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

An ion pair reverse phase high performance liquid chromatography (RP-HPLC) coupled with inductively coupled plasma mass spectrometry (ICP-MS) method was developed for determination of deoxyribonucleotide in plasmid DNA. The optimum separation conditions for the four deoxyribonucleotides including 2′-deoxycytidine-5′-monophosphate (dCMP), 2′-deoxythymidine-5′-monophosphate (dTMP), 2′-deoxyguanosine-5′-monophosphate (dGMP), and 2′-deoxyadenosine-5′-monophosphate (dAMP) were obtained using a mobile phase containing 10 mM ammonium acetate (pH4.8) and 2.5% methanol. The hexapole collision/reaction cell technique (CCT) by adding O2 was used for the detection of 31P16O+ to avoid the potential interferences of polyatomic isobaric at m/z 31 by directly detecting 31P+. Using such HPLC-ICP-MS hyphenation technique, the detection limits of the four deoxyribonucleotides are 0.211 μM for dCMP, 0.204 μM for dTMP, 0.173 μM for dGMP, and 0.225 μM 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) μM of dCMP, (228.2 ± 4.0) μM of dTMP, (125.3 ± 3.0) μM of dGMP, and (222.9 ± 3.3) μM of dAMP, respectively.