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

Abstract

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 Including 2'-deoxycytidine-5'-monophosphate (dCMP), 2'-deoxythymidine-5'-monophosphate (dCMP), 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 O-2 is employed for the detection of (PO+)-P-31-O-16 to avoid potential interferences of polyatomic isobaric at m/z 31 by directly detecting P-31(+). By such HPLC-TCP-MS hyphenation technique, detection limits of four deoxyribonucleotides are 0.211 mu mol/L for dCMP, 0.204 mu mol/L for dTMP, 0.173 mu mol/L for dGMP and 0.225 mu mol/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 mu mol/L of dCMP, 228.2 +/- 4.0 mu mol/L of dTMP, 125.3 +/- 3.0 mu mol/L of dGMP and 222.9 +/- 3.3 mu mol/L of dAMP.