Automated multicolumn screening workflow in ultra-high pressure hydrophilic interaction chromatography for streamlined method development of polar analytes.

The pharmaceutical industry is rapidly advancing toward new drug modalities, necessitating the development of advanced analytical strategies for effective, meaningful, and reliable assays. Hydrophilic Interaction Chromatography (HILIC) is a powerful technique for the analysis of polar analytes. Despite being a well-established technique, HILIC method development can be laborious owing to the multiple factors that affect the separation mechanism, such as the selection of stationary phase chemistry, mobile phase eluents, and optimization of column equilibration time.

Unified chromatography in drug development: Exploiting chaotropic/kosmotropic salts for an accelerated method development.

Recent trends in supercritical fluid chromatography (SFC) introduced an innovative gradient profile called Unified Chromatography (UC), which pushes the amount of liquid modifier up to 80-100 % of the total mobile phase composition. These new conditions allow the full transition from a supercritical to a liquid state, unifying the benefits of both SFC and liquid chromatography. However, to facilitate the use of UC for industrial drug development, a stronger effort is needed to streamline and simplify its method development and optimization.

Automated Hydrophobic Interaction Chromatography Screening Combined with Optimization as a Framework for Nondenaturing Analysis and Purification of Biopharmaceuticals.

The mounting complexity of new modalities in the biopharmaceutical industry entails a commensurate level of analytical innovations to enable the rapid discovery and development of novel therapeutics and vaccines. Hydrophobic interaction chromatography (HIC) has become one of the widely preferred separation techniques for the analysis and purification of biopharmaceuticals under nondenaturing conditions. Inarguably, HIC method development remains very challenging and labor-intensive owing to the numerous factors that are typically optimized by a "hit-or-miss" strategy (e.

Dual-Gradient Unified Chromatography: A New Paradigm for Versatility in Simultaneous Multicomponent Analysis.

Generality in analytical chemistry can be manifested in impactful platforms that can streamline modern organic synthesis and biopharmaceutical processes. We herein introduce a hybrid separation technique named Dual-Gradient Unified Chromatography (DGUC), which is built upon an automated dynamic modulation of CO , organic modifier, and water blends with various buffers. This concept enables simultaneous multicomponent analysis of both small and large molecules across a wide polarity range in single experimental runs.

Deuterated Modifiers in Sub/Supercritical Fluid Chromatography for Streamlined NMR Structure Elucidation.

Isolation and chemical characterization of target components in fast-paced pharmaceutical laboratories can often be challenging, especially when dealing with mixtures of closely related, possibly unstable species. Traditionally, this process involves intense labor and manual intervention including chromatographic method development and optimization, fraction collection, and drying processes prior to NMR analyses for unambiguous structure elucidation. To circumvent these challenges, a foundational framework for the proper utilization of supercritical carbon dioxide (scCO) and deuterated modifiers (CDOD) in sub/supercritical fluid chromatography (SFC) is herein introduced.

Sub/supercritical fluid chromatography versus liquid chromatography for peptide analysis.

The aim of this study was to evaluate the potential of ultra-high performance supercritical fluid chromatography (UHPSFC) for peptide analysis by comparing its analytical performance to several chromatographic approaches based on reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC) and mixed-mode liquid chromatography. First, the retention behavior of synthetic peptides with 3 to 30 amino acids and different isoelectric points (acid, neutral, and basic) was evaluated. For all the tested conditions (13 peptides in 8 conditions), only 4 results were not exploitable (not retained or not eluted), confirming that all the tested chromatographic conditions can be successfully applied when analyzing a wide range of diverse peptides.

Parallel chiral sub/supercritical fluid chromatography screening as a framework for accelerated purification of pharmaceutical targets.

Chiral sub/supercritical fluid chromatography (SFC) has established itself as one of the preferred techniques for enantioseparations at both analytical and preparative scale. Herein, we introduce a parallel multicolumn SFC screening for automated chiral method development in fast-paced settings. The practicality and speed advantages of this approach are illustrated with parallel screening of a diverse set of chiral molecules across ten columns with five different organic modifiers/CO based eluents enabling rapid identification of suitable enantioseparation conditions for accelerated purification of pharmaceutical targets.

