Gas Chromatographic Determination of Purines and Pyrimidines from DNA Using Ethyl Chloroformate as Derivatizing Reagent

Abstract

An analytical method has been proposed for the separation and determination of guanine, adenine, cytosine, thymine and uracil by gas chromatography (GC) following precolumn derivatization using ethyl chloroformate. The GC separation was achieved from HP-5 (30 m × 0.32 mm id) column with layer thickness 0.25 μm. The linear calibrations were observed within 0.5-50.0 μmol/L for each of the compound and limits of detection were within 0.1-0.17 μmol/L. The derivatization, separation and quantitation was repeatable with intra (n=5) and inter (n=5) variation in terms of peak height/peak area and retention time with relative standard deviation (RSD) within 4.70-6.43%. The method was applied for the analysis of isolated DNA from human blood and plant leaves after acid hydrolysis. The concentration of thymine, adenine, cytosine and guanine in blood samples were observed within 0.602-2.135 μmol/L of each compounds with RSD 2.60- 6.00%. The recovery of the nucleobases by standard addition was calculated within 98-108% with RSD 2.5-7.8%.