A Rapid and Reliable Method for Separation of Ribonucleotides, Deoxynucleotides, and Cyclic Nucleotides by Reversed Phase HPLC
We have recently reported a rapid method of separating ribonucleotides on a C18-reversed phase column, using isocratic elution. The elution profiles were dependent on both pH and ionic strength (1). To our knowledge, none of the existing methods affords simultaneous detection of major ribo, deoxyribo and cyclic nucleotides using isocratic elution conditions. Accordingly, some protocols provide the separation of purine nucleotides alone (2), pyridine ones alone (3), exclusive separation of either ribonucleotides (4), or deoxynucleotides (5). Using an isocratic elution buffer and a reversed phase column, we now report the resolution of the deoxynucleotides and cyclic nucleotides from each other and from ribonucleotides. The deoxynucleotides and cyclic nucleotides elute, in general, later than the corresponding ribonucleotides, and depending on the analysis desired, their elution could be expedited using the same buffer fortified with 10% methanol (V/V). Combining periodate treatment to remove ribonucleotides, deoxynucleotides can be easily distinguished from ribonucleotides in biological samples. It is hoped that this protocol will be of use in the analysis of ribotides, deoxyribotides and cyclic ribotides from various cell types, cells under different pathological state and cells in different energy levels with varying metabolic conditions.
KeywordsReverse Phase HPLC Cyclic Nucleotide Isocratic Elution Purine Nucleotide Sodium Periodate
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