title: Automated assay of methylmalonic acid in serum and urine by derivatization with 1-pyrenyldiazomethane, liquid chromatography, and fluorescence detection.
abstract: Determination of methylmalonic acid (MMA) in serum has been established as an accurate test for the diagnosis of cobalamin deficiency. We describe here the development and performance of a liquid-chromatographic assay of MMA in blood and urine. The assay is based on our recent finding that one of the carboxylic acid moieties of some short-chain dicarboxylic acids reacts with the fluorogenic reagent 1-pyrenyldiazomethane in an aqueous medium, whereas the other remains underivatized (Anal Chem 1992; 63:315-9). The pH-dependent ionization of the free carboxylic acid group of 1-pyrenylmethyl monoesters permits retention on anion-exchange columns, which are used for solid-phase extraction. The analysis is done with a cyanopropyl column coupled in series with an octadecyldimethylsilyl column. Solid-phase extraction and sample injection are carried out automatically by a Gilson ASPEC sample processor. The assay response varies linearly with MMA concentration in the range 0.1-1000 mumol/L in serum. The within-day and between-day CVs are 2.8-10.9%, and the detection limit of 5 fmol injected (approximately 20 nmol/L in serum) is sufficiently low to determine MMA in serum (mean 0.187 mumol/L, SD 0.084, range 0.044-0.431, n = 44) and urine from healthy subjects.
authors.affiliation: Department of Pharmacology and Toxicology, University of Bergen, Haukeland Hospital, Norway.
authors.forename: J
authors.initials: J
authors.lastname: Schneede
authors.forename: P M
authors.initials: PM
authors.lastname: Ueland
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D005260:Female; D016231:Fluorescence Polarization Immunoassay; D005456:Fluorescent Dyes; D006801:Humans; D008297:Male; D008764:Methylmalonic Acid / Q000097:blood* / Q000652:urine; D008875:Middle Aged; D011721:Pyrenes / Q000737:chemistry; D014806:Vitamin B 12 Deficiency / Q000097:blood* / Q000652:urine
chemical_list: D005456:Fluorescent Dyes; D011721:Pyrenes; C058827:1-pyrenyldiazomethane; D008764:Methylmalonic Acid