Ion-pair reversed-phase and hydrophilic interaction chromatography methods for analysis of phosphorothioate oligonucleotides.

In this study we investigated the separation of a 25 mer fully phosphorothioated oligonucleotide from its truncated n-1 (24 mer) species and selected phosphodiester 25 mer impurities using ion-pair reversed-phase chromatography. The hydrophobicity of ion-pairing agents (alkylamines) impacts n-1 separation selectivity. 25 mer impurities with single and double phosphodiester bonds eluted prior to the parent phosphorothioate oligonucleotide in the same region as 24 mer impurities, which complicated the chromatographic separation.

Impact of mobile and stationary phases on siRNA duplex stability in liquid chromatography.

Nucleic acid duplexes are typically analyzed in non-denaturing conditions. Melting temperature (T) is the property used to measure duplex stability; however, it is not known how the chromatographic conditions and mobile phase composition affect the duplex stability. We employed differential scanning calorimetry (DSC) method to measure the melting temperature of chemically modified silencing RNA duplex (21 base pairs, 0.

Impact of ion-pairing systems choice on diastereomeric selectivity of phosphorothioated oligonucleotides in reversed-phase liquid chromatography.

Ion-pairing reversed-phase liquid chromatography was utilized for the analysis of native and phosphorothioated oligonucleotides differing in the length (2-6mers and 21mer) and the number and position of phosphorothioate modifications. We investigated the influence of counterion (acetate vs. hexafluoroisopropanol) on the adsorption of eleven alkylamines on the stationary phases.

Hydrophilic interaction liquid chromatography with mass spectrometry for the separation and identification of antisense oligonucleotides impurities and nusinersen metabolites.

With the development of therapeutic oligonucleotides for antisense and gene therapies, the demand for analytical methods also increases. For the analysis of complex samples, for example plasma samples, where the use of mass detection is essential, hydrophilic interaction liquid chromatography is a suitable choice. The aim of the present work was to develop a method for separation and identification of the oligonucleotide impurities and metabolites by hydrophilic interaction liquid chromatography.

Liquid Chromatography Methods for Analysis of mRNA Poly(A) Tail Length and Heterogeneity.

Messenger RNA (mRNA) is a new class of therapeutic compounds. The current advances in mRNA technology require the development of efficient analytical methods. In this work, we describe the development of several methods for measurement of mRNA poly(A) tail length and heterogeneity.

Mixed-mode column allows simple direct coupling with immobilized enzymatic reactor for on-line protein digestion.

Liquid chromatography coupled with mass spectrometry is widely used in the field of proteomic analysis after off-line protein digestion. On-line digestion with chromatographic column connected in a series with immobilized enzymatic reactor is not often used approach. In this work we investigated the impact of chromatographic conditions on the protein digestion efficiency.

Phosphorothioate oligonucleotides separation in ion-pairing reversed-phase liquid chromatography: effect of temperature.

Analysis of diastereomers of phosphorothioate oligonucleotides in ion-pairing reversed-phase liquid chromatography is affected not only by the character and concentration of ion-pairing system, but also by the separation temperature. In this work, eight ion-pairing systems at two concentrations buffered with acetic acid were used with octadecyl column to investigate the effects of temperature (in the range from 20 °C to 90 °C) on retention, diastereomeric separation, resolution of mers of different length and resolution of oligonucleotides with different number of phosphorothioate linkages. It was observed that elevated temperature suppresses the diastereomeric separation and oligonucleotide peaks become narrower.

Phosphorothioate oligonucleotides separation in ion-pairing reversed-phase liquid chromatography: Effect of ion-pairing system.

Ion-pairing reversed-phase liquid chromatography was utilized for the analysis of native and phosphorothioate oligonucleotides of the identical sequence but different amount and position of phosphorothioate modifications. The effects of ion-pairing amines nature (alkyl chain length, hydrophobicity) and concentration on retention, n/n-1 resolution, and diastereomeric separation of phosphorothioate oligonucleotides were investigated using octadecyl column. Eight different ion-pairing agents at two concentrations (10 mM and 100 mM) buffered with acetic acid were investigated.

Contribution of ionic interactions to stationary phase selectivity in hydrophilic interaction chromatography.

We compared the separation selectivities of 19 different hydrophilic interaction chromatography columns. The stationary phases included underivatized silica and hybrid particles, cyano-bonded silica, materials with neutral ligands such as amide, diol, pentahydroxy, and urea, zwitterionic sorbents, and mixed-mode materials with amine functionalities. A set of 77 small molecules was used to evaluate the columns.

Effect of ion-pairing reagent hydrophobicity on liquid chromatography and mass spectrometry analysis of oligonucleotides.

We performed a systematic study of thirteen alkylamines used as ion-pairing reagents for ion-pair reversed-phase liquid chromatography (IP RP LC) separations of oligonucleotides on a C18 column. We proposed a method to classify the hydrophobicity of alkylamines by their retention in RP LC. The IP reagent hydrophobicity correlated with the retention and resolution of oligonucleotides in the corresponding IP mobile phases.

Impact of Nonspecific Adsorption to Metal Surfaces in Ion Pair-RP LC-MS Impurity Analysis of Oligonucleotides.

Nonspecific adsorption has been a consistent challenge in the analysis of oligonucleotides. Nonspecific adsorption is a result of interactions between charged acidic analytes and adsorption sites present in metallic surfaces located in the fluidic path of chromatography systems. Due to their high surface area, adsorption to column frits is especially concerning.

