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目的:建立液相色谱-三重四级杆质谱联用法(LC-MS/MS)测定猪内脏和肌肉组织中唾液酸N-乙酰神经氨酸(Neu5Ac)、N-羟乙酰神经氨酸(Neu5Gc)和脱氨神经氨酸(KDN)浓度的分析方法。方法:将内脏肉组织匀浆后,利用三氟乙酸(TFA)将结合态的唾液酸从糖脂或糖蛋白链上解离下来后进样分析。色谱条件:色谱柱为ZORBAX Eclipse Plus C18(5μm,4.6×250 mm,Waters),流动相为0.1%醋酸铵-100%乙腈(9:1),柱温:25℃,流速800μL·min~(-1),进样量20μL。质谱条件:离子源为电喷雾化离子源(ESI),扫描方式为多重反应监测(MRM),监测离子对:Neu5Ac:307.7→87.0,Neu5Gc:323.3→116.0,KDN:266.7→87.0,13C3Neu5Ac内标品:310.9→90.0。结果:Neu5Ac、Neu5Gc和KDN分别在0.1~20.0、0.05~10.00和0.005~1.000μmol·L~(-1)的浓度范围内和对照品与内标峰面积比值线性关系良好,重复性平均RSD为1.2%,稳定性平均RSD为1.9%,日间和日内精密度试验RSD均小于6.7%,平均回收率为92.9~106.4%。结论:本方法简便、快速、灵敏度高,可广泛运用于组织和体液中唾液酸的测定。
OBJECTIVE: To establish a method for the determination of sialic acid N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc) in porcine viscera and muscle tissue by liquid chromatography-triple quadrupole mass spectrometry (LC-MS / MS) And deamination of neuraminic acid (KDN) concentration analysis method. METHODS: After the visceral meat tissue was homogenized, trifluoroacetic acid (TFA) was used to dissociate the bound sialic acid from glycolipid or glycoprotein chains and then injected for analysis. The chromatographic conditions were as follows: the column was ZORBAX Eclipse Plus C18 (5 μm, 4.6 × 250 mm, Waters), the mobile phase consisted of 0.1% ammonium acetate and 100% acetonitrile (9: 1) -1), injection volume 20μL. Mass spectrometry conditions: the ion source was electrospray ionization (ESI) and the scanning mode was multi-reaction monitoring (MRM). The ion pairs monitored: Neu5Ac: 307.7 → 87.0, Neu5Gc: 323.3 → 116.0, KDN: 266.7 → 87.0, 13C3Neu5Ac internal standard Product: 310.9 → 90.0. Results: Neu5Ac, Neu5Gc and KDN had a good linear relationship with the ratio of standard peak area to internal standard within the concentration range of 0.1-20.0,0.05-10.00 and 0.005-1.000μmol·L-1, respectively. The average RSD was 1.2%. The average RSD of stability was 1.9%. The RSDs of intra-day and intra-day precision were less than 6.7%, and the average recoveries were 92.9% -106.4%. Conclusion: The method is simple, rapid, sensitive and can be widely used in the determination of sialic acid in tissues and body fluids.