficient: r=0.999700, r^2=0.999400Calibration curve: 0.238484* x + 0.00160461Response type: Internal Std (Ref 6), Height*(IS Conc./IS Height)Curve type: Linear, Origin: Exclude, Weighing: 1/x, Axis trans: None

MRM of 6 Channels ES+155.2 > 123.2 (d4 3MT)

1.68e6

LLOQ 0.0375 nmol/L

LLOQ 0.075 nmol/L

LLOQ 0.0375 nmol/L

Metanephrine

Deuterated Internal

Standard Metanephrine

Deuterated Internal

Standard Normetanephrine

Normetanephrine

3-Methoxytyramine

Deuterated 3-MT

Internal Standard

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

SUMMA RY

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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©2013 Waters Corporation. Produced in the U.S.A. September 2013 720004613EN IH-PDF

For research use only. Not intended for use in diagnostic procedures.