Shimadzu Review Vol.77[1・2](2020)
Life Science


Development of an LC/MS Assay Method for a Therapeutic Oligonucleotide and Its analogs

by Tairo Ogura, Ph.D.1Toshiya Matsubara, Ph.D.1Noriko Kato 2

Shimadzu Review 77[1・2] (2020.7)


The purpose of this study is to establish a qualitative and quantitative analytical method for a phosphorothioate oligonu-cleotide. Therapeutic oligonucleotides are synthetic oligonucleotides composed of 10–30 nucleic acid bases. They are  molecularly targeted drugs that bind to the mRNA or miRNA of a gene or directly to a target protein involved in disease pathogenesis. Compared to ELISA and other immunological detection methods, mass spectrometry (MS) is simpler in terms of sample preparation and method development. MS is also the only methodology capable of distinguishing slight differences in chemical modification and of using accurate mass data to determine the molecular mass of a target compo-nent and identify structurally similar impurities. 
This article describes using a liquid chromatograph/mass spectrometer to determine molecular mass and construct a quantitative assay for a therapeutic oligonucleotide and its analogs. The compounds used in the analysis were the therapeu-tic oligonucleotide mipomersen and three mipomersen analogs. Based on mass spectral deconvolution of multivalent ions, we determined the molecular weight of the oligonucleotides to a mass error of less than 1 ppm. To develop a quantitative method using LC-QTOF-MS and LC-TQ-MS, we selected a product ion derived from phosphorothioate as the MRM   transition and then constructed a highly sensitive MRM-based quantitative assay. Given that phosphorothioate modifica-tion is the most common method of enhancing oligonucleotide stability in vivo, we expect MRM-based assays constructed by a similar methodology to be applicable across a variety of therapeutic oligonucleotides. 

1MS Business Unit, Life Science Business Department, Analytical & Measuring Instruments Division, Shimadzu Corporation, Kyoto, Japan
2Global Application Development Center, Analytical & Measuring Instruments Division, Shimadzu Corporation, Kyoto, Japan

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