The Role of Fluorinated Alcohols as Mobile Phase Modifiers for LC-MS Analysis of Oligonucleotides

Basiri, Babak; Hattum, Hilde van; Dongen, W. D. van; Murph, Mandi M.; Bartlett, Michael G.

doi:10.1007/s13361-016-1500-3

Cited by 67 publications

(71 citation statements)

References 42 publications

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“…These findings have been extended by the systematic investigation of modifiers for the quantification of phosphorothioate DNA and recently other fluorinated alcohols alongside HFIP have been proposed [22,38]. Furthermore, the impact of different ion-pairing reagents has also been reported for the LC-MS/MS analysis of specific ODNs [39][40][41].…”

Section: Lc-ms/ms Methods Development Lc Solventsmentioning

confidence: 80%

“…MS has become a method of choice to study structural properties [19][20][21], as well as for quantitative analyses of ODNs. Other chromatographic strategies (utilizing capillary electrophoresis or ion exchange in combination with fluorescence or UV detection [22][23][24][25]) are also reported, although these methods are often incompatible with MS. There are several technical issues associated with LC-MS analysis of ODNs as they are large acidic molecules with limited ionization efficiency in ESI-MS. High polarity of ODNs leads to limited chromatographic retention.…”

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confidence: 99%

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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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Section: Lc-ms/ms Methods Development Lc Solventsmentioning

confidence: 80%

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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“…Several attempts have also been made to replace HFIP with other fluoroalcohols . Until very recently, Basiri and co‐workers compared the performance of HFIP, mainly ESI‐MS detection, with another four fluoroalcohols, pentafluoropropanol (PFP), 1,1,1,3,3,3‐hexafluoro‐2‐methyl‐2‐propanol (HFTP), trifluoroethanol (TFE), and nonafluoro‐ tert ‐butyl alcohol (NFTB) . They concluded that HFTP can significantly outperform HFIP when used with more hydrophobic ion‐pairing reagents in respect of MS sensitivity.…”

Section: Introductionmentioning

confidence: 99%

“…TFE and PFP were excluded from the study due to their high pKa values, inapplicable to IP‐RP analysis. NFTB was also not included in the work because it can cause considerable MS signal suppression . As a result, only a comparison of the aforementioned seven IPRs buffered with HFIP and HFTP on the IP‐RP separation and MS detection of oligonucleotides was conducted in the study.…”

Section: Introductionmentioning

confidence: 99%

“…NFTB was also not included in the work because it can cause considerable MS signal suppression. 40 As a result, only a comparison of the aforementioned seven IPRs buffered with HFIP and HFTP on the IP-RP separation and MS detection of oligonucleotides was conducted in the study. TTT TTT TTT TTT (dT15), TTT TTT TTT TTT TTT TTT TTT TTT T (dT25), TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT T (dT40).…”

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The role of fluoroalcohols as counter anions for ion‐pairing reversed‐phase liquid chromatography/high‐resolution electrospray ionization mass spectrometry analysis of oligonucleotides

Liu

Ruan

Liu

et al. 2019

Rapid Comm Mass Spectrometry

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Rationale Hexafluoroisopropanol (HFIP) has been widely used as a counter anion in the mobile phase for ion‐pairing reversed‐phase liquid chromatography/mass spectrometry (IP‐RP‐LC/MS) analysis of oligonucleotides. However, researchers are still searching for improvements to counter anions for LC/MS analysis of oligonucleotides. This study aimed to find alternatives to HFIP for analyzing oligonucleotides. Methods The study was performed using an Agilent 1290 ultra‐high‐performance liquid chromatography (UHPLC) system coupled to an Agilent 6540 mass spectrometer by using an oligonucleotide BEH C18 column (100 × 2.1 mm, 1.7 μm). Buffer systems containing ion‐pairing reagents (triethylamine, tripropylamine, hexylamine, dimethylbutylamine, diisopropylethylamine, N,N‐dimethylcyclohexylamine, and octylamine) and fluoroalcohols (HFIP and hexafluoro‐2‐methyl‐2‐propanol (HFTP)) were compared chromatographically and mass spectrometrically. Results Results showed that HFTP has better desalting ability than HFIP, but both HFIP and HFTP have comparable effects on the separation of oligonucleotides sized from 10mer to 40mer for most of ion‐pairing reagents, with the exception of triethylamine and N,N‐dimethylcyclohexylamine, where HFIP performed better than HFTP. Conclusions The choice of fluoroalcohols in IP‐RP‐LC/MS analysis of oligonucleotides depends on the type of ion‐pairing reagents used in the mobile phase. As a guideline, we would recommend to use either HA‐HFIP or HA‐HFTP for small oligonucleotides, but TPA‐HFTP for large oligonucleotides for IP‐RP‐LC/MS analysis of synthetic oligonucleotides.

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Sample Preparation for LC‐MS Bioanalysis of Oligonucleotides

Bartlett¹,

Kim²,

Basiri³

et al. 2019

Sample Preparation in LC‐MS Bioanalysis

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The Role of Fluorinated Alcohols as Mobile Phase Modifiers for LC-MS Analysis of Oligonucleotides

Cited by 67 publications

References 42 publications

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

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

The role of fluoroalcohols as counter anions for ion‐pairing reversed‐phase liquid chromatography/high‐resolution electrospray ionization mass spectrometry analysis of oligonucleotides

Sample Preparation for LC‐MS Bioanalysis of Oligonucleotides

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