2134/13512
Claire L. Camp
Claire L.
Camp
Barry Sharp
Barry
Sharp
Helen Reid
Helen
Reid
John Entwisle
John
Entwisle
Heidi Goenaga-Infante
Heidi
Goenaga-Infante
Analysis of mono-phosphate nucleotides as a potential method for quantification of DNA using high performance liquid chromatography-inductively coupled plasma-mass spectrometry
Loughborough University
2013
Chemistry
Quantification
DNA
ICP-MS
Mass balance
Nucleotides
Chemical Sciences not elsewhere classified
2013-11-06 14:12:44
Journal contribution
https://repository.lboro.ac.uk/articles/journal_contribution/Analysis_of_mono-phosphate_nucleotides_as_a_potential_method_for_quantification_of_DNA_using_high_performance_liquid_chromatography-inductively_coupled_plasma-mass_spectrometry/9393866
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.