Two-dimensional liquid chromatography coupled to mass spectrometry for impurity analysis of dye-conjugated oligonucleotides

Koshel, Brooke M.; Birdsall, Robert E.; Chen, Weibin

doi:10.1016/j.jchromb.2019.121906

Cited by 14 publications

(9 citation statements)

References 27 publications

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“…Two-dimensional liquid chromatography (2D-LC) methods have been developed to provide a comprehensive oligonucleotide impurity profiling and desalted fractions collected from 1 D analysis for MS identification. For example, anion-exchange chromatography has been coupled to ion-pairing reversed-phase liquid chromatography (IPRP) or HILIC for the characterization of chemically modified oligonucleotides. , Alternatively, an IPRP–IPRP 2D-LC method was developed to achieve an optimal separation of impurities from dye-conjugated oligonucleotides in the first dimension and optimal MS detection in the second dimension …”

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confidence: 99%

“…12,13 Alternatively, an IPRP−IPRP 2D-LC method was developed to achieve an optimal separation of impurities from dyeconjugated oligonucleotides in the first dimension and optimal MS detection in the second dimension. 14 Beyond preventing LC column and MS instrument contamination, immobilized enzymes present significant advantages in combination with multidimensional LC−MS (mD-LC−MS) setups. First, the on-line digestion and analysis significantly reduce the sample amount needed and digestion time.…”

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confidence: 99%

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On-line Sequencing of CRISPR Guide RNAs and Their Impurities via the Use of Immobilized Ribonuclease Cartridges Attached to a 2D/3D-LC–MS System

Goyon¹,

Scott²,

Kurita³

et al. 2021

Anal. Chem.

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In this study, for the first time, the automated digestion and sequencing of an RNA molecule via the use of immobilized RNase cartridges attached to a multidimensional liquid chromatography (LC)−mass spectrometry (MS) system are presented. We first developed an on-line digestion-HILIC twodimensional (2D)-LC−MS method in order to sequence CRISPR guide RNAs for gene editing. Three RNases (T1, A, and U2) were immobilized on polyetheretherketone cartridges, and their performance was evaluated. Ultrafast digestions were performed within 2.3 min with the on-line approach versus 30 min via the conventional off-line approach. The higher sequence coverage was achieved by the RNase T1 (71%), which is the same as the off-line mode. A 20fold reduction in the gRNA sample amount was achieved with the on-line digestion approach (6.5 μg) in comparison to that with the off-line approach (130 μg). In the second step, a three-dimensional (3D)-LC−MS method was developed for the sequencing of fractions collected on-line across the main peak and the partially separated tail by the reference ion-pairing RPLC method. Additional insights were gained in order to better understand the cause of the main peak tailing.

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confidence: 99%

On-line Sequencing of CRISPR Guide RNAs and Their Impurities via the Use of Immobilized Ribonuclease Cartridges Attached to a 2D/3D-LC–MS System

Goyon¹,

Scott²,

Kurita³

et al. 2021

Anal. Chem.

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“…If needed, further purification in the second dimension can be easily implemented by programming a shallow eluent gradient rather than a step gradient and by narrowing the time window for fraction collection. By using identical column dimensions in the first dimension as in quality control, a very high F I G U R E 2 Flow scheme of the instrumental 2D-UHPLC set-up for preparative small-scale separations (System 2), adopted from Koshel et al 11 T A B L E 1 Overview of timed events to control the 2D-UHPLC system for small-scale preparative separations (System 2); bold typeface indicates the justification for the respective step.…”

Section: Resultsmentioning

confidence: 99%

“…Another possibility is to use trapping cartridges as an interface 10 . A third option is the direct transfer, with or without modulation, from the first separation column to the second separation column 11 . This direct transfer approach, according to Koshel et al 11 is adopted in this manuscript.…”

Section: Resultsmentioning

confidence: 99%

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Small‐scale two‐dimensional liquid chromatography for a preparative re‐purification of a highly labile tritium‐labeled compound

Sandvoß

Klaus

Weck

et al. 2023

Labelled Comp Radiopharmac

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Tritium‐labeled compounds are generally less stable than their non‐labeled counterparts. This requires storage at low temperatures, a constant workflow of quality checks, and subsequent re‐purifications. As the amount of tritium‐labeled material is typically purified in the μg range, repeated injections on analytical‐scale ultra high‐performance liquid chromatography systems can provide high‐resolution re‐purification results. Yet, degradants can be undesirably included in the compound isolation because the amount of decomposition can vary dramatically depending on the structure. We report a case of a sensitive molecule that could not be isolated in pure form even though the chromatographic separation was successful. In this case, the use of a small‐scale two‐dimensional preparative liquid chromatography approach with a direct transfer interface to a second (trapping) column resulted in a highly pure compound (>98% radiochemical purity). This approach combines high chromatographic resolution, accurate control over the re‐purification process, minimal sample manipulation, and higher overall safety for the handling of radioactive samples.

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2 D ‐ HPLC – Method Development for Successful Separations

Stoll

2021

Optimization in HPLC

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Two-dimensional liquid chromatography coupled to mass spectrometry for impurity analysis of dye-conjugated oligonucleotides

Cited by 14 publications

References 27 publications

On-line Sequencing of CRISPR Guide RNAs and Their Impurities via the Use of Immobilized Ribonuclease Cartridges Attached to a 2D/3D-LC–MS System

On-line Sequencing of CRISPR Guide RNAs and Their Impurities via the Use of Immobilized Ribonuclease Cartridges Attached to a 2D/3D-LC–MS System

Small‐scale two‐dimensional liquid chromatography for a preparative re‐purification of a highly labile tritium‐labeled compound

2 D ‐ HPLC – Method Development for Successful Separations

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