High-resolution gas chromatography of methylated ribonucleosides and hypermodified adenosines : Evaluation of trimethylsilyl derivatization and split and splitless operation modes

Abstract

Methylated ribonucleosides and hypermodified adenosines were trimethylsilylated on chromatographed by high-resolution gas chromatography on a fused-silica capillary column operated in split and splitless modes. Evaluation of micro-silylation (50-μl volume) of methylated ribonucleosides showed that N,O-bis(trimethylsilyl)trifluoracetamide (BSTFA) and pyridine at 150°C gave greater yields than silylation with either BSTFA alone or BSTFA and pyridine at 75%C. N-Methyl-N-trimethylsilyltrifluoracetamide gave lower yields of derivatives of N 6-substituted adenosines, such as N 6-methyladenosine, relative to those obtained with BSTFA. Methylated ribonucleosides generally gave sharp, symmetrical peaks on the SE-54 column operated in the split mode; however, the compound were not as well resolved as the cytokinin-active hypermodified adenosines on the relatively non-polar SE-54 stationary phase. The splitless operation mode employing a cold trapping procedure (40°C initial temperature) yielded sharp peaks and nanogram quantities of N 6-methyladenosine were detectable. Most hypermodified adenosines separated well from other compounds, although several peaks of unknown composition eluted in the same chromatographic region as the methylated ribonucleosides when the cold trapping splitless technique was used.