Image: Representative chromatogram of dNTP levels at the LLOQ. Ion chromatograms showing relative abundance for dGTP, ATP, dATP, labeled C-13 dATP, dTTP, and dCTP upon injection of 62.5 fmol dNTPs, 1250 fmol ATP, and 2.5 pmol of labeled C-13 dATP.
Sigurast Olafsson, Dale Whittington, Jason Murray, Michael Regnier, Farid Moussavi-Harami. Journal of Chromatography B, Volumes 1068–1069, 15 November 2017, Pages 90-97.
Abstract
Deoxyribonucleoside triphosphates (dNTPs) are used in DNA synthesis and repair. Even slight imbalances can have adverse biological effects. This study validates a fast and sensitive HPLC–MS/MS method for direct quantification of intracellular dNTPs from tissue. Equal volumes of methanol and water were used for nucleotide extraction from mouse heart and gastrocnemius muscle and isolated cardiomyocytes followed by centrifugation to remove particulates. The resulting supernatant was analyzed on a porous graphitic carbon chromatography column using an elution gradient of ammonium acetate in water and ammonium hydroxide in acetonitrile with a run time of just 10 min. Calibration curves of all dNTPs ranged from 62.5 to 2500 fmol injections and demonstrated excellent linearity (r2 > 0.99). The within day and between day precision, as measured by the coefficient of variation (CV (%)), was < 25% for all points, including the lower limit of quantification (LLOQ). The inter-day accuracy was within 12% of expected concentration for the LLOQ and within 7% for all other points on the calibration curve. The intra-day accuracy was within 22% for the LLOQ and within 11% for all points on the curve. Compared to existing methods, this study presents a faster and more sensitive method for dNTP quantification.