Automated ion exchange chromatography screening combined with in silico multifactorial simulation for efficient method development and purification of biopharmaceutical targets.

Bioprocess development of increasingly challenging therapeutics and vaccines requires a commensurate level of analytical innovation to deliver critical assays across functional areas. Chromatography hyphenated to numerous choices of detection has undeniably been the preferred analytical tool in the pharmaceutical industry for decades to analyze and isolate targets (e.g.

Method Development of Achiral and Chiral Tandem Column Reversed-phase Liquid Chromatography for Multicomponent Pharmaceutical Mixtures.

Tandem column liquid chromatography (LC) is a convenient, cost-effective approach to resolve multicomponent mixtures by serially coupling columns on readily available one-dimensional separation systems without specialized user training. Yet, adoption of this technique remains limited, mainly due to the difficulty in identifying optimal selectivity out of many possible tandem column combinations. At this point, method development and optimization require laborious "hit-or-miss" experimentation and "blind" screening when investigating different column selectivity without standard analytes.

Trapping-Enrichment Multi-dimensional Liquid Chromatography with On-Line Deuterated Solvent Exchange for Streamlined Structure Elucidation at the Microgram Scale.

At the forefront of chemistry and biology research, development timelines are fast-paced and large quantities of pure targets are rarely available. Herein, we introduce a new framework, which is built upon an automated, online trapping-enrichment multi-dimensional liquid chromatography platform (TE-Dt-mDLC) that enables: 1) highly efficient separation of complex mixtures in a first dimension ( D-UV); 2) automated peak trapping-enrichment and buffer removal achieved through a sequence of H O and D O washes using an independent pump setup; and 3) a second dimension separation ( D-UV-MS) with fully deuterated mobile phases and fraction collection to minimize protic residues for immediate NMR analysis while bypassing tedious drying processes and minimizing analyte degradation. Diverse examples of target isolation and characterization from organic synthesis and natural product chemistry laboratories are illustrated, demonstrating recoveries above 90 % using as little as a few micrograms of material.

Enantioselective UHPLC Screening Combined with Modeling for Streamlined Development of Ultrafast Enantiopurity Assays.

Enantioselective chromatography has been the preferred technique for the determination of enantiomeric excess across academia and industry. Although sequential multicolumn enantioselective supercritical fluid chromatography screenings are widespread, access to automated ultra-high-performance liquid chromatography (UHPLC) platforms using state-of-the-art small particle size chiral stationary phases (CSPs) is an underdeveloped area. Herein, we introduce a multicolumn UHPLC screening workflow capable of combining 14 columns (packed with sub-2 μm fully porous and sub-3 μm superficially porous particles) with nine mobile phase eluent choices.

Selective Plate-Based Assay for Trace EDTA Analysis via Boron Trifluoride-methanol Derivatization UHPLC-QqQ-MS/MS Enabling Biologic and Vaccine Processes.

The employment of ethylenediaminetetraacetic acid (EDTA) across several fields in chemistry and biology has required the creation of a high number of quantitative assays. Nonetheless, the determination of trace EDTA, especially in biologics and vaccines, remains challenging. Herein, we introduce an automated high-throughput approach based on EDTA esterification in 96-well plates using boron trifluoride-methanol combined with rapid analysis by ultra-high-performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS).

Using 1.5 mm internal diameter columns for optimal compatibility with current liquid chromatographic systems.

This article describes the use of a new prototype column hardware made with 1.5 mm internal diameter (i.d.

Expanding the range of sub/supercritical fluid chromatography: Advantageous use of methanesulfonic acid in water-rich modifiers for peptide analysis.

The aim of this work was to expand the applicability range of UHPSFC to series of synthetic and commercialized peptides. Initially, a screening of different column chemistries available for UHPSFC analysis was performed, in combination with additives of either basic or acidic nature. The combination of an acidic additive (13 mM TFA) with a basic stationary phase (Torus DEA and 2-PIC) was found to be the best for a series of six synthetic peptides possessing either acidic, neutral or basic isoelectric points.

