Separation and identification of phosphorothioate oligonucleotides by HILIC-ESIMS

Easter, Renee N.; Barry, Colin; Caruso, Joseph A.; Limbach, Patrick A.

doi:10.1039/c3ay26519f

Cited by 27 publications

(9 citation statements)

References 17 publications

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“…According to the literature, IPC mode allows for selective separation of N-deleted ASO metabolites; however, quantification levels in MS detection (LOQ > 2.5 ng/mL) need further improvement [2,19,21]. On the other hand, HILIC mode provided lower limits of quantification (LOQ > 0.02 ng/mL) compared to IPC; however, selective separation of shorter metabolites is difficult [22][23][24][25]. For this reason, we decided to compare the sensitivity of Q-TOF-MS for these two chromatographic modes regarding ASO TIC peak areas.…”

Section: Resultsmentioning

confidence: 99%

Studying in vitro metabolism of the first and second generation of antisense oligonucleotides with the use of ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

2020

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The aim of the present investigation was the analysis and identification of antisense oligonucleotide metabolism products after incubation with human liver microsomes regarding four different oligonucleotide modifications. Separation and detection methods based on the use of liquid chromatography coupled with quadrupole time-of-flight mass spectrometry were developed for this purpose. Firstly, the optimization of mass spectrometer parameters was done to select those which ensure the highest possible sensitivity of oligonucleotide analysis. This step was conducted for two chromatographic modes—ion pair chromatography and hydrophilic interaction liquid chromatography—due to their common application in oligonucleotide analysis. Based on sensitivity results, ion pair chromatography coupled with mass spectrometry was selected for the separation of model oligonucleotide mixtures in order to verify its selectivity for N-deleted metabolite separation. Next, the developed method was applied in the examination of oligonucleotides in vitro metabolism. First, wide optimization of incubation parameters was conducted including the concentration of the reaction buffer components. Obtained results indicated that both 3′-exonucleases and 5′-exonucleases contributed to the biotransformation of oligonucleotides. Moreover, it may be concluded that the number of metabolites depends on oligonucleotide modification and consequently its resistance to enzymatic attack. Thus, the number of the oligonucleotide metabolites decreased with the decrease of the resultant polarity of oligonucleotide caused by chemical modification.

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Section: Resultsmentioning

confidence: 99%

Studying in vitro metabolism of the first and second generation of antisense oligonucleotides with the use of ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

2020

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“…Hydrophilic interaction liquid chromatography (HILIC) is another method that can be used to analyze therapeutic oligonucleotides. There are only a handful of published results surrounding this methodology for application to oligonucleotides (Alpert, 1990; Easter et al, 2010, 2013; Holdsvendova et al, 2007; Gong & McCullagh, 2011; Studzińska, Łobodziński, & Buszewski, 2017; Lobue et al, 2019). Currently the most significant challenge facing this approach the reduced sensitivity when compared to ion‐pair chromatography.…”

Section: Lc‐ms Bioanalytical Methodsmentioning

confidence: 99%

“…Lobue et al (2019) have shown that for their method the ion‐pair method is more sensitive for three out of four oligonucleotides present, including a phosphorothioate. Another challenge is that HILIC may have greater difficulty achieving similar chromatographic resolution to ion‐pair chromatography since the mechanism of retention for different column chemistries is currently still under investigation (Gong & McCullagh, 2011; Li et al, 2012; Easter et al, 2013; Gong, 2017; Studzińska, Łobodziński, & Buszewski, 2017; Lobue et al, 2019). Studzinska provides the greatest insight into how column chemistries impact HILIC performance comparing several columns indirectly (Studzińska, Łobodziński, & Buszewski, 2017).…”

Section: Lc‐ms Bioanalytical Methodsmentioning

confidence: 99%

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

Sutton

Kim

Zahar

et al. 2020

Mass Spectrometry Reviews

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Since 2016, eight new oligonucleotide therapies have been approved which has led to increased interest in oligonucleotide analysis. There is a particular need for powerful bioanalytical tools to study the metabolism and biotransformation of these molecules. This review provides the background on the biological basis of these molecules as currently used in therapies. The article also reviews the current state of analytical methodology including state of the art sample preparation techniques, liquid chromatography‐mass spectrometry methods, and the current limits of detection/quantitation. Finally, the article summarizes the challenges in oligonucleotide bioanalysis and provides future perspectives for this emerging field. © 2020 John Wiley & Sons Ltd.

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“…In order to couple AEX or size exclusion chromatography (SEC) to MS, two-dimensional liquid chromatography (2D-LC) approaches have recently been used with the use of IPRP in the second dimension. , Despite some hardware limitations (only a 40 μL loop was available at the time with the 2D-LC setup), the AEX-IPRP allowed the separation and identification of a “ n +16 dithioate impurity” . In parallel, preliminary work has suggested the promising potential of HILIC for the analysis of oligonucleotides but surprisingly only a limited number of studies have been published. ,− The very polar nature of therapeutic oligonucleotides makes them ideal candidates for HILIC analysis in the absence of ion-pairing agents for unprecedented MS sensitivity in oligonucleotide impurity identification.…”

mentioning

confidence: 99%

Characterization of Antisense Oligonucleotide Impurities by Ion-Pairing Reversed-Phase and Anion Exchange Chromatography Coupled to Hydrophilic Interaction Liquid Chromatography/Mass Spectrometry Using a Versatile Two-Dimensional Liquid Chromatography Setup

Goyon¹,

Zhang²

2020

Anal. Chem.

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Determination of phosphorothioate oligonucleotide purity and impurity profile is commonly performed by ion-pairing reversed-phase liquid chromatography (IPRP) with a mobile phase containing triethylamine (TEA) and hexafluoro-2-propanol (HFIP). However, ion-suppressing effects of TEA hamper mass spectrometry (MS) instrumentation sensitivity and HFIP can affect the robustness of the mass spectrometer due to its corrosive nature. Anion exchange chromatography (AEX) is an orthogonal separation mode to IPRP but typically cannot be directly coupled to MS. In this work, we developed a multiple heart-cutting IPRP-, AEX-hydrophilic interaction liquid chromatography(HILIC)/MS method for quantification and high sensitivity identification of antisense oligonucleotide (ASO) impurities using a Q-Exactive mass spectrometer. Notably, both AEX-HILIC and IPRP-HILIC modes could be operated on a versatile two-dimensional liquid chromatography (2D-LC) setup including several column selectors. The HILIC mobile phase contained 25 mM ammonium acetate and allowed identifying impurities at levels down to 0.3%. Careful selection of the sample loop volume and the 2 D HILIC column dimension allowed straightforward coupling of HILIC for both IPRP and AEX without the need to use any solvent modulation. Overall, the 2 D HILIC allowed online desalting of AEX and IPRP modes and further separation of additional impurities.

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Separation and identification of phosphorothioate oligonucleotides by HILIC-ESIMS

Cited by 27 publications

References 17 publications

Studying in vitro metabolism of the first and second generation of antisense oligonucleotides with the use of ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

Studying in vitro metabolism of the first and second generation of antisense oligonucleotides with the use of ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

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

Characterization of Antisense Oligonucleotide Impurities by Ion-Pairing Reversed-Phase and Anion Exchange Chromatography Coupled to Hydrophilic Interaction Liquid Chromatography/Mass Spectrometry Using a Versatile Two-Dimensional Liquid Chromatography Setup

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