Optimizing conditions in online RPLC × SFC for the analysis of complex samples containing neutral compounds: Solving injection issues.

The online combination of reversed-phase liquid chromatography and supercritical fluid chromatography (online RPLC × SFC) is an attractive technique for the characterization of complex samples containing neutral compounds as the two techniques are highly complementary, especially with a polar stationary phase in supercritical fluid chromatography (SFC). However, the setup is challenging due to the presence of hydro-organic solvents in RPLC, which become injection solvent in SFC. In this study, numerous key experimental parameters were identified and found to have a major effect on peak shape under RPLC × SFC conditions.

Online Supercritical Fluid Chromatography Hyphenated to Fourier Transform Ion Cyclotron Resonance Mass Spectrometry with Quadrupole Detection for Microalgae Bio-Oil Characterization.

Microalgae are an attractive feedstock for biofuel production thanks to their renewable nature, high growth rate, and ability to use anthropogenic CO. The conversion of microalgae by hydrothermal liquefaction (HTL) leads to a solid residue, a gaseous phase, and a biocrude. However, the bio-oil is rich in heteroatoms and requires upgrading processes to be used as biofuels.

Interplatform comparison between three ion mobility techniques for human plasma lipid collision cross sections.

The implementation of ion mobility spectrometry (IMS) in liquid chromatography-high-resolution mass spectrometry (LC-HRMS) workflows has become a valuable tool for improving compound annotation in metabolomics analyses by increasing peak capacity and by adding a new molecular descriptor, the collision cross section (CCS). Although some studies reported high repeatability and reproducibility of CCS determination and only few studies reported good interplatform agreement for small molecules, standardized protocols are still missing due to the lack of reference CCS values and reference materials. We present a comparison of CCS values of approximatively one hundred lipid species either commercially available or extracted from human plasma.

Theoretical and practical guidelines for solvent dilution between the two dimensions in online comprehensive two-dimensional liquid chromatography.

Online comprehensive two-dimensional liquid chromatography (online LC x LC) has become increasingly popular. Among the different chromatographic modes that can be combined, hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) are particularly interesting because they offer a high degree of orthogonality. However, this combination remains complex due to the incompatibility of the solvents in the two dimensions.

How the COVID-19 pandemic and related school closures reduce physical activity among children and adolescents in the WHO European Region: a systematic review and meta-analysis.

Background: Emerging research suggests that physical activity among children and adolescents decreased during the COVID-19 pandemic. However, a differentiated overview of European youth is lacking. In particular, no systematic analysis has been conducted to date on the impact of heterogeneous pandemic restrictions and school closures within European countries, and with regard to potentially vulnerable groups.

Physical activity and physical fitness among children and adolescents after the onset of the COVID-19 pandemic in the WHO European Region: a systematic review protocol.

Introduction: The implementation of COVID-19 pandemic-related restrictions resulted in limitations for physical activity (PA) opportunities, which may have initiated a longer-term behavioural change. The protocol describes the methodology for a planned systematic review that aims to summarise changes in PA and physical fitness (PF) in children and adolescents in the WHO European Region after the onset of the COVID-19 pandemic.

Methods And Analysis: The protocol adheres to the 'Preferred Reporting Items for Systematic Review and Meta-Analysis for Protocols' (PRISMA-P) statement.

Reversed HILIC Gradient: A Powerful Strategy for On-Line Comprehensive 2D-LC.

The aim of the present work is to evaluate the possibilities and limitations of reversed hydrophilic interaction chromatography (revHILIC) mode in liquid chromatography (LC). This chromatographic mode consists of combining a highly polar stationary phase (bare silica) with a gradient varying from very low (1-5%) to high (40%) acetonitrile content (reversed gradient compared to HILIC). The retention behavior of revHILIC was first compared with that of reversed-phase LC (RPLC) and HILIC using representative mixtures of peptides and pharmaceutical compounds.

On-line reversed-phase liquid chromatography x supercritical fluid chromatography coupled to high-resolution mass spectrometry: A powerful tool for the characterization of advanced biofuels.

Bio-oils obtained by thermochemical or biochemical conversion of biomass represent a promising source of energy to complement fossil fuels, in particular for maritime or air transport for which the use of hydrogen or electricity appears complicated. As these bio-oils are very rich in water and heteroatoms, additional treatments are necessary before they can be used as biofuel. In order to improve the efficiency of these treatments, it is important to have a thorough knowledge of the composition of the bio-oil.

Theoretical and practical comparison of RPLC and RPLC × RPLC: how to consider dilution effects and sensitivity in addition to separation power?

The objective of this work was to provide an unbiased comparison of one-dimensional reversed-phase liquid chromatography (1D-RPLC) and comprehensive two-dimensional RPLC (RPLC × RPLC), through calculations and experimental verifications. For this purpose, various quality descriptors were evaluated, including peak capacity, analysis time, dilution factor, number of runs in the second dimension, and injection volume. The same strategy was applied to small pharmaceuticals and peptides.

