Gas-liquid chromatography of nucleosides : Effect of silylating reagents and solvents

Abstract

The aims of this investigation were to study the completeness of silylation of nucleosides with three different reagents, bis(trimethylsilyl)trifluoroacetamide (BSTFA), bis(trimethylsilyl)acetamide (BSA) and trimethylsilylimidazole (TMSI), and to investigate the effect of different solvents (acetonitrile, pyridine, dimethylformamide, chloroform, methylene chloride, hexane, benzene, and toluene) on quantitation of derivatization. Closed-tube silylations of the nucleosides were performed with BSTFA, BSA, and TMSI, and for the most complete silylation, the optimal time, temperature, and molar excess of reagent were: for BSTFA, 150°-15 min and 225 molar excess; for TMSI, 60°-3 and 1000 molar excess; and for BSA, 120°-2 h and 250 molar excess. Also, silylations of seven major and minor nucleosides were carried out using a 1000 molar excess of BSTFA, BSA, and TMSI at 25° with 5 min sonication, and at optimal silylation conditions as described above for the three reagents. The silylating strengths were determined by the increase in RWR (= weight response of nucleoside/weight response of pyrene) values, and are summarized for the amino group containing nucleosides silylated at room temperature as BSTFA > TMSI > BSA, and for silylation under optimal conditions as BSTFA > BSA > TMSI. The efficiency of silylation for the hydroxyl group-containing nucleosides silylated at room temperature was BSTFA > TMSI > BSA, and for silylation under optimal conditions BSTFA > TMSI = BSA. Significantly lower RWR values were obtained for all the nucleosides when silylations were made at 25°-5 min, 1000 molar excess, sonication, and a comparison was made with silylation at 150°-15 min, 225 molar excess. Room temperature silylations using BSA and BSTFA also gave two peaks for cytidine at retention temperatures (RT) of 238° and 250° on a 4 2/2% OV-11 on 100–200 mesh Supelcoport 1 m × 4 mm I.D. glass column. Solvents also had a pronounced effect on the trimethylsilylation of nucleosides. The RWR values obtained for TMS nucleosides in different solvents were compared and acetonitrile was found to be best. When the derivatized samples in certain solvents were further diluted and chromatographed an increase in RWR R/P value was observed. From this, it was concluded that the solubility was the primary factor for incomplete derivatization in these experiments. When the amount of water was above 10 ppt, all of the nucleosides showed a significant decrease in RWR N/P values. Cytidine gave two peaks (RT = 238° and RT = 250°) in the presence of 0.5 ppt water. The peak with an RT at 250° was used in the gas-liquid chromatographic analysis of cytidine in a mixture of nucleosides; thus the presence of 0.5 ppt of water has a significant effect on derivatization and stability of the cytidine derivatives. Also, silylation of nucleosides with BSTFA containing 1% TMCS, and non-solvent silylations, showed no significant differences in RWR N/P values.