High performance liquid chromatography (HPLC) Determination of nitrogenous bases Cytosine, Adenine and Guanine by derivatization with 2- hydroxynaphthaldehyde.
Analytical procedure has been developed to determine nitrogenous bases from DNA samples using 2- hydroxynaphthaldeyde (HN) as derivatizing reagent from pharmaceutical and from DNA samples. Three nitrogenous bases adenine, cytosine and guanine were derivatized with (HN) at pH 8 and separated from HPLC column Phenomenexμm C18, 5μm (150x4.6mm id), the determination was done on photodiode array at wavelength 260 nm. The gradient elution was done with methanol, water and acetonitrile CH3CN (58:4:38 volume/volume) with flow rate 1 ml/minutes. The rate of elution of three nitrogenous bases was very rapid within 12 minutes. The method was reiteration with relative standard deviation (RSD) within1, 8-3.7% for each of the compound. The limits of quantification (LoQ) & limits of detection (LoD) were determined at the range of 0.191-0.298μg/ml & 0.063-0.097μg/ml respectively. This method was used for determination of nitrogenous from DNA samples. The results were further confirmed by standard addition technique. The method reports the separation and determination of nitrogenous bases for possible clinical analysis from DNA samples..