Analysis of mono-phosphate nucleotides as a potential method for quantification of DNA using high performance liquid chromatography-inductively coupled plasma-mass spectrometry

The determination of total deoxyribonucleic acid (DNA) concentration is of great importance in many biological and bio-medical analyses. The quantification of DNA is traditionally performed by UV spectroscopy; however the results can be affected greatly by the sample matrix. The proposed method quantifies phosphorus in digested calf thymus DNA and human DNA by high performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICP-MS). The method presented showed excellent baseline separation between all 4 DNA mono-nucleotides and 5’UMP. Column recoveries ranging from 95% to 99% for phosphorus resulted in a mass balance of 95% ± 0.5% for standard nucleotides, determined by LC-ICP-MS, compared to total DNA determined by flow injection coupled to ICP-MS (FI-ICP-MS). The ability of LC-ICPMS to act as an internal check that only DNA derived phosphorus was counted in the assay was demonstrated by establishing a mass balance between the total phosphorous signal from undigested DNA and that from the speciated DNA. The method for quantification was evaluated by analysis of NIST SRM 2372; a total speciated DNA recovery of 52.1 ng/μL, compared with an expected value of 53.6 ng/μL, was determined by external calibration. From repeat measurements a mass balance of 97% ± 0.5% for NIST DNA was achieved. The method limits of detection for individual nucleotides were determined between 0.8 to 1.7 μg L-1 (31P) for individual nucleotides by LC-ICP-MS, and 360 ng L-1 for 5’AMP by direct nebulisation.