Simultaneous Determination of Methionine and Homocysteine by on-column derivatization with o-phtaldialdehyde

A fast and simple HPLC-based assay has been developed for the simultaneous determination of homocysteine (Hcy) and methionine (Met) in plasma and urine samples, utilizing as small volume of sample as 10 μL. The assay uses on-column derivatization with o-phthaldialdehyde. The separation of Hcy and Met was achieved in 14 min on a reversed phase C-18 column, followed by fluorescence detection (excitation at 348 nm and emission at 438 nm for Met; excitation at 370 nm and emission at 480 nm for Hcy). Linearity of the detector response was observed in the range of 2–60 μmol L−1 for Met and 2–40 μmol L−1 for Hcy. The method was successfully applied for Met and Hcy quantification in human and mouse plasma and urine samples from cystathionine β-synthase deficient and unaffected individuals.