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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C_(18)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0.21μg/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。
Objective To establish a method for the determination of N-acetylneuraminic acid in breast milk by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). Methods Acidic hydrolysis of the sample with hydrochloric acid liberated bound N-acetylneuraminic acid in human milk. After centrifugation at high speed, C_ (18) column was used for separation. The gradient elution was carried out with 0.1% formic acid-acetonitrile solution and 0.1% formic acid-water as mobile phase, and electrospray ionization mass spectrometry (ESI-MS / MS) and positive ion multiple reaction monitoring (MRM) were used for qualitative and quantitative analysis. Results Under the optimal conditions, N-acetyl neuraminic acid showed a good linear relationship (r = 0.9997) in the range of 0.01-0.5 mg / L. The average recoveries of N-acetylneuraminic acid were 95.0% ~ 98.7% with relative standard deviations of 11.6% ~ 14.5% at three levels of 10, 50 and 100 mg / kg. The detection limit of amino acid was 0.21μg / kg. Conclusion The method is simple, rapid, reproducible and sensitive. It can be used in the determination of N-acetylneuraminic acid in breast milk.