Ultra-high performance supercritical fluid chromatography coupled to tandem mass spectrometry for antidoping analyses: Assessment of the inter-laboratory reproducibility with urine samples.

The aim of this study was to assess the interlaboratory reproducibility of ultra-high performance supercritical fluid chromatography coupled with tandem mass spectrometry method for routine antidoping analyses. To do so, a set of 21 doping agents, spiked in urine and analyzed after dilute and shoot treatment, was used to assess the variability of their retention times between four different laboratories, all equipped with the same chromatographic system and with the same ultra-high performance supercritical fluid chromatography stationary phase chemistry. The average relative standard deviations (RSD%) demonstrated a good reproducibility of the retention times for 19 out of 21 analytes, with RSD% values below 3.

Investigating the use of unconventional temperatures in supercritical fluid chromatography.

The use of unorthodox temperatures, ranging from -5 °C up to 80 °C, have been thoroughly investigated in supercritical fluid chromatography. To this purpose, an initial evaluation of the kinetic and thermodynamic performance has been made with a set of 4 analytes eluting at different percentages of organic co-solvent in the mobile phase (3%-10% - 45%-80%). The van Deemter plots have demonstrated how, at low organic modifier presence, the use of low temperatures did not necessarily translate into worse performance, while high temperatures could pose more issues due to the poor handling of the super/subcritical mobile phase by the chromatographic system.

Applicability of Supercritical fluid chromatography-Mass spectrometry to metabolomics. II-Assessment of a comprehensive library of metabolites and evaluation of biological matrices.

In this work, the impact of biological matrices, such as plasma and urine, was evaluated under SFCHRMS in the field of metabolomics. For this purpose, a representative set of 49 metabolites were selected. The assessment of the matrix effects (ME), the impact of biological fluids on the quality of MS/MS spectra and the robustness of the SFCHRMS method were each taken into consideration.

Supercritical fluid chromatography-mass spectrometry in routine anti-doping analyses: Estimation of retention time variability under reproducible conditions.

The aim of this study was to estimate the retention time variability under reproducible conditions of an SFC-MS analytical method for routine anti-doping analyses. For this purpose, a set of 51 doping agents, as neat standards and spiked in diluted urine, was used to assess their retention times variability over a period of four months, as well as the column inter-batch reproducibility. Three UHPSFC stationary phases have been employed, the Acquity UPC Torus 2-Picolylamine (2-PIC), UPC Viridis BEH and Acquity UPLC HSS C18 SB.

Unmatched Kinetic Performance in Enantioselective Supercritical Fluid Chromatography by Combining Latest Generation Whelk-O1 Chiral Stationary Phases with a Low-Dispersion in-House Modified Equipment.

This proof-of-concept work investigates the ultimate kinetic limits reachable in chiral supercritical fluid chromatography (SFC) with modern columns and advanced technological solutions. A commercial equipment (Waters Acquity UPC) has been in-house modified to minimize its overall extra-column variance through a series of technical adjustments including low-volume connecting tubings, reduced-volume flow cell, an in-house made external column oven, external low-dispersion injection system, and electronic temperature controller. Compared to the original (as-shipped) configuration, the extra-column variance on the low-dispersion equipment was reduced by more than 97%, from about 85 to slightly more than 2 μL (measured at 2.

Applicability of supercritical fluid chromatography - mass spectrometry to metabolomics. I - Optimization of separation conditions for the simultaneous analysis of hydrophilic and lipophilic substances.

The aim of this study was to evaluate the suitability of SFC-MS for the analysis of a wide range of compounds including lipophilic and highly hydrophilic substances (log P values comprised between -6 and 11), for its potential application toward human metabolomics. For this purpose, a generic unified chromatography gradient from 2 to 100% organic modifier in CO was systematically applied. In terms of chemistry, the best stationary phases for this application were found to be the Agilent Poroshell HILIC (bare silica) and Macherey-Nagel Nucleoshell HILIC (silica bonded with a zwitterionic ligand).