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

关键词:  液相色谱-质谱  核苷(酸)化合物  基线分离  质谱平台

Key words:  LC-MS  nucleosides and nucleotides compounds  baseline separation  mass spectrometry platform

摘要:

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(120mmol·L^-1 Na₂HPO₄，120mmol·L^-1 KH₂PO₄和3mmol·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4000V(正离子模式)/3500V(负离子模式)，雾化气压力45psi，干燥气流量10L·min^-1，去溶剂温度350℃。碎裂电压为180V，碰撞能量设置为5~30eV，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。

Abstract:

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 Na₂HPO₄, 120 mmol·L^-1 KH₂PO₄ and 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 4 000 V (positive ion mode)/3 500 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 was 180 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 16 representative 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.