高效液相色谱-电感耦合等离子体质谱联用测定DNA分子中4种脱氧核苷酸

Analysis of Deoxynucleotide in Plasmid Deoxyribonucleic Acid Using High Performance Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry

建立了用反相离子对色谱和电感耦合等离子体质谱的联用技术同时测定DNA分子中4种脱氧核苷酸的含量。液相流动相最佳条件是:pH4.8,甲醇2.5%,10mmol/LNH4Ac。为了避免直接测定31P时14N16O1H+,15N16O+等复合离子的干扰造成信噪较差的问题,利用碰撞/反应池技术(CCT)加入O2和31P生成31P16O复合离子后测定该复合离子的信号可提高测定磷的信噪比。使用该联用技术测定4种脱氧核苷酸的检出限分别为0.211μmol/L(dCMP)、0.204μmol/L(dTMP)、0.173μmol/L(dGMP)和0.225μmol/L(dAMP),并且测定经过核酸酶酶解的质粒DNA后生成的4种脱氧核苷酸的含量分别是:152.9±2.4μmol/L(dCMP)、228.2±4.0μmol/L(dTMP)、125.3±3.0μmol/L(dGMP)和222.9±3.3μmol/L(dAMP)。

An ion pair reverse phase high performance liquid chromatography （RP-HPLC） coupled with inductively coupled plasma mass spectrometry （ICP-MS） method has been developed for the determination of deoxyribonucleotide in plasmid DNA. Optimum separation conditions for the four deoxyribonucleotides inclu- ding 2＇-deoxycytidine-5＇-monophosphate （ dCMP）, 2＇-deoxythymidine-5＇-monophosphate （ dTMP）, 2＇-deoxyguanosine-5＇-monophosphate （dGMP） and 2＇-deoxyadenosine-5＇-monophosphate （dAMP） are obtained using a mobile phase containing 10 mmoL/L ammonium acetate （pH 4.8） and 2.5% methanol. The hexapole collision/reaction cell technique （CCT） by adding O2 is employed for the detection of ^31p^16O^＋ to avoid potential interferences of polyatomic isobaric at m/z 31 by directly detecting ^31p ＋. By such HPLC-ICP-MS hyphenation technique, detection limits of four deoxyribonucleotides are 0. 211 μmol/L for dCMP, 0.204 μmol/L for dTMP, 0. 173 μmol/L for dGMP and 0. 225 μmol/L for dAMP, respectively. This hyphenated HPLC-ICP-MS method has been successfully applied to the analysis of four deoxyribonucleotides in plasmid DNA, which contained 152.9 ±2.4 μmol/L of dCMP, 228.2 ±4.0 μmol/L of dTMP, 125.3 ±3.0 μmol/L of dGMP and 222.9 ± 3.3 μmol/L of dAMP.