Sample Preparation for LC‐MS Bioanalysis of Oligonucleotides

Bioanalysis and Biotransformation of Oligonucleotide Therapeutics by Liquid Chromatography‐mass Spectrometry

“…There have been many approaches used successfully to isolate therapeutic oligonucleotides and their metabolites prior to liquid chromatography‐mass spectrometry (LC‐MS) analysis (Fig. 5) (Bartlett, Kim, Basiri, 2019). They can be divided into several different categories.…”

In vitro metabolism of 2′‐ribose unmodified and modified phosphorothioate oligonucleotide therapeutics using liquid chromatography mass spectrometry

“…Among these diverse synthetic oligonucleotides, which include an aptamer, antisense oligonucleotides (ASOs), and small interfering ribonucleic acids, six products are ASOs that are small fragments of a single strand of DNA or RNA (Geary, Rosie, & Levin, 2007; Mansoor & Melendez, 2008). ASOs are typically 15–25 nucleotides in length (Bartlett, Kim, Basiri, & Li, 2019; Dias & Stein, 2002) and bind to complementary sequences on the target messenger RNA (mRNA). This binding causes the downregulation of expression of a specific gene related to a disease (Sannes‐Lowery & Hofstadler, 2003) by supporting RNase H activity followed by the decay of the target mRNA (Crooke & Geary, 2013; Geary et al, 2007).…”

“…Unmodified ASOs with phosphodiester (PO) backbones (Figure 1) are extremely susceptible to degradation by nucleases in biological fluids (Bartlett et al, 2019; Blackburn, 2006) and can barely penetrate the cell membrane which has a net negative charge (Chan, Lim, & Wong, 2006; Lysik & Wu‐Pong, 2003), thereby making them further challenging to use for therapeutic applications. The need to circumvent nuclease‐mediated degradation, improve tissue half‐life, and increase the affinity and potency of ASOs have led to various chemical modifications (Chan et al, 2006; Faria & Ulrich, 2008).…”

“…Hence, we proposed an appropriate LC–MS method to identify metabolites and successfully applied the method to understand the impact of modifications of 2′‐ribose and backbones on the generation of metabolites after in vitro incubation with purified nucleases, mouse liver homogenates, and liver microsomes. In the method, we used a mobile phase system consisting of an alkylamine and a fluorinated alcohol that are commonly applied to enhance the response of oligonucleotides when using LC–MS with electrospray ionization (ESI; Bartlett et al, 2019; Basiri & Bartlett, 2014; Beverly, Hartsough, & Machemer, 2005; Chen & Bartlett, 2012; Elzahar, Magdy, El‐Kosasy, & Bartlett, 2018; Ewles, Goodwin, Schneider, & Rothhammer‐Hampl, 2014; Ye & Beverly, 2011).…”