Vanillylmandelic Acid, Homovanillic Acid, and 5-Hydroxyindoleacetic Acid in Human Urine (Quantitative Control Level 1) on Raptor Biphenyl

	Peaks	t_R (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).

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

0.1% Formic acid, 5 mM ammonium formate in water

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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