Determination of free N-acetylneuraminic acid in urine by high-performance liquid chromatography using 3-[(1-[[4-(5,6-dimethoxy-1-oxoisoindolin-2-yl)-2-methoxyphenyl]sulfonyl]pyrrolidin-2-yl)carbonylamino]phenylboronic acid as a fluorescent labeling reagent

A highly sensitive high-performance liquid chromatography (HPLC) method for the determination of urinary N-acetylneuraminic acid (NeuAc) using 3-(1-4-(5,6-dimethoxy-1-oxoisoindolin-2-yl)-2-methoxyphenyl]sulfonyl]pyrrolidin-2-yl)carbonylamino]phenylboronic acid as a fluorescent labeling reagent was developed. The labeling reaction was carried out at 30 degrees C for 30 min in the presence of pyridine. The derivative was monitored at Ex 314 nm and Em 388 nm. The detection limit of NeuAc was about 48 fmol per injection. The relative standard deviations of within-day and between-day precisions were 2.6-3.3 and 1.7-3.3%, respectively. Urine diluted 10 times with distilled water was analyzed by employing the standard-addition method. The concentrations were 8-89 nmol/mg creatinine (30+/-28 nmol/mg creatinine, n=9).