1. Hydroxyl amine hydrochloride -- Sigma H-2391 (ACS reagent.) Keep in desiccator.
2. Acetic anhydride ¨C Supelco 3-3085
3. Pyridine 99.9% HPLC grade ¨C Sigma-Aldrich 27,040-7
4. Ethyl acetate ¨C Fisher OPTIMA E196-4
Standards:
unlabeled ribose.
Procedure:
1. Prepare ribose ¨C unlabeled standard or acid hydrolized RNA. Dry 100 mL of 100-200 mg % ribose solution.
2. Prepare hydroxylamine hydrochloride solution in pyridine,
20 mg / 1 ml.
3. Add 100 mL of hydroxylamine solution to each tube. Heat to 100oC for 30 min in
heating block.
4. Add 75 mL of acetic anhydride, heat for 1 hour at 100oC.
5. Evaporate to dryness with N2.
6. Add 50-200mL of ethyl acetate as solvent just before GC/MS analysis.
7.
GC/MS:
GC column: HP5, length 30m, 250mm ID.
Inlet temp. 250oC, split ratio 1:100 adjusted depending on sample concentration
Relevant
ion clusters for SIM analysis:
CI mode, m/z 256 cluster for C1-C5. (monitor 254 to 262)