Assessing the impact of nonspecific binding on oligonucleotide bioanalysis.

Accurate and reliable quantification of oligonucleotides can be difficult, which has led to an increased focus on bioanalytical methods for more robust analyses. Recent advances toward mitigating sample losses on liquid chromatography (LC) systems have produced recovery advantages for oligonucleotide separations. LC instruments and columns constructed from MP35N metal alloy and stainless steel columns were compared against LC hardware modified with hybrid inorganic-organic silica surfaces.

Mitigation of analyte loss on metal surfaces in liquid chromatography.

The adsorptive loss of acidic analytes in liquid chromatography was investigated using metal frits. Repetitive injections of acidic small molecules or an oligonucleotide were made on individual 2.1 or 4.

Characterization and comparison of mixed-mode and reversed-phase columns; interaction abilities and applicability for peptide separation.

In this work, two mixed-mode columns from a different manufacturers and one marketed as a reversed-phase column were characterized and compared in the terms of their interaction abilities, retentivity, peak symmetry, and applicability for peptide separation. All the tested columns contain octadecyl ligand and positively charged modifier, i.e.

Using Hybrid Organic-Inorganic Surface Technology to Mitigate Analyte Interactions with Metal Surfaces in UHPLC.

Interactions of analytes with metal surfaces in high-performance liquid chromatography (HPLC) instruments and columns have been reported to cause deleterious effects ranging from peak tailing to a complete loss of the analyte signal. These effects are due to the adsorption of certain analytes on the metal oxide layer on the surface of the metal components. We have developed a novel surface modification technology and applied it to the metal components in ultra-HPLC (UHPLC) instruments and columns to mitigate these interactions.

Method for evaluation of ionic interactions in liquid chromatography.

In this work we utilized basic and acidic analytes to investigate the ionic interaction participation in retention behavior of selected reversed-phase and polar columns. The test analytes included nitrate, benzenesulfonate and trimethylphenylammonium ions. The fully aqueous mobile phase comprising 10 mM dichloroacetic acid buffered with ammonia solution to desirable pH was used for retention experiments.

Evaluating MISER chromatography as a tool for characterizing HILIC column equilibration.

Hydrophilic Interaction Liquid Chromatography (HILIC) is a technique for retaining polar analytes that uses polar stationary phases and acetonitrile-rich mobile phases. While this technique has several advantages over reversed-phase liquid chromatography (RPLC), one main drawback is the reported need for longer column equilibration. The reason for this is not fully understood and is a topic of current investigation.

Utility of linear and nonlinear models for retention prediction in liquid chromatography.

Linear solvation strength model in reversed-phase liquid chromatography assumes linear relationship between ln k and Φ. In this work we show that this assumption is true only in narrow range of mobile phase strength. The ln k versus Φ relationship could be more accurately described by three-parametric non-linear model in a wide range of eluent strength.

Retention loss of reversed-phase chromatographic columns using 100% aqueous mobile phases from fundamental insights to best practice.

This work deals with experimental investigations pertaining to the impact of chemical (electrolyte concentration from 0 to 100 mM, dissolved nitrogen gas from 0 to 6.7  ×  10 M in water; surface chemistry including hexylphenyl, polyphenyl, C, C, and C; surface coverage in C-bonded chains from 1.5 to 3.

Mismatch between sample diluent and eluent: Maintaining integrity of gradient peaks using in silico approaches.

The mismatch of elution strength between the sample diluent and the eluent causes undesirable peak deformations for large sample volumes in gradient liquid chromatography. The solution to that problem consists in diluting the sample solution in a weak solvent. But the minimum dilution factor has to be determined by the user given some performance objectives.

Kinetic mechanism of water dewetting from hydrophobic stationary phases utilized in liquid chromatography.

An experimental protocol was designed to accurately measure the dewetting kinetics of aqueous mobile phases from reversed-phase liquid chromatography (RPLC) columns. The protocol enables the determination of the losses in the wetted surface area and internal pore volume (leading to undesirable retention losses) of RPLC columns as a function of the dewetting time. It is used to evaluate the impact of the buffer/salt concentration in water (0-100 mM), nitrogen concentration dissolved in water (0-6.

Impact of frit dispersion on gradient performance in high-throughput liquid chromatography.

The impact of porous frits (2.1 mm i.d.

Semi-preparative high-resolution recycling liquid chromatography.

A semi-preparative high-resolution system based on twin column recycling liquid chromatography was built. The integrated system includes a binary pump mixer, a sample manager, a two-column oven compartment, two low-dispersion detection cells, and a fraction manager (analytical). It addresses challenges in drug/impurity purification, which involve several constraints simultaneously: (1) small selectivity factors (α < 1.

Chromatographic performance of microfluidic liquid chromatography devices: Experimental evaluation of straight versus serpentine packed channels.

We prepared a series of planar titanium microfluidic (μLC) columns, each 100 mm long, with 0.15, 0.3 and 0.

Applications of high-resolution recycling liquid chromatography: From small to large molecules.

A twin-column recycling separation process (TCRSP) is assembled and used to generate higher speed and/or higher resolution levels than those of the usual non-recycling process at the same back pressure. It enables the users to solve very challenging separation problems caused by too small selectivity factors and/or too low column efficiencies. The relative gain in speed-resolution performance increases with increasing the number of cycles in the TCRSP, decreasing the maximum allowable pressure imposed by the LC system, decreasing the column permeability, and with reducing the separation speed.