A Fast Dilute-And-Shoot Method for Simultaneous 5-Hydroxyindoleacetic Acid (5-HIAA), Vanillylmandelic Acid (VMA), and Homovanillic Acid (HVA) LC-MS/MS Analysis in Human Urine

Figure 1: Structures of VMA, HVA, and 5-HIAA.

Experimental

To evaluate linearity, eight standard solutions (0.2, 0.4, 1.0, 5.0, 10, 25, 50, and 100 µg/mL) were prepared in synthetic human urine (Surine negative urine control, Cerilliant). In addition, two levels of urine control samples (Bio-Rad Lyphochek quantitative urine control, bilevel minipak #375X) were used as QC samples for accuracy and precision analysis. Controls 1 and 2 were described by Bio-Rad Laboratories as normal and abnormal levels, respectively, and had labeled concentration ranges (Table II) for 5-HIAA, VMA, and HVA that were determined by Bio-Rad using a reversed-phase HPLC method.

All prepared standards and samples were diluted 10x by mixing 40 µL of prepared standard or sample in urine with 360 µL of water and 10 µL of internal standard solution (33.3 µg/mL of 5-HIAA-D5, VMA-D3, and HVA-D5 in methanol) in a 0.45 µm Thomson SINGLE StEP PVDF filter vial (Restek, cat.# 25896). Standards and samples were filtered and injected for analysis.

5-HIAA, VMA, and HVA LC analysis was performed on a Shimadzu Nexera XR System coupled with a Sciex API 4000 mass spectrometer. Instrument conditions were as follows and analyte transitions are provided in Table I.

Table I: Analyte Transitions

Results and Discussion

To develop a quantitative dilute-and-shoot method, it is important to ensure chromatographic separation of all matrix interferences from the target analytes. Upon initial method development, it was observed that the response of HVA internal standard suffered from urine matrix suppression and resulted in inaccurate measurement of HVA concentration. To solve this problem, it was necessary to modify the additive type in the mobile phase and select an LC column with the proper phase chemistry. The final method demonstrated that the Raptor Biphenyl column is unique in that it offered excellent chromatographic performance for simultaneous accurate and precise quantification of 5-HIAA, VMA, and HVA in diluted human urine samples (see Figure 3, 4, and 5 for representative chromatograms).

Linear responses were obtained from 0.2–100 µg/mL for 5-HIAA, VMA, and HVA. A 1/x weighted quadratic regression provided the best fit for the standard calibration curves of all three analytes. The deviations were all ≤10% (except for the lowest concentration, which was ≤15%) and the R² values all were 0.999–1.000 (see examples in Figure 2). As estimated from the signal-to-noise value of the 0.2 µg/mL standard injection, the LODs were 0.03, 0.007, and 0.02 µg/mL for 5-HIAA, VMA, and HVA, respectively.

Figure 2: Calibration Curves for 5-HIAA, VMA, and HVA LC Analyses.

Three sets of samples were prepared and analyzed to evaluate method accuracy and precision. Method accuracy was demonstrated to be acceptable as the quantitative concentrations fell within the nominal concentration ranges for both the low and high urine control samples (Table II). The %RSD ranged from 0.2–2.8% and 0.2–2.9% for intraday and interday, respectively (data not shown), indicating acceptable method precision.

Table II: Accuracy and Precision of Urine Control Samples

	QC Level 1 (n = 3)			QC Level 2 (n = 3)
Analyte	Range (µg/mL)*	Average Conc. (µg/mL)	%RSD	Range (µg/mL)*	Average Conc. (µg/mL)	%RSD
5-HIAA	2.69–4.49	3.59	2.2	21.6–32.7	27.5	1.7
VMA	2.12–3.90	3.34	1.0	10.9–18.2	16.3	0.2
HVA	1.33–2.46	1.95	2.4	12.9–18.9	17.5	2.9

*The QC sample concentration ranges were supplied by the vendor.

Conclusion

Commercial kits for the analysis of 5-hydroxyindoleacetic acid (5-HIAA), vanillylmandelic acid (VMA), and homovanillic acid (HVA), require extensive sample preparation and chromatographic analysis times up to 16 minutes. In contrast, using Restek’s dilute-and-shoot method and a Raptor Biphenyl column, these serotonin and catecholamine metabolites can be measured in a quick 5-minute analysis. This method is applicable to clinical 5-HIAA, VMA, and HVA LC-MS/MS analysis in urine and provides the benefits of both fast analysis times and low-cost sample preparation procedures.

Figure 3: 5-HIAA, VMA, and HVA LC Analysis in Fortified Synthetic Human Urine.

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Peaks

	Peaks	tR (min)	Conc. (µg/mL)	Precursor Ion	Product Ion
1.	4-Hydroxy-3-methoxymandelic acid-D3 (VMA-D3)	0.90	0.83	200.1	139.9
2.	Vanillylmandelic acid (VMA)	0.92	1	197.0	137.9
3.	5-Hydroxyindole-4,6,7-D3-3-acetic-D2 acid (5-HIAA-D5)	1.80	0.83	195.1	148.0
4.	5-Hydroxyindole-3-acetic acid (5-HIAA)	1.82	1	190.0	145.9
5.	4-Hydroxy-3-methoxyphenyl-D3-acetic-D2 acid (HVA-D5)	2.10	0.83	186.1	127.0
6.	Homovanillic acid (HVA)	2.12	1	181.0	121.9

