Mobile Site Full Version
share:

article_image

Trace-Level Analysis of Metanephrines in Plasma by HILIC LC-MS/MS

Abstract

Highly sensitive analysis of metanephrines in plasma is critical in the diagnosis and treatment of pheochromocytoma and paraganglioma. Here, a HILIC LC-MS/MS method was developed using a Raptor HILIC-Si column because this approach provides retention of all target compounds in highly organic mobile phases, thus providing the increased sensitivity needed to reach low detection limits. Data from linearity, accuracy, and precision testing demonstrate that accurate results are consistently obtained—even at trace levels—for these important clinical biomarkers.

Introduction

The metanephrines—metanephrine (MN), normetanephrine (NMN), and 3-methoxytyramine (3-MT)—are methylated metabolites of epinephrine, norepinephrine, and dopamine, respectively (Figure 1). These catecholamine metabolites are released from the adrenal medulla and sympathetic nervous cells and are normally maintained at very low concentrations in the bloodstream. Elevated levels of these compounds in circulation can indicate the presence of pheochromocytoma or paraganglioma, so highly sensitive measurement of in vivo concentrations is critical for correct diagnosis, treatment, and long-term patient monitoring.

Trace-level analysis of metanephrines in plasma using typical reversed-phase LC can be difficult due to limited chromatographic retention and lack of sensitivity. As an alternative, we developed an LC-MS/MS method based on hydrophilic interaction liquid chromatography (HILIC) using a Raptor HILIC-Si column. Combined with a simple solid phase extraction (SPE) procedure, an accurate and precise analysis of these important biomarkers can be achieved in a fast, 5-minute analysis time, making this a beneficial assay for high-throughput clinical labs.

Figure 1: Structures of Metanephrine (MN), Normetanephrine (NMN), and 3-Methoxytyramine (3-MT)


Experimental

Sample Preparation

Internal standard (IS) solution was prepared at 4 ng/mL for metanephrine-d3, and at 8 ng/mL for normetanephrine-d3 and 3-methoxytyramine-d4 in methanol. A plasma sample aliquot (200 μL) was mixed with 10 μL of IS solution and 600 μL of 50 mM ammonium acetate. The mixture was loaded onto an EVOLUTE EXPRESS WCX 96-well plate (30 mg) and washed with 1 mL water and 1 mL methanol:acetonitrile (50:50). The elution was performed twice with 0.9 mL of 5% formic acid in methanol:acetonitrile (50:50) and samples were then evaporated to dryness at 55 °C under a gentle stream of nitrogen. Dried extracts were reconstituted with 100 μL of 100 mM ammonium formate in water (pH 3.0):acetonitrile (10:90) and injected (10 μL) for analysis.

Calibration Standards and Quality Control Samples

Charcoal stripped human plasma (BioreclamationIVT) was fortified with metanephrine, normetanephrine, and 3-methoxytyramine to prepare calibration standards and QC samples. The linearity ranges were 0.051-20.28 nmol/L (10-4,000 pg/mL) for metanephrine; 0.14-21.83 nmol/L (24- 4,000 pg/mL) for normetanephrine; and 0.060-23.92 nmol/L (10-4,000 pg/mL) for 3-methoxytyramine. Three QC levels were prepared at 40, 400, and 2,500 pg/mL for all three analytes. The fortified standard and QC samples were prepared using the SPE procedure described above.

LC-MS/MS analysis of metanephrines in plasma was performed on an ACQUITY UPLC instrument coupled with a Waters Xevo TQ-S mass spectrometer. Instrument conditions were as follows and analyte transitions are provided in Table I.

Analytical column:

Raptor HILIC-Si (2.7 µm, 50 mm x 2.1 mm; cat.# 9310A52)

Mobile phase A:

100 mM ammonium formate in water, pH 3.0

Mobile phase B:

Acetonitrile

Gradient

Time (min)     %B

 

0.00              90

 

5.00              90

Flow rate:

0.3 mL/min

Injection volume:

10 µL

Column temp.:

30 °C

Ion mode:

Positive ESI


Table I: Analyte Transitions for Plasma Free Metanephrines LC-MS/MS Analysis

Analyte

Precursor Ion

Product Ion
Quantifier

Product Ion
Quantifier

Metanephrine-d3

183.00

151.15

Metanephrine

179.94

148.22

165.01

Normetanephrine-d3

169.00

136.96

Normetanephrine

166.00

134.02

121.01

3-Methoxytyramine-d4

155.07

122.93

3-Methoxytyramine

151.00

119.00

91.02


Results and Discussion

Chromatographic Performance

Using a HILIC approach and Raptor HILIC-Si column, good retention was obtained for all analytes. A fast, 5-minute chromatographic analysis (Figure 2) was achieved with injection of reconstituted sample extract. No chromatographic interferences were observed in the analysis of blank plasma samples (Figure 3), indicating that the simple SPE procedure was effective and specific. In addition, the isocratic elution with HILIC showed consistent results over multiple injections indicating that the method was robust (Figure 4).

