Image
Oligonucleotides Absolute quantification PO PS ratio in a single ICPMS run

Oligonucleotides: Absolute quantification and P=O/P=S in a single ICP-MS/MS run

File

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.
     
Date
Authors
Juliusz Bianga
Philippe De Raeve
Arnaud Delobel
Caroline Cajot
Resource type
Location
TIDES 2018