Conditions

Column	Raptor Biphenyl (cat.# 9309512)
Dimensions:	100 mm x 2.1 mm ID
Particle Size:	5 µm
Pore Size:	90 Å
Guard Column:	Raptor Biphenyl guard column cartridge 5 mm, 2.1 mm ID, 5 µm (cat.# 930950252)
Temp.:	30 °C
Standard/Sample
Diluent:	Water
Inj. Vol.:	5 µL
Mobile Phase
A:	0.1% Formic acid, 5 mM ammonium formate in water
B:	Methanol
	Time (min) Flow (mL/min) %A %B 0.00 0.5 85 15 3.00 0.5 20 80 3.01 0.5 85 15 5.00 0.5 85 15

Detector	MS/MS
Ion Mode:	ESI-
Mode:	MRM
Instrument	UHPLC
Sample Preparation	The synthetic urine (Surine) was fortified with VMA, HVA, and 5-HIAA to a concentration of 10 μg/mL. A 40 μL aliquot of fortified urine was mixed with 360 μL of water and 10 μL of internal standard solution (33.3 μg/mL in methanol) in a Thomson 0.45 μm PVDF filter vial and injected for analysis after filtration.

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Figure 4: 5-HIAA, VMA, and HVA LC Analysis in Human Urine (Control Level 1).

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Peaks

	Peaks	tR (min)	Conc. (µg/mL)	Precursor Ion	Product Ion
1.	4-Hydroxy-3-methoxymandelic acid-D3 (VMA-D3)	0.90	0.83	200.1	139.9
2.	Vanillylmandelic acid (VMA)	0.92	2.12–3.90*	197.0	137.9
3.	5-Hydroxyindole-4,6,7-D3-3-acetic-D2 acid (5-HIAA-D5)	1.80	0.83	195.1	148.0
4.	5-Hydroxyindole-3-acetic acid (5-HIAA)	1.82	2.69–4.49*	190.0	145.9
5.	4-Hydroxy-3-methoxyphenyl-D3-acetic-D2 acid (HVA-D5)	2.10	0.83	186.1	127.0
6.	Homovanillic acid (HVA)	2.12	1.33–2.46*	181.0	121.9

*These concentration ranges were supplied by the vendor (see notes).

Conditions

Column	Raptor Biphenyl (cat.# 9309512)
Dimensions:	100 mm x 2.1 mm ID
Particle Size:	5 µm
Pore Size:	90 Å
Guard Column:	Raptor Biphenyl guard column cartridge 5 mm, 2.1 mm ID, 5 µm (cat.# 930950252)
Temp.:	30 °C
Standard/Sample
Diluent:	Water
Inj. Vol.:	5 µL
Mobile Phase
A:	0.1% Formic acid, 5 mM ammonium formate in water
B:	Methanol
	Time (min) Flow (mL/min) %A %B 0.00 0.5 85 15 3.00 0.5 20 80 3.01 0.5 85 15 5.00 0.5 85 15

Detector	MS/MS
Ion Mode:	ESI-
Mode:	MRM
Instrument	UHPLC
Sample Preparation	The human urine sample is the Bio-Rad Lyphochek quantitative urine control, level 1. A 40 μL aliquot of urine was mixed with 360 μL of water and 10 μL of internal standard solution (33.3 μg/mL in methanol) in a Thomson 0.45 μm PVDF filter vial and injected for analysis after filtration.

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Figure 5: 5-HIAA, VMA, and HVA LC Analysis in Human Urine (Control Level 2)

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Peaks

	Peaks	tR (min)	Conc. (µg/mL)	Precursor Ion	Product Ion
1.	4-Hydroxy-3-methoxymandelic acid-D3 (VMA-D3)	0.90	0.83	200.1	139.9
2.	Vanillylmandelic acid (VMA)	0.91	10.9–18.2*	197.0	137.9
3.	5-Hydroxyindole-4,6,7-D3-3-acetic-D2 acid (5-HIAA-D5)	1.81	0.83	195.1	148.0
4.	5-Hydroxyindole-3-acetic acid (5-HIAA)	1.83	21.6–32.7*	190.0	145.9
5.	4-Hydroxy-3-methoxyphenyl-D3-acetic-D2 acid (HVA-D5)	2.11	0.83	186.1	127.0
6.	Homovanillic acid (HVA)	2.14	12.9–18.9*	181.0	121.9

*These concentration ranges were supplied by the vendor (see notes).

Conditions

Column	Raptor Biphenyl (cat.# 9309512)
Dimensions:	100 mm x 2.1 mm ID
Particle Size:	5 µm
Pore Size:	90 Å
Guard Column:	Raptor Biphenyl guard column cartridge 5 mm, 2.1 mm ID, 5 µm (cat.# 930950252)
Temp.:	30 °C
Standard/Sample
Diluent:	Water
Inj. Vol.:	5 µL
Mobile Phase
A:	0.1% Formic acid, 5 mM ammonium formate in water
B:	Methanol
	Time (min) Flow (mL/min) %A %B 0.00 0.5 85 15 3.00 0.5 20 80 3.01 0.5 85 15 5.00 0.5 85 15

Detector	MS/MS
Ion Mode:	ESI-
Mode:	MRM
Instrument	UHPLC
Sample Preparation	The human urine sample is the Bio-Rad Lyphochek quantitative urine control, level 2. A 40 μL aliquot of urine was mixed with 360 μL of water and 10 μL of internal standard solution (33.3 μg/mL in methanol) in a Thomson 0.45 μm PVDF filter vial and injected for analysis after filtration.

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Products Mentioned

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Catalog No. 9309512

Colonna HPLC Raptor bifenilica, 5 µm, 100 x 2,1 mm

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Catalog No. 930950252

Cartuccia per precolonna Raptor bifelinica EXP, 5 µm, 5 x 2.1 mm, 3 pz.

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Catalog No. 25808

Holder EXP a connessione diretta per cartucce per precolonne EXP, comprende raccordo e ferrule

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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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• 
  Sue Steinike

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CFAN2465

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