Sense and nonsense of shifting gradients in on-line comprehensive reversed-phase LC × reversed-phase LC.

In on-line comprehensive two-dimensional liquid chromatography (LC × LC), the combination of similar chromatographic modes such as reversed-phase liquid chromatography × reversed-phase liquid chromatography (RPLC × RPLC) usually leads to the partial occupation of the available separation space. A possible solution to circumvent this issue may be to dynamically adjust the gradient elution in the second dimension (D) throughout the LC × LC analysis. This allows the gradient elution to be tailored to the elution conditions of each fraction instead of using a conventional full gradient program in which the same gradient profile is repeated for each D-fraction.

A simple mathematical treatment for predicting linear solvent strength behavior in gradient elution: Application to biomolecules.

This paper describes an approach to rapidly and easily calculate the linear solvent strength parameters, namely log k and S, under reversed-phase liquid chromatography conditions. This approach, which requires two preliminary gradient experiments to determine the retention parameters, was applied to various representative compounds including small molecules, peptides, and proteins. The retention time prediction errors were compared to the ones obtained with a commercial HPLC modeling software, and a good correlation was found between the values.

Development of a sub-hour on-line comprehensive cation exchange chromatography x RPLC method hyphenated to HRMS for the characterization of lysine-linked antibody-drug conjugates.

This study details the development of on-line two-dimensional liquid chromatography (2D-LC) methods combining cation-exchange chromatography (CEX) and reversed-phase liquid chromatography (RPLC) for the separation of the charge variants of a lysine-linked antibody-drug conjugate (ADC). This combination gives an excellent example of the potential benefits of 2D-LC approaches for the analysis of such complex protein formats. CEX is considered the reference technique for the separation of protein charge variants but its retention mechanism usually requires the use of a high concentration of non-volatile salts, which impedes its compatibility with MS detection.

On-line 2D-RPLC x RPLC - HRMS to assess wastewater treatment in a pharmaceutical plant.

Pharmaceutical effluents are complex media containing hundreds of compounds including active ingredients, intermediate products and unknown impurities. Bringing an industrial wastewater treatment plant (WWTP) into compliance with European directives requires a thorough analysis of the effluent. In this study, we demonstrate how online comprehensive two-dimensional liquid chromatography (on-line LC × LC) hyphenated to high resolution mass spectrometry (HRMS) can be a powerful analytical methodology to monitoring the outlet water, by analysing the content of known molecules while characterizing unknown compounds.

Strategies to circumvent the solvent strength mismatch problem in online comprehensive two-dimensional liquid chromatography.

On-line comprehensive two-dimensional liquid chromatography is a powerful technique for the separation of highly complex samples. Due to the addition of the second dimension of separation, impressive peak capacities can be obtained within a reasonable analysis time compared to one-dimensional liquid chromatography. In online comprehensive two-dimensional liquid chromatography, the separation power is maximized by selecting two separation dimensions as orthogonal as possible, which most often requires the combination of different mobile phases and stationary phases.

A comprehensive study on the phenomenon of total breakthrough in liquid chromatography.

Differences in elution strength between the sample solvent and the mobile phase usually give rise to undesirable effects on the chromatographic separation, which may range from slight broadening to severe peak deformation or even splitting. In the most extreme case, the retention factor of the analyte at the head of the column is so small at the time of injection that part of the analyte goes through the column with very little interaction with the stationary phase and hence elutes very close to the column dead time. This phenomenon is known as breakthrough.

Interlaboratory study of a supercritical fluid chromatography method for the determination of pharmaceutical impurities: Evaluation of multi-systems reproducibility.

Modern supercritical fluid chromatography (SFC) is now a well-established technique, especially in the field of pharmaceutical analysis. We recently demonstrated the transferability and the reproducibility of a SFC-UV method for pharmaceutical impurities by means of an inter-laboratory study. However, as this study involved only one brand of SFC instrumentation (Waters®), the present study extends the purpose to multi-instrumentation evaluation.

Characterization of positional isomers of drug intermediates by off-line RPLC x SFC hyphenated to high resolution MS.

Many steps are needed in the synthesis of a new active pharmaceutical ingredient (API). In a practical case proposed by a French pharmaceutical company, an intermediate synthesis step, needed to protect 8 hydroxyl groups before oxidation, could produce a mixture of neutral compounds containing up to 652 structures being positional isomers of 18 molecular formulas. Some mixtures allowed obtaining the desired API, others did not.

Detailed numerical study of the peak shapes of neutral analytes injected at high solvent strength in short reversed-phase liquid chromatography columns and comparison with experimental observations.

We report on a numerical investigation of the different steps in the development of the spatial concentration profiles developing along the axis of a liquid chromatography column when injecting large relative volumes (>10 to 20% of column volume) of analytes dissolved in a high solvent strength solvent band as can be encountered in the second dimension (D) column of a two-dimensional liquid chromatography (2D-LC) system. More specifically, we made a detailed study of the different retention and the axial band broadening effects leading to the double-headed peak shapes or strongly fronting peaks that can be experimentally observed under certain conditions in 2D-LC. The establishment of these intricate peak profiles is discussed in all its fine, mechanistic details.

Comparison of existing strategies for keeping symmetrical peaks in on-line Hydrophilic Interaction Liquid Chromatography x Reversed-Phase Liquid Chromatography despite solvent strength mismatch.

In two-dimensional liquid chromatography, the combination of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) is very attractive due to the complementarity of their separation mechanisms. On-line comprehensive HILIC x RPLC is well-known to give rise to a large retention space coverage when dealing with ionisable compounds. However, method development in on-line HILIC x RPLC is challenging due to the reversed solvent strength between both dimensions, which can greatly affect the peak shapes in the second RPLC dimension, and thus the separation quality and the method sensitivity.

Suspect screening of environmental contaminants by UHPLC-HRMS and transposable Quantitative Structure-Retention Relationship modelling.

A Quantitative Structure-Retention Relationship (QSRR) model is proposed and aims at increasing the confidence level associated to the identification of organic contaminants by Ultra-High Performance Liquid Chromatography hyphenated to High Resolution Mass Spectrometry (UHPLC-HRMS) in environmental samples under a suspect screening approach. The model was built from a selection of 8 easily accessible physicochemical descriptors, and was validated from a set of 274 organic compounds commonly found in environmental samples. The proposed predictive figure approach is based on the mobile phase composition at solute elution (expressed as % acetonitrile), that has the major advantage of making the model reusable by other laboratories, since the elution composition is independent of both the column geometry and the UHPLC-system.

Pushing the limits of resolving power and analysis time in on-line comprehensive hydrophilic interaction x reversed phase liquid chromatography for the analysis of complex peptide samples.

In the present work, we have investigated the combination of hydrophilic interaction liquid chromatography (HILIC) and reversed phase liquid chromatography (RPLC) for the separation of peptides in on-line HILIC x RPLC. This combination usually leads to significant solvent strength mismatch, since a weak solvent in HILIC becomes a strong solvent in RPLC. This may result in band broadening, peak distortion, and breakthrough phenomena.

Comprehensive two-dimensional liquid chromatography with inductively coupled plasma mass spectrometry detection for the characterization of sulfur, vanadium and nickel compounds in petroleum products.

The petroleum industry is increasingly concerned with the conversion of vacuum residues as a consequence of decreased conventional crude oil availability. The compositional analysis of heavy oil products has become a key step in conversion processes, but the complexity of these oil matrices tends to increase with their boiling point. In this study, comprehensive two-dimensional liquid chromatography (LCxLC) coupled to inductively coupled mass spectrometry (ICP-MS/MS) is considered with a view to meet new requirements and to bring additional information regarding the species present in these matrices.

Drug Loading and Distribution of ADCs After Reduction or IdeS Digestion and Reduction.

High-resolution native mass spectrometry (MS) provides accurate mass measurements (within 30 ppm) of intact ADCs and can also yield drug load distribution (DLD) and average drug to antibody ratio (DAR) in parallel with hydrophobic interaction chromatography (HIC). Native MS is furthermore unique in its ability to simultaneously detect covalent and noncovalent species in a mixture and for HIC peak identity assessment offline or online.As an orthogonal method described in this chapter, LC-MS following ADC reduction or IdeS (Fabricator) digestion and reduction can also be used to measure the DLD of light chain and Fd fragments for hinge native cysteine residues such as brentuximab vedotin.

Two-Dimensional Liquid Chromatography Coupled to High-Resolution Mass Spectrometry for the Analysis of ADCs.

From a structural point of view, the complete characterization of ADCs is a challenging task due to their high complexity. ADCs combine the heterogeneity of the initial antibody to the variability associated with the conjugation strategy, the manufacturing process, and the storage. Given the inherent complexity of these biomolecules, online comprehensive two-dimensional liquid chromatography (LC × LC) is an attractive technique to address the challenges associated with ADC characterization.

Suitable interface for coupling liquid chromatography to inductively coupled plasma-mass spectrometry for the analysis of organic matrices. 2 Comparison of Sample Introduction Systems.

Liquid chromatography (LC) coupled with a specific detection such as inductively coupled plasma-mass spectrometry (ICP-MS/MS) is a technique of choice for elementary speciation analysis for complex matrices. The analysis of organic matrices requires the introduction of volatile solvents into the plasma which is an analytical challenge for this coupling technique. Detection sensitivity can be significantly affected by instrumental limitations.