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Unit Code 9254:
Vanillylmandelic Acid (VMA) and Homovanillic Acid (HVA), Pediatric, Urine

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Method Description

HVA is measured in urine by liquid chromatography-tandem mass

spectrometry (LC-MS/MS). Octadecyl (C18) solid-phase extraction is

performed on a 1-mL aliquot of urine. The solid phase extraction column

is eluted with 1 mL of methanol. The eluate is evaporated at 30 degrees C

to 40 degrees C under nitrogen and the residue is reconstituted in 1-mL

LC-MS/MS mobile phase. LC-MS/MS is performed by injecting 10 uL

of the reconstituted specimen onto an amide-C16 high-pressure liquid

chromatography (HPLC) column. The mobile phase (30% acetonitrile

in 0.05% aqueous acetic acid) is pumped over the HPLC analytical

column at the rate of 1 mL/min, with the flow diverted to the MS/MS

electrospray probe tip by 1:5. HVA elutes apart from the bulk of the

specimen matrix at a retention time of approximately 1.5 minutes.

HVA is quantitated by using a custom-synthesized stable

isotope-labeled internal standard ([13]C[6]-4-hydroxy-[18]O-HVA)

from calibration over a concentration range of 1.04 mg/L to 16.7 mg/L

(Magera MJ, Stoor A, Helgeson JK, et al: Determination of

homovanillic acid in urine by stable isotope dilution and electrospray

tandem mass spectrometry. Clin Chim Acta 2001;306:35-41)

 

VMA is measured by solid-phase extraction (SPE) of a 1-mL

aliquot of urine. A known amount of stable isotope-labeled VMA (IS) is

added to each urine specimen prior to SPE. VMA and IS are eluted

from the SPE column with methanol. The methanol is evaporated and

the VMA and IS are redissolved in LC-MS/MS mobile phase. A portion

of this prepared extract is injected onto a LC column that separates VMA

and IS from the bulk of any remaining specimen matrix. The VMA and IS

are measured by MS/MS using the selected reaction monitoring mode.

VMA is quantified using the ratio to IS versus urine calibrators over the

concentration range 1.03 mg/L to 20 mg/L. (Magera MJ, Thompson AL,

Stoor AL, et al: Determination of vanillylmandelic acid in urine by stable

isotope dilution and electrospray tandem mass spectrometry.

Clin Chem 2003;49:825-826)

Performing Laboratory Location

Rochester

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