Abstract:

A pre-column derivatization RP-HPLC method with detection with a photodiode array detector (DAD) or a fluorescecse detector (FLD) was developed for the determination of N-acetylneuraminic acid in edible birds’ nest (EBN). Samples were dissolved in 0.5 mol/L sodium bisulfate aqueous solution and kept for 30 min in 80 ℃ water bath. After cooling the derivatization was carried out using O-phenylenediamine · 2HCl as derivative. The chromatographic separation was achieved on a C18 column using a mobile phase composed of 1.0% tetrahydrofuran aqueous solution (containing phosphoric acid and 1- butylamine at the levels of 0.5% and 0.15%, respectively) and acetonitrile (95:5, V/V) at a flow rate of 1.0 mL/min in the isocratic elution mode. The DAD wavelength was set at 230 nm and the FLD Ex and Em wavelengths at 230 and 425 nm, respectively. The relationship between HPLC peak area and N-acetylneuraminic acid concentration over the range of 0.1－750 μg/mL exhibited good linearity (r ＞ 0.9990). A complete separation of N-acetylneuraminic acid was observed within 20 min. Mean recoveries of 6 replicates for N-acetylneuraminicacid in a real sample spiked at 3 levels ranged from 85.03% to 97.14%. The limit of detection (LOD) of the method was 0.2μg /mL with a DAD and 0.005μg /mL with a FLD. Clearly, the LOD with a FLD was two orders of magnitude lower than that with a DAD. Meanwhile, less miscellaneous small peaks were observed in the FLD detection. However, a DAD presented higher detection sensitivity than a FLD at the same concentrations over the LOD resulting from DAD detection. The method demonstrates the advantages of high sensitivity, good repeatability and rapidity, and is consequently most suitable for the determination of N-acetylneuraminic acid in some samples like EBN.

Key words: edible bird s nest, N-acetylneuraminic acid, high-performance liquid chromatography, determination