高效液相色谱-串联质谱法测定母乳中的 N-乙酰神经氨酸

Determination of N-acetylneuraminic acid in breast milk by high performance liquid chromatography-tandem mass spectrometry

中文关键词:  高效液相色谱-串联质谱法  N-乙酰神经氨酸  母乳

英文关键词:high performance liquid chromatography-tandem mass spectrometry  N-acetylneuraminic acid  breast milk

中文摘要:

      目的 建立高效液相色谱-串联质谱(HPLC-MS/MS)法测定母乳中N-乙酰神经氨酸的分析方法。方法 用盐酸对样品进行酸水解释放出母乳中的结合态N-乙酰神经氨酸, 高速离心后, 采用C18柱进行分离。以0.1%甲酸-乙腈溶液和0.1%甲酸-水溶液作为流动相进行梯度洗脱, 电喷雾质谱检测, 正离子多反应监测模式(MRM)进行定性和定量分析。结果 在优化的条件下, N-乙酰神经氨酸在0.01~0.5 mg/L范围线性关系良好(r=0.9997)。以10、50、100 mg/kg 3个添加水平进行加标回收试验, N-乙酰神经氨酸的平均回收率为95.0%~98.7%, 相对标准偏差为11.6%~14.5%, N-乙酰神经氨酸的检出限为0.21 ?g/kg。结论 该方法简单、快速、重复性好、灵敏度高, 可广泛用于母乳中N-乙酰神经氨酸的测定。

英文摘要:

      Objective To develop a method for the determination of sialic acid (N-acetylneuraminic acid, NANA) in breast milk by high performance liquid-tandem mass spectrometry (HPLC-MS/MS). Methods The NANA was released through the hydrolysis of samples by hydrochloric acid, and purified by the high-speed centrifugation. The HPLC separation process was performed on a C18 column (150 mm×4.6 mm, 5 μm) utilizing a gradient elution program of acetonitrile (containing 0.1% formic acid) and water (containing 0.1% formic acid) as the mobile phases. The identification and quantification were achieved by using electrospray ionization (ESI)-MS/MS in positive ion mode and multiple reactions monitoring (MRM) mode. Result The results showed that the NANA had a good linear relationship in the range of 0.01~0.5 mg/L (r=0.9997). The average recoveries spiked at the 3 concentration levels of 10, 50, 100 mg/kg were in the range of 95.0%~98.7% with the relative standard deviations from 11.6% to 14.5%. The method was sensitive with the detection limit (S/N=3) of 0.21 ?g/kg．Conclusion The established method has the characteristics of simple operation, rapidity, high reproducibility and sensitivity, and can be widely applied to the quantitative determination of NANA in breast milk.