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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(dT-MP)、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ZORBAXSBAq-C18柱(4.6mm×250mm,5μm);以20mmol.L-1醋酸铵缓冲盐溶液(A)-乙腈(B)为流动相进行梯度洗脱(0～10min,B相从5%→10%;10～11min,B相从10%→90%;11～15min,B相为90%;15～16min,B相从90%→5%;16～18min,B相为5%),流速0.6mL·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/z332.1/81.1和332.1/136.2)、5′-胸腺嘧啶脱氧核苷酸(dTMP)(m/z323.1/81.1和323.1/207.2)、5′-鸟嘌呤脱氧核苷酸(dGMP)(m/z348.1/152.1348.1/313.3)和5′-胞嘧啶脱氧核苷酸(dCMP)(m/z308.1/81.1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34.8～1160.0ng.mL-1(r=0.9997),32.8～820.0ng.mL-1(r=0.9997),60.0～1200.0ng.mL-1(r=0.9990),37.6～940.0ng.mL-1(r=0.9998),检出限分别为9.4,8.48,11.43,10.35ng·mL-1;定量限分别为31.35,28.28,38.10,34.50ng·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 a rapid and sensitive method for the simultaneous determination of 5’-thymidine (dT-MP), 5’-guanine deoxyribonucleic acid (dAMP) (DGMP), 5’-cytosine deoxynucleotide (dCMP) by ultra performance liquid chromatography mass spectrometry (UPLC-MS) method. METHODS: The mobile phase was eluted with 20 mmol·L-1 ammonium acetate-buffered saline (A) -acetonitrile (B) using UPLC-MS system with Agilent ZORBAXSBAq-C18 column (4.6 mm × 250 mm, (0-10 min, B phase from 5% → 10%; 10-11 min, B phase from 10% → 90%; 11-15 min, B phase 90% 16-18 min, phase B 5%) at a flow rate of 0.6 mL · min-1. In the electrospray positive ion mode, 5 ’-Adenine deoxyribonucleotide (dAMP) was selectively monitored by multiple reaction monitoring (MRM) (m / z 332.1 / 81.1 and 332.1 / 136.2), 5’- thymidine (dTMP) (m / z 323.1 / 81.1 and 323.1 / 207.2), 5’-guanine deoxynucleotide dGMP) (m / z 348.1 / 152.1348.1 / 313.3) and 5’-cytosine deoxynucleotide (dCMP) (m / z 308.1 / 81.1 and 308.1 / 112.1). Results: 5’-adenosine deoxyribonucleotide (dAMP), 5’-thymidine (dTMP), 5’-guanine deoxynucleotide (dGMP) and 5’-cytosine deoxynucleotide The linear range of dCMP was 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 The detection limits were 9.4, 8.48, 11.43 and 10.35 ng · mL-1, respectively. The limits of quantification were 31.35, 28.28, 38.10 and 34.50 ng · mL-1, respectively. The samples were 37.6-940.0 ng.mL-1 (r = 0.9998) The spiked recoveries ranged from 71.1% to 117.2%. The RSDs of intra-day and inter-day precision tests ranged from 0.94% to 4.99%. Conclusion: The method is rapid, sensitive, specific and reproducible. It can be used for the analysis of four deoxynucleotides in DNA hydrolysates.