Determination of homovanillic acid in urine by stable isotope dilution and electrospray tandem mass spectrometry.
We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of homovanillic acid (HVA), a biochemical marker for catecholamine and neurotransmitter metabolism. Urine specimens are spiked with 5 microg of a stable-isotope labeled internal standard, 13C(6)18O-HVA, and prepared by automated solid phase extraction. Residues were dissolved in acetonitrile: 0.05% aqueous acetic acid and analyzed by MS/MS in the selected reaction monitoring mode (HVA: m/z 181 to m/z 137; 13C(6)18O-HVA: m/z 189 to m/z 145) after separation using a Discovery RP Amide C16 column. Consecutive calibrations (n=7) between 0.52 and 16.7 mg/l exhibited consistent linearity and reproducibility. At a urine concentration of 0.51 mg/l, the signal-to-noise ratio for HVA was 21:1. Inter- and intra-assay CVs ranged from 0.3% to 11.4%, at mean concentrations ranging 1.8 to 22.7 mg/l. Recovery of HVA added to urine ranged between 94.7% and 105% (1.25 mg/l added), 92.0% and 102% (5.0 mg/l), and 96.0% and 104% (10 mg/l). LC-MS/MS is well suited to replace an HPLC method for routine HVA determination, by providing positive identification, faster turn around time, virtually no repeat analyses and a 44% reduction of personnel necessary to perform the testing.
Biochemical Genetics Laboratory-Hilton 330, Department of Laboratory Medicine and Pathology, Mayo Clinic and Foundation, 200 First Street SW, Rochester, MN 55905, USA.
M J
MJ
Magera
A L
AL
Stoor
J K
JK
Helgeson
D
D
Matern
P
P
Rinaldo
D006719:Homovanillic Acid / Q000652:urine*; D006801:Humans; D007554:Isotopes; D012015:Reference Standards; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D007554:Isotopes; D006719:Homovanillic Acid