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目的建立超高效液相色谱-三重四级杆串联质谱法测定乳粉类产品中5种核苷酸,包括胞嘧啶核苷酸(cytidine monophosphate,CMP)、尿嘧啶核苷酸(uridine monophosphate,UMP)、腺嘌呤核苷酸(adenosine monophosphate,AMP)、鸟嘌呤核苷酸(guanosinemonophosphate,GMP)、次黄嘌呤核苷酸(inosinemonophosphate,IMP)含量的分析方法。方法样品经温水溶解后,经1%(V:V)甲酸沉淀蛋白离心后,分别通过固相萃取SPE法和超滤离心法有效净化其他干扰成分,从而进行前处理方法评价;采用Waters ACQUITY UPLC HSS T3(2.1mm×50 mm,1.8μm)为分析柱,0.1%(V:V)甲酸水溶液和乙腈作为流动相,等度洗脱分离。质谱采用正离子模式,多反应监测模式(MRM)进行检测,对纯水配制标准溶液和阴性基质溶液配制标准溶液进行比较,外标法定量分析。结果 5种核苷酸在4.5 min内达到基线分离,AMP、IMP和GMP在10~1000.0 ng/mL浓度范围内线性关系良好,CMP和UMP分别在25~1000 ng/mL和100~1000 ng/mL浓度范围内线性关系良好,相关系数均在0.9990以上,方法检出限分别为UMP 5 mg/kg、CMP 1 mg/kg、AMP0.5 mg/kg、IMP 0.5 mg/kg、GMP 0.1 mg/kg。在10.0、50.0、100.0mg/kg加标水平下的超滤离心法的平均回收率在73.6%~110.9%之间,相对标准偏差(RSD)为3.56%~15.89%;SPE法的平均回收率在86.4%~109.5%之间,相对标准偏差(RSD)为2.32%~14.45%。结论该方法灵敏度高,可以快速准确鉴别并定量乳粉产品中的核苷酸。
OBJECTIVE To establish a method for the determination of five kinds of nucleotides in milk powder products by ultra performance liquid chromatography-triple quadrupole mass spectrometry, including cytidine monophosphate (CMP), uridine monophosphate (UMP) ), Adenosine monophosphate (AMP), guanosinemonophosphate (GMP), and inosine monophosphate (IMP) content were determined. Methods After the sample was dissolved in warm water, the protein was precipitated by 1% (V: V) formic acid, and then the other interference components were effectively purified by SPE and ultrafiltration respectively to evaluate the pretreatment method. Waters ACQUITY UPLC HSS T3 (2.1 mm × 50 mm, 1.8 μm) was used as analytical column, 0.1% (V: V) formic acid in water and acetonitrile as the mobile phase. The mass spectra were detected by positive ion mode and multi-reaction monitoring mode (MRM). The standard solution of pure water and negative matrix solution were compared, and the external standard method was used for quantitative analysis. Results The five nucleotide sequences were separated within 4.5 min. The linearities of AMP, IMP and GMP in the concentration range of 10 ~ 1000.0 ng / mL were good. The concentrations of CMP and UMP were 25 ~ 1000 ng / mL and 100 ~ 1000 ng / mL. The detection limits were UMP 5 mg / kg, CMP 1 mg / kg, AMP 0.5 mg / kg, IMP 0.5 mg / kg, GMP 0.1 mg / kg. The average recoveries were between 73.6% and 110.9% with relative standard deviations (RSDs) of 3.56% ~ 15.89% at the spiked levels of 10.0, 50.0 and 100.0 mg / kg. The average recoveries of SPE The relative standard deviations (RSDs) ranged from 2.32% to 14.45% between 86.4% and 109.5%. Conclusion The method has high sensitivity and can quickly and accurately identify and quantify nucleotides in milk powder products.