Quantitation of a low level coeluting impurity present in a modified oligonucleotide by both LC–MS and NMR

• A co-eluting mono-phosphate impurity present in modified oligonucleotides can be analysed by both NMR Spectroscopy and LC–MS.
• LC–MS is a more rapid technique and complements the quantitative nature of NMR Spectroscopy.
• Both techniques were used to analyse a series of oligonucleotide samples spiked with increasing levels of the impurity.
• Excellent agreement was found between the quantitative results obtained by the two orthogonal techniques with a correlation coefficient of 0.993 obtained.
• This gives confidence in the quantitative results obtained using LC–MS and demonstrates that this more rapid technique can be used in future studies.

This paper describes the use of two complementary techniques, LC–MS and NMR, to quantify a low level mono phosphate substituted impurity in an oligonucleotide drug substance. This impurity is the result of a sulphurisation failure, leading to the production of a sequence where a phosphorothioate linkage is replaced by a phosphate. Few quantitative methods are possible to analyse these challenging molecules especially if reversed phase ion pair chromatography, one of the most commonly used techniques for the separation of oligonucleotides, is unable to resolve the impurity in question. With the use of a standard addition method it could be demonstrated that both analytical techniques show equivalency and furthermore, the LC–MS method alone with additional validation has the potential to perform this quantitative assay with a high degree of accuracy.

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