Quantification of Oligonucleotides by LC–MS/MS: The Challenges of Quantifying A Phosphorothioate Oligonucleotide and Multiple Metabolites

Ewles, Matthew; Goodwin, Lee; Schneider, Anneliese; Rothhammer-Hampl, Tanja

doi:10.4155/bio.13.319

Cited by 43 publications

(25 citation statements)

References 35 publications

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“…It has been reported that the detection sensitivity of nucleic acids changes when additives, such as citrate, are added to the matrix solution . Moreover, amines such as TEA are commonly added when oligonucleotides are analyzed by LC/MS . Therefore, in this study, we selected optimized amine additives such as MA, DMA or TEA to improve the detection sensitivity of LNA‐A.…”

Section: Resultsmentioning

confidence: 99%

Development of a detection method for antisense oligonucleotides in mouse kidneys by matrix‐assisted laser desorption/ionization imaging mass spectrometry

Yokoi

Kasahara

Obika

et al. 2018

Rapid Comm Mass Spectrometry

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Section: Resultsmentioning

confidence: 99%

Development of a detection method for antisense oligonucleotides in mouse kidneys by matrix‐assisted laser desorption/ionization imaging mass spectrometry

Yokoi

Kasahara

Obika

et al. 2018

Rapid Comm Mass Spectrometry

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“…Initially, a similar extraction approach as described by the authors of the 18-mer Trabedersen assay was assessed [33]. This method combined an LLE method using chloroform-phenol-isoamylalcohol with a hydrophilic-lipophilicbalanced SPE.…”

Section: Mixed-mode Anion Exchange Spe Methods Developmentmentioning

confidence: 99%

“…Various approaches for ODN extraction from the biological matrix have been used including protein precipitation, liquid-liquid extraction (LLE) and SPE, though each method has its own shortcomings and compound-dependent success rates [27,[29][30][31][32][33].…”

mentioning

confidence: 99%

Quantitative Analysis of Imetelstat in Plasma with LC–MS/MS Using Solid-Phase or Hybridization Extraction

et al. 2017

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Aim: Imetelstat, a 13-mer oligonucleotide with a lipid tail is being evaluated for treating hematologic myeloid malignancies. This report describes the development of extraction and quantification methods for imetelstat. Methodology & results: Imetelstat was extracted using SPE (rat plasma) or by hybridization using a biotinylated capture probe (human plasma) and was quantified by LC-MS/MS. Calibration curves were established (0.1-50 μg/ml). Stability of imetelstat in plasma was demonstrated. Concentrations of imetelstat extracted using either of the methods and quantified with LC-MS/MS were comparable with a validated ELISA. Conclusion: Two extraction methods (solid phase and hybridization) were developed for quantifying imetelstat in plasma using LC-MS/MS. The hybridization extraction in combination with LC-MS/MS is a novel extraction approach. In recent years, oligonucleotide drugs (ODNs) have gained popularity owing to their ability to target specific genes and provide specificity. ODNs comprise an oligonucleotide sequence, containing 10-50 nucleotides [1,2]. Numerous ODNs have been investigated so far [1][2][3][4]. But only six ODNs (fomivirsen, pegaptanib, mipomersen, eteplirsen, nusinersen and defibrotide sodium) have been approved by the US FDA since 1998 [5].Pharmaceutical development of ODNs has several technical challenges such as maintaining in vivo stability, ensuring delivery and bioavailability, and minimizing off-target effects. These challenges can be minimized by chemical modifications of ODNs to enhance their pharmacokinetic and pharmacodynamic properties [4,6]. These chemical modifications include: sulfurization of the phosphodiester bond to avert degradation by enlindogenous exonucleases, addition of methoxy or methoxyethyl groups to sugar moieties [7,8], locked and unlocked nucleic acids, and alterations in the internucleotide linkage (e.g., amide linkage) resulting in peptide nucleic acids [1]. Sulfurization results in phosphorothioate analogs that are more hydrophobic, exhibit more complex secondary structures with higher protein binding and greater accumulation in organs than phosphodiester analogs [9][10][11].Imetelstat is a novel, first-in-class telomerase inhibitor ODN [12], being investigated for the treatment of hematologic myeloid malignancies [13,14] and certain solid tumors [12,15,16]. Imetelstat is a 13-mer oligonucleotide N3 -P5 thio-phosphoramidate (NPS) with a covalently linked C16 (palmitoyl) lipid moiety at the 5 -end (Figure 1; Supplementary Table 1). Addition of a lipid chain and the modified oligonucleotide backbone enables imetelstat to penetrate cells and tissues, with high biodistribution into normal and malignant cells [12].Imetelstat differs from antisense oligonucleotides in its mechanism of action. Imetelstat is complementary to the template region of the RNA component of telomerase, to which it binds with high affinity and specificity, and directly competes with telomere binding, thereby inhibiting telomerase activity [17]. Thus, imetelstat acts as a classical ac...

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“…A similar issue has been encountered by Zhang et al, 80 who determined PS ASOs in rat plasma at relatively low LLOQ value (5 ng mL À1 ), but without complete separation. Such a problem has been largely solved by Ewles et al 81 by the application of accurate sample clean up, careful optimization of MS parameters, and selection of chromatographic parameters. The separation of 20 mer PS and 3 0 N-1-3 0 N-3 as well as 5 0 N-1-5 0 N-3, and their quantication with LLOQ values in the range of 2-1000 ng mL À1 were obtained.…”

Section: Golodirsen (Vyondys 53™)mentioning

confidence: 99%

In vivoandin vitrostudies of antisense oligonucleotides – a review

Kilanowska

Studzińska

2020

RSC Adv.

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Quantification of Oligonucleotides by LC–MS/MS: The Challenges of Quantifying A Phosphorothioate Oligonucleotide and Multiple Metabolites

Cited by 43 publications

References 35 publications

Development of a detection method for antisense oligonucleotides in mouse kidneys by matrix‐assisted laser desorption/ionization imaging mass spectrometry

Development of a detection method for antisense oligonucleotides in mouse kidneys by matrix‐assisted laser desorption/ionization imaging mass spectrometry

Quantitative Analysis of Imetelstat in Plasma with LC–MS/MS Using Solid-Phase or Hybridization Extraction

In vivoandin vitrostudies of antisense oligonucleotides – a review

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