燕窝中唾液酸的DAD/FLD串联HPLC测定方法研究

Determination of Sialic Acid in Edible Birds Nest Using Pre-column Derivatization Reversed Phase High Performance Liquid Chromatography with Photodiode Array or Fluorescence Detection

建立燕窝中唾液酸的柱前衍生二极管阵列/荧光检测器(DAD/FLD)串联的反相高效液相色谱检测方法。用硫酸氢钠溶液水解样品,以邻苯二氨盐酸盐为衍生化试剂,采用C18柱分离,四氢呋喃溶液(含体积分数0.5%磷酸和体积分数0.15%正丁胺)-乙腈为流动相等度洗脱,流速1.0mL/min,二极管阵列检测波长230nm,荧光激发波长(Ex)230nm,发射波长(Em)425nm。结果表明,唾液酸在0.1～750μg/mL范围内线性良好(r>0.9990),20min内完全分离,回收率在85.03%～97.14%之间。检出限0.2μg/mL(DAD),0.005μg/mL(FLD),FLD检出限比DAD低两个数量级且杂峰少,在DAD检出限以上的相同浓度下,DAD比FLD更灵敏。本方法具有灵敏度高,重复性好,分析速度快等特点,可准确测定燕窝等样品中唾液酸含量。

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.