Determination of methylmalonic acid after diazonium derivatization by high-performance liquid chromatography

The use of a single prederivatization step in conjunction with high-performance liquid chromatography (h.p.l.c.) is described for the determination of methylmalonic acid (MMA). The method is based on the reaction of MMA and 4-diazobenzenesulfonic acid, which produces a derivative that has a molar absorptivity of about 9 × 10³ l mol^?1 cm^?1 at 353 nm. The derivatization reaction is optimized for various parameters. A reagent concentration of 3.3 mM at a reaction pH of 4.6 and a temperature of 100°C are optimal. The reaction product is separated from the excess of reagent and other interfering components by using a polystyrene-divinylbenzene column and a highly aqueous mobile phase. After a simple clean-up step, it is possible to quantify MMA in urine at about 0.8 mg l^?1 with linear response up to 32 mg l^?1.