Determination of methylmalonic acid in urine by HPLC with intramolecular excimer-forming fluorescence derivatization.
We developed and validated an HPLC method with intramolecular excimer-forming fluorescence derivatization to determine methylmalonic acid, a unique biochemical marker for methylmalonic aciduria. Methylmalonic acid in urine and an internal standard were derivatized with pyrenebutyric hydrazide and separated on a C8 column. The derivatives were detected by monitoring the fluorescence at 475 nm (excitation wavelength 345 nm). At a signal-to-noise ratio of 3, the detection limit was 0.33 pmol on the column and the calibration curve was linear up to 1 mmol[sol ]L in urine. In a retrospective study on a relatively large number of known methylmalonic aciduria cases (n = 48), the method enabled us to differentiate methylmalonic aciduria cases from healthy controls (n = 52), regardless of age of patients at sampling or years of specimen storage. No interference was observed from isomeric or other dicarboxylic acids, or other urine constituents. As described, the method can be used retrospectively or prospectively for the diagnosis of methylmalonic aciduria and can be easily adopted by laboratories with no access to gas chromatography-mass spectrometry.
Al-Dirbashi
Osama Y
OY
Department of Genetics, King Faisal Specialist Hospital and Research Centre, PO Box 3354, Riyadh 11211, Saudi Arabia.
Jacob
Minnie
M
Al-Hassnan
Zuhair
Z
Chabayta
Reem W
RW
El-Badaoui
Fahad
F
Rashed
Mohamed S
MS
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry; D008764:Methylmalonic Acid / Q000652:urine*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D008764:Methylmalonic Acid