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

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.

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

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).

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Peaks

	Peaks	tR (min)	Conc. (pg/mL)	Precursor Ion	Product Ion
1.	3-Methoxytyramine-d4 (IS)	1.80	400	155.07	122.93
2.	3-Methoxytyramine	1.80	50	151.00	119.00
3.	Metanephrine-d3 (IS)	2.03	200	183.00	151.15
4.	Metanephrine	2.03	50	179.94	148.22
5.	Normetanephrine-d3 (IS)	2.11	400	169.00	136.96
6.	Normetanephrine	2.11	50	166.00	134.02

Conditions

Column	Raptor HILIC-Si (cat.# 9310A52)
Dimensions:	50 mm x 2.1 mm ID
Particle Size:	2.7 µm
Pore Size:	90 Å
Temp.:	30 °C
Standard/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 0.00 0.3 10 90 5.00 0.3 10 90

Detector	MS/MS
Ion Mode:	ESI+
Mode:	MRM
Instrument	UHPLC
Sample Preparation	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.

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Figure 3: Blank Human Plasma.

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Peaks

	Peaks	tR (min)	Conc. (pg/mL)	Precursor Ion	Product Ion
1.	3-Methoxytyramine-d4 (IS)	1.80	400	155.07	122.93
2.	3-Methoxytyramine	1.80	50	151.00	119.00
3.	Metanephrine-d3 (IS)	2.03	200	183.00	151.15
4.	Metanephrine	2.03	50	179.94	148.22
5.	Normetanephrine-d3 (IS)	2.11	400	169.00	136.96
6.	Normetanephrine	2.11	50	166.00	134.02

Conditions

Column	Raptor HILIC-Si (cat.# 9310A52)
Dimensions:	50 mm x 2.1 mm ID
Particle Size:	2.7 µm
Pore Size:	90 Å
Temp.:	30 °C
Standard/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 0.00 0.3 10 90 5.00 0.3 10 90

Detector	MS/MS
Ion Mode:	ESI+
Mode:	MRM
Instrument	UHPLC
Sample Preparation	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.

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

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Peaks

	Peaks	Conc. (ng/mL)	Precursor	Product Ion	Product Ion	1st Injection (tR)	135th Injection (tR)
1.	3-Methoxytyramine	1	151.00	119.00	91.02	1.80	1.83
2.	Metanephrine	1	179.94	148.22	165.01	2.03	2.07
3.	Normetanephrine	1	166.00	134.02	121.01	2.11	2.15

Conditions

Column	Raptor HILIC-Si (cat.# 9310A52)
Dimensions:	50 mm x 2.1 mm ID
Particle Size:	2.7 µm
Pore Size:	90 Å
Temp.:	30 °C
Standard/Sample
Diluent:	Mobile phase A:mobile phase B (10:90)
Conc.:	1 ng/mL
Inj. Vol.:	10 µL
Mobile Phase
A:	Water, 100 mM ammonium formate, pH 3.0
B:	Acetonitrile
	Time (min) Flow (mL/min) %A %B 0.00 0.3 10 90 5.00 0.3 10 90

Detector	MS/MS
Ion Mode:	ESI+
Mode:	MRM

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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

Analyte	QC Level 1 (40 pg/mL)			QC Level 2 (400 pg/mL)			QC Level 3 (2,500 pg/mL)
	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.

Products Mentioned

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Catalog No. 9310A52

Colonna HPLC Raptor HILIC-Si, 2,7 µm, 50 x 2,1 mm

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Authors

• 
  Shun-Hsin Liang, PhD

  Shun-Hsin is a senior principal scientist in LC Solutions at Restek. He received his bachelor’s degree from the National Taiwan University in 1988 and obtained his PhD from Michigan State University in 1996. He performed postdoctoral research at the University of Michigan from 1996 to 2000 for oncology studies. In 2001, he was appointed as research faculty at The Pennsylvania State University and focused on molecular toxicology research. In 2006, he joined MPI Research Inc. as a senior research scientist and was a study director for GLP analytical projects. In 2013, Dr. Liang joined the LC Solutions department at Restek and specialized in developing application methods across the fields of environmental, food safety, and life sciences.

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• 
  Frances Carroll

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