正源负源比较液质联用测定酶解液中脱氧核苷酸含量

目的: 建立一种快速灵敏的同时测定DNA酶解液中5'-腺嘌呤脱氧核苷酸(dAMP)、5'-胸腺嘧啶脱氧核苷酸(dTMP)、5'-鸟嘌呤脱氧核苷酸(dGMP)、5'-胞嘧啶脱氧核苷酸(dCMP)的超高效液相色谱质谱(UPLC-MS)方法。 方法: 采用UPLC-MS色谱质谱系统,安捷伦ZORBAX SB Aq-C₁₈柱(4.6 mm×250 mm,5 μm);以20 mmol·L^-1醋酸铵缓冲盐溶液(A)-乙腈(B)为流动相进行梯度洗脱(0~10 min,B相从5%→10%;10~11 min,B相从10%→90%;11~15 min,B相为90%;15~16 min,B相从90%→5%;16~18 min,B相为5%),流速0.6 mL·min^-1;在电喷雾正离子模式下,采用多反应监测模式(MRM)选择性监测5'-腺嘌呤脱氧核苷酸(dAMP)(m/z 332.1/ 81.1和332.1/136.2)、5'-胸腺嘧啶脱氧核苷酸(dTMP)(m/z 323.1/ 81.1和323.1/207.2)、5'-鸟嘌呤脱氧核苷酸(dGMP)(m/z 348.1/152.1 348.1/313.3)和5'-胞嘧啶脱氧核苷酸(dCMP)(m/z 308.1 /81.1和308.1 /112.1)。 结果: 5'-腺嘌呤脱氧核苷酸(dAMP)、5'-胸腺嘧啶脱氧核苷酸(dTMP)、5'-鸟嘌呤脱氧核苷酸(dGMP)和5'-胞嘧啶脱氧核苷酸(dCMP)的线性范围分别为34.8~1160.0 ng· mL^-1(r=0.9997),32.8~820.0 ng· mL^-1(r=0.9997),60.0~1200.0 ng· mL^-1(r=0.9990),37.6~940.0 ng· mL^-1(r=0.9998),检出限分别为9.4,8.48,11.43,10.35 ng·mL^-1;定量限分别为31.35,28.28,38.10,34.50 ng·mL^-1;样品加标回收率为71.1%~117.2%;日内、日间精密度试验的RSD为0.94%~4.99%。 结论: 该方法快捷,灵敏度高,专一性强,重复性好,可用于DNA酶解液中4种脱氧核苷酸的分析。

Objective: To establish an UPLC-MS method for simultaneous determination of dAMP,dTMP,dGMP and dCMP in enzymatically digested DNA. Method: Four deoxyribonucleotides were determined by UPLC with Agilent ZORBAX SB-Aq C₁₈(4.6 mm×250 mm,5 μm)column using 20 mmol· L^-1 ammonium acetate (A) and acetonitrile (B) as mobile phase,with gradient elution (0-10 min,5%→10% B;10-11 min,10%→90% B;11-15 min,90% B;15-16 min,90%→5% B;16-18 min,5% B) at a flow rate of 0.6 mL·min^-1.The analytes were detected by positive electrospray ionization (ESI+) under multi-reaction monitoring (MRM) mode,the parent ions and product ions m/z was 332.1/81.1,332.1/136.2 for dAMP,323.1/81.1,323.1/207.2 for dTMP,348.1/152.1,348.1/313.3 for dGMP and 308.1/81.1,308.1/136 for dCMP. Results: The linear ranges were 34.8-1160.0 ng· mL^-1(r=0.9997),32.8-820.0 ng· mL^-1(r=0.9997)、60.0-1200.0 ng· mL^-1(r=0.9990)and 37.6-940.0 ng· mL^-1(r=0.9998),with detection limits of 9.4,8.48,11.43,10.35 ng· mL^-1,with quantitation limits of 31.35,28.28,38.10,34.50 ng· mL^-1 for dAMP,dTMP,dGMP and dCMP,respectively.The average recoveries ranged from 71.1% to 117.2%.The relative standard deviations of intra-day and inter-day ranged from 0.94% to 4.99%. Conclusion: The method is rapid,sensitive,specific and suitable for the quality control of four deoxyribonucleotides in enzymatically digested DNA.