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

Rafi O Zaman Brohi*, Muhammad Yar Khuhawar, Azizullah Channa, Abdul Jabbar Laghari and Kulsoom Abbasi Institute of Advenced Research Studies in Chemical Sciences, University of Sindh, Jamshoro, Sindh, Pakistan. Institute of Environmental Engineering, Mehran University of Engineering & Technology. Jamshoro, Pakistan. Dr. M.A Kazi Institute of Chemistry, University of Sindh, Jamshoro, Pakistan. *Corresponding Author Email: rafi.brohi@gmail.com Received 26 January 2016, Revised 21 June 2016, Accepted 28 June 2016 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 2.606.00%. The recovery of the nucleobases by standard addition was calculated within 98-108% with RSD 2.5-7.8%.