Oligonucleotide quantification is conventionally performed by UV absorption on basis of a theoretically calculated absorption coefficient; such a method suffers from inaccuracy of the predicted coefficient and potential bias introduced by other UV absorbing molecules. Fluorescence spectroscopy or PCR may also be used, but these techniques require a well-characterised oligonucleotide reference material that often is not available.
A sensitive method was developed to determine the phosphorus content by ICP-MS/MS, after microwave digestion of the sample: accurate quantification of oligonucleotides can be performed using the fixed stoichiometry of phosphorus in the sample and a simple phosphoric acid solution as standard.
Sulphur may also be measured in the same run, allowing the simultaneous accurate determination of the P=O/P=S ratio, a very important parameter for the characterisation of therapeutic phosphorothioates, that can only be measured by 31P-NMR, a technique not readily available in pharmaceutical labs.
ICP-MS/MS was shown to be a suitable and very convenient method for the absolute quantification of an oligonucleotide and the simultaneous determination of the P=O/P=S ratio in the case of a phosphorothioate. Sodium may also be determined with the same method.
The methods characteristics are:
- Good sensitivity: 5-6 mg of oligo are enough for one determination
- High reproducibility: RSDs < 2 %
- Great accuracy: < 3% bias for quantification and P/S ratio
- Does not require any reference substance other than S & P standard solutions
It is suitable for:
- Assay of a single oligonucleotide (powder or solution)
- Accurate determination of an oligonucleotide standard solution
- Accurate determination of the molar extinction coefficient.