16种核苷与核苷酸LC检测方法的建立及质谱裂解规律的研究

Establishment of LC Methods for 16 Nucleosides and Nucleotides and the Study of Cleavage Law by Mass Spectrometry

目的建立通用LC-MS法用于分离和鉴定核苷(酸)化合物。方法选用ODS-AQ柱,流动相A为缓冲液1(120 mmol·L^(-1)NaHPO,120 mmol·L^(-1)KHPO和3 mmol·L^(-1)TBAOH),流动相B为水,流动相C为甲醇,梯度洗脱,对16种核苷和核苷酸化合物进行分离分析。采用电喷雾离子化(ESI)检测,喷雾电压4000 V(正离子模式)/3500 V(负离子模式),雾化气压力45 psi,干燥气流量10 L·min^(-1),去溶剂温度350℃。碎裂电压为180 V,碰撞能量设置为5~30 eV,对4种不同类型的核苷和核苷酸化合物的质谱碎片进行分析。结果建立的LC方法对16个代表性核苷(酸)化合物达到了基线分离(R>2.0),基于质谱碎片规律,构建核苷(酸)化合物的质谱平台。结论建立的LC-MS方法可为未知的核苷(酸)化合物的分离与鉴定提供参考。

OBJECTIVE To establish a general LC-MS method for the separation and identification of nucleoside and nucleotide compounds.METHODS The ODS-AQ column was picked,the mobile phase A was buffer 1(120 mmol·L^(-1)NaHPO,120 mmol·L^(-1)KHPOand 3 mmol·L^(-1)TBAOH),the mobile phase B was water,and the mobile phase C was methanol,gradient elution.The 16 nucleoside and nucleotide compounds were separated and analyzed.Electrospray ionization(ESI)detection was adopted,spray voltage was 4000 V(positive ion mode)/3500 V(negative ion mode),atomizing gas pressure was 45 psi,drying gas flow rate was 10 L·min^(-1),and solvent removal temperature was 350℃.The fragmentation voltage was180 V,and the collision energy was set to 5-30 eV.Mass spectrometry fragments of 4 different types of nucleoside and nucleotide compounds were analyzed.RESULTS The established LC method achieved baseline separation(R>2.0)for 16representative nucleoside and nucleotide compounds.Based on the mass spectrum fragmentation rule,a mass spectrometry platform for nucleoside and nucleotide compounds was constructed.CONCLUSION The established LC-MS method can provide reference for the separation and identification of unknown nucleoside and nucleotide compounds.