GC-FID determination of nucleobases guanine, adenine, cytosine, and thymine from DNA by precolumn derivatization with isobutyl chloroformate

Abstract

Adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) are building blocks and major constituents of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The metabolic pathway of DNA and RNA is strongly related to A, G, C, T, and U. Gas chromatography (GC) has been examined for the ease of separation of the nucleobases guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U) after precolumn derivatization with isobutyl chloroformate. GC was carried out from the column HP-5 (30 m × 0.32 mm id) with layer thickness 0.25 μm. The linear calibration curves were obtained with 0.5–50 μmol L−1 with limits of detection (LOD) 0.08–0.10 μmol L−1. The isolated DNA after acid hydrolysis from human blood and plants was analyzed, and amounts of T, A, C, and G were found in human blood within the ranges 0.521–2.625, 0.559–2.655, 0.490–2.915, and 0.450–2.739 μmol L−1, respectively, with relative standard deviation (RSD) 0.77–5.66 %. The ratios between T:A and G:C were 0.94–1.05 and 0.94–1.06, respectively. The values of (T + A)/(G + C) were within 0.90–1.07. Similarly from plant, T, A, C, and G were found within 0.48–2.915 nmolg−1 with RSD 0.88–5.00 %. The method was applicable for the analysis of DNA nucleobases from biological samples.