GAS-LIQUID-CHROMATOGRAPHY OF NUCLEOSIDES DERIVATIZATION AND CHROMATOGRAPHY

The optimum reaction conditions for silylation of nucleosides with bis(trimethylsilyl)trifluoroacetamide (BSTFA) and the chromatographic properties of adenosine, guanosine, cytidine, thymine, inosine, xanthosine and uridine were investigated. Closed tube silylations were performed with a 1000 molar excess of BSTFA at 25, 75, 120, 150 and 175.degree. for 15, 30, 60, 120 and 240 min. The optimal time and temperature for derivatization were found to be 150.degree. and 15 min. Using these reaction conditions, samples were then silylated with 50, 100, 200, 500 and 1000 molar excess of BSTFA; a molar excess of 225 was best. The stability of the nucleoside derivatives on standing at room temperature for 1-7 days was investigated. Quantitative GLC of trimethylsilyl (TMS) nucleosides can be performed if samples are analyzed within 48 h, after which time the relative weight response for the nucleosides decreased somewhat. Chromatographic column studies were made using various liquid phases and supports. With methylsiloxanes as the liquid phase, Supelcoport as support was found to be superior. Resolution of TMS guanosine from TMS cytidine was attempted at different column lengths using 3% (wt/wt) SE-30 on Supelcoport, different loadings (%, wt/wt) of SE-30 on Supelcoport, and different polarity liquid phases, 4% (wt/wt) OV-11 or 3% (wt/wt) OV-17 or 4% (wt/wt) Dexsil-300 on Supelcoport. A complete separation of the 6 ribonucleosides including guanosine and cytidine was obtained with 1 m .times. 4 mm I.D. [inside diameter] glass columns of 4% (wt/wt) OV-11 and 3% (wt/wt) OV-17 on 100-120 mesh Supelcoport. With 2''-deoxycytidine the TMS cytosine peak (retention temperature 150.degree.) plus another peak with a retention temperature of 120.degree. are obtained under all derivatization conditions with BSTFA and bis(trimethylsilyl)acetamide. Similar formation of bases from other 2''-deoxyribonucleosides occurred when the molar excess of BSTFA was greater than 500. The minimal detectable amounts obtained for all the nucleosides ranged from 5-10 ng injected with a signal-to-noise ratio of 3. The relative SD for all nucleosides and deoxynucleosides ranged from 1.2-4.8% on different methylsiloxanes on Supelcoport columns.