Figure 2: Human Plasma Fortified with Metanephrine, Normetanephrine, and 3-Methoxytyramine (50 pg/mL Calibration Standard)

PeakstR (min)Conc. (pg/mL)Precursor IonProduct Ion
1.3-Methoxytyramine-d4 (IS)1.80400155.07122.93
2.3-Methoxytyramine1.8050151.00119.00
3.Metanephrine-d3 (IS)2.03200183.00151.15
4.Metanephrine2.0350179.94148.22
5.Normetanephrine-d3 (IS)2.11400169.00136.96
6.Normetanephrine2.1150166.00134.02
Metanephrine, Normetanephrine, and 3-Methoxytyramine in Human Plasma on Raptor HILIC-Si
LC_CF0689
ColumnRaptor HILIC-Si (cat.# 9310A52)
Dimensions:50 mm x 2.1 mm ID
Particle Size:2.7 µm
Pore Size:90 Å
Temp.:30 °C
Sample
Diluent:Mobile phase A:mobile phase B (10:90)
Inj. Vol.:10 µL
Mobile Phase
A:Water, 100 mM ammonium formate, pH 3.0
B:Acetonitrile
Time (min)Flow (mL/min)%A%B
0.000.31090
5.000.31090
DetectorMS/MS
Ion Mode:ESI+
Mode:MRM
InstrumentUHPLC
NotesSolid Phase Extraction Procedure:
Charcoal stripped human plasma was fortified with analytes at 50 pg/mL (0.25, 0.27, and 0.30 nmol/L for metanephrine, normetanephrine, and 3-methoxytyramine, respectively). Internal standard (IS) was prepared at 4 ng/mL for metanephrine-d3 and at 8 ng/mL for normetanephrine-d3 and 3-methoxytyramine-d4 in methanol. The plasma sample (200 μL) was mixed with 10 μL of IS solution and 600 μL of 50 mM ammonium acetate solution. The mixture was loaded in an EVOLUTE EXPRESS WCX 96-well plate (30 mg) and washed with 1 mL water and 1 mL methanol:acetonitrile (50:50). The elution was performed twice with 0.9 mL of 5% formic acid in methanol:acetonitrile (50:50) and evaporated to dryness at 55 °C under a gentle stream of nitrogen. Dried extract was reconstituted with 100 μL of diluent and injected (10 μL) for analysis.

Figure 3: Blank Human Plasma

PeakstR (min)Conc. (pg/mL)Precursor IonProduct Ion
1.3-Methoxytyramine-d4 (IS)1.80400155.07122.93
2.3-Methoxytyramine--151.00119.00
3.Metanephrine-d3 (IS)2.04200183.00151.15
4.Metanephrine--179.94148.22
5.Normetanephrine-d3 (IS)2.12400169.00136.96
6.Normetanephrine--166.00134.02
Analysis of Blank Human Plasma for Metanephrine, Normetanephrine, and 3-Methoxytyramine on Raptor HILIC-Si
LC_CF0695

Figure 4: Robust Column Performance for the Analysis of Metanephrines in Plasma

PeaksConc.
(ng/mL)
PrecursorProduct IonProduct Ion1st Injection (tR)135th Injection (tR)
1.3-Methoxytyramine1151.00119.0091.021.801.83
2.Metanephrine1179.94148.22165.012.032.07
3.Normetanephrine1166.00134.02121.012.112.15
Raptor HILIC-Si Injection Consistency for Metanephrine, Normetanephrine, and 3-Methoxytyramine
LC_CF0701

Linearity

Using 1/x weighted linear regression, all three analytes showed acceptable linearity with r2 values of 0.999 or greater, and deviations of <10% (Figure 5). The established limits of quantitation (LOQ) were 0.051 nmol/L (10 pg/mL), 0.14 nmol/L (24 pg/mL), and 0.060 nmol/L (10 pg/mL) for metanephrine, normetanephrine, and 3-methoxytyramine, respectively.

Figure 5: Calibration Curves


Accuracy and Precision

Precision and accuracy testing was performed on three different days. Method accuracy was demonstrated by recovery values that were within 5% of the nominal concentration for all QC levels. The %RSD was 0.2-4.5% and 1.1- 4.2% for intraday and interday comparisons, respectively, indicating good method precision (Table II).

Table II: Accuracy and Precision of QC Samples for the Analysis of Metanephrines in Plasma

 

QC Level 1 (40 pg/mL)

QC Level 2 (400 pg/mL)

QC Level 3 (2,500 pg/mL)

Analyte

Average Conc. (pg/mL)

Average Accuracy (%)

%RSD

Average Conc. (pg/mL)

Average Accuracy (%)

%RSD

Average Conc. (pg/mL)

Average Accuracy (%)

%RSD

Metanephrine

39.5

98.7

3.39

411

103

1.10

2,480

99.2

2.89

Normetanephrine

39.3

98.2

4.21

401

100

1.92

2,440

97.3

2.34

3-Methoxytyramine

40.0

100

1.81

407

102

2.07

2,470

98.9

2.02


Conclusion

As demonstrated here, using HILIC for the analysis of metanephrines in plasma is a viable approach that provides excellent method performance at clinically relevant concentrations. The Raptor HILIC-Si column ensures good retention of metanephrine, normetanephrine, and 3-methoxytyramine, which is generally difficult to achieve at low detection limits using reversed-phase chromatography. Accurate results were consistently obtained without matrix interference even at trace levels. With a fast, simple sample preparation procedure and 5-minute analysis time, the established method provides a reliable high-throughput assay for the clinical diagnosis of pheochromocytoma and paraganglioma.

Related Products