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. We used three different commercial ion mobility technologies from different laboratories, drift tube IMS (DTIMS), travelling wave IMS (TWIMS) and trapped IMS (TIMS), to evaluate both instrument repeatability and interlaboratory reproducibility. We showed that CCS discrepancies of 0.3% (average) could occur depending on the data processing software tools. Moreover, eleven CCS calibrants were evaluated yielding mean RSD below 2% for eight calibrants, ESI Low concentration tuning mix (Tune Mix) showing the lowest RSD (< 0.5%) in both ion modes. Tune Mix calibrated CCS from the three different IMS instruments proved to be well correlated and highly reproducible (R2 > 0.995 and mean RSD ≤ 1%). More than 90% of the lipid CCS had deviations of less than 1%, demonstrating high comparability between techniques, and the possibility to use the CCS as molecular descriptor. We highlighted the need of standardized procedures for calibration, data acquisition, and data processing. This work demonstrates that using harmonized analytical conditions are required for interplatform reproducibility for CCS determination of human plasma lipids.

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. To avoid this problem, it is possible to dilute the first dimension (1D) effluent with (zdilution -1) volumes of a weaker solvent added to one volume of 1D-effluent, where zdilution represents the extent to which the fraction volume has been multiplied. This can be done using either active solvent modulation technology or an additional pump, prior to the second dimension analysis. The objective of this study was to develop theoretical models to predict whether or not dilution can be effective, and, if so, what is the minimum zdilution value required. This approach is based on the calculation of the ratio (called xdilution) between the peak standard deviation due to the injection process and the peak standard deviation in the absence of extra-column dispersion. xdilution was calculated from theoretical relationships and plotted as a function of zdilution, to predict the value required to obtain good peak shapes for the compound of interest. The maximum xdilution value was found to be of the order of 1 for chromatographically acceptable peak shapes. The proposed theoretical approach was experimentally validated on a number of representative small molecules and peptides. Agreement between experimental results and theoretical models was very high, especially for small molecules. Finally, it is shown that this approach helps to predict the most appropriate set of conditions in HILIC x RPLC, depending on the compounds to be separated.

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. It appears that the achievable selectivity can be ranked in the order RPLC > revHILIC > HILIC with the two different samples. Next, two-dimensional liquid chromatography (2D-LC) conditions were evaluated by combining RPLC, revHILIC, or HILIC with RPLC in an on-line comprehensive (LC × LC) mode. evHILIC × RPLC not only showed impressive performance in terms of peak capacity and sensitivity, but also provided complementary selectivity compared to RPLC × RPLC and HILIC × RPLC. Indeed, both the elution order and the retention time range differ significantly between the three techniques. In conclusion, there is no doubt that revHILIC should be considered as a viable option for 2D-LC analysis of small molecules and also peptides.

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. The characterization of bio-oils is difficult because they are very complex mixtures with thousands of compounds covering a very wide range of molecular weight and polarity. Due to the high degree of orthogonality between the two chromatographic dimensions, the on-line combination of reversed-phase liquid chromatography and supercritical fluid chromatography (on-line RPLC x SFC) can significantly improve the characterization of such complex matrices. The hyphenation was optimized by selecting, in SFC, the stationary phase, the co-solvent, the make-up solvent prior to high resolution mass spectrometry (HRMS) and the injection solvent. Additionally, a new interface configuration is described. Quality descriptors such as the occupation of the separation space, the peak shapes and the signal intensity were considered to determine the optimal conditions. The best results were obtained with bare silica, a co-solvent composed of acetonitrile and methanol (50/50, v/v), a make-up solvent composed of methanol (90%) and water (10%) with formic acid (0.1%), an addition of co-solvent through an additional pump for SFC separation in a 2.1 mm column, and an hydro-organic solvent as injection solvent. The optimized setup was used to analyze two microalgae bio-oils: the full bio-oil coming from hydrothermal liquefaction and Soxhlet extraction of microalgae, and the gasoline cut obtained after distillation of the full bio-oil. Results in on-line RPLC x SFC-qTOF were particularly interesting, with very good peak shapes and high reproducibility. Moreover, the high degree of orthogonality for microalgae bio-oils of RPLC and SFC was highlighted by the very large occupation of the separation space. Isomeric profiles of compound families could be obtained in RPLC x SFC-qTOF and many isomers not separated in SFC alone were separated in RPLC and vice versa, thus showing the complementarity of the two chromatographic techniques.

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. Whatever the analysis time between 30 and 200 min, short columns of only 30 × 2.1 mm packed with sub-2-µm particles should be selected in both dimensions of the 2D-LC setup to obtain the best compromise in terms of peak capacity and sensitivity. The peak capacity in RPLC × RPLC vs. RPLC was significantly improved for analysis times beyond 5 min. However, extra-column volume located after the second-dimension column was found to be particularly critical for peptides, and up to 50% lower peak capacity was observed with MS vs. UV detection. Contrary to common belief, higher dilution is not always observed in RPLC × RPLC. With adequate analytical conditions, better sensitivity (in theory fivefold and in practice three- to fivefold) could be achieved in RPLC × RPLC compared to 1D-RPLC, regardless of the analysis time.

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 (2D) 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 2D-fraction. In this study, an online RPLC × RPLC method is optimized with shifting gradients in 2D. The logic behind implementing such programs in on-line LC × LC is explained. The optimized method consists of a combination of segmented and shifted gradient modes. It is shown that the retention space coverage can be increased by 50 % compared to a conventional full gradient program, leading to a significant increase in peak capacity (about 35 %). However, such an increase comes at the expense of larger peak widths in 2D and thus lower peak intensities. It is shown here that the use of shifting gradients raises another serious issue related to the repeatability of retention times between two successive 2D-separations.

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 k0 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. However, two important constraints have to be accounted for to maintain good predictions with this new approach: i) the retention factor at the initial composition of the preliminary gradient series have to be large enough (i.e., log ki above 2.1) and ii) the retention models have to be sufficiently linear. While these two conditions are not always met with small molecules or even peptides, the situation is different with large biomolecules. This is why our simple calculation method should be preferentially applied to calculate the linear solvent strength parameters of protein samples.

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. In this context, the use of an on-line 2D-LC-MS approach not only allows on-line desalting and indirect coupling of CEX with mass spectrometry (MS) detection but it also provides increased and complementary information within a single analysis. The first part of this study was devoted to the choice of stationary phases and the optimization of chromatographic conditions in both dimensions. Based on the results obtained in 1D-CEX with ultraviolet detection (UV) and 1D-RPLC with UV and high-resolution mass spectrometry (HRMS) detections, an on-line comprehensive two-dimensional liquid chromatography method combining CEX and RPLC was developed. The last part of this study was devoted to the identification of the separated species using HRMS detection and in the comparison of three ADC samples exposed to different durations of thermal stress.

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. The online transfer of a given solvent from the first dimension to the second dimension may cause severe injection effects in the second dimension, mostly due to solvent strength mismatch. Those injection effects may include peak broadening, peak distortion, peak splitting or breakthrough phenomenon. They are often found to reduce significantly the peak capacity and the peak intensity. To overcome such effects, arising specifically in online comprehensive two-dimensional liquid chromatography, different methods have been developed over the years. In this review, we focused on the most recently reported ones. A critical discussion, supported by a theoretical approach, gives an overview of their advantages and drawbacks.

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. Reversed phase liquid chromatography (RPLC) was used in both dimensions, with a penta-fluoro-phenyl silica-based column at neutral pH in the first dimension (1D) and a C18 column at acidic pH in the second one (2D). The conditions were optimized for a total analysis time of 60 min. The variability of both retention times and peak areas was evaluated. The average standard deviation on retention times was found to be less than 0.1 s in 2D. The relative standard deviation on peak area was about 7% for run-to-run analysis. This analytical approach, applied to the pharmaceutical effluents before (inlet) and after (outlet) wastewater treatment permitted to detect 240 compounds. These included 27 priority pharmaceutical products, 8 of which were of very high priority and their concentrations could be compared to target values. The comparison of 2D-LC and 1D-LC approaches clearly highlights the power of on-line RPLC x RPLC technique, which allows both targeted quantitative analysis and non-targeted qualitative analysis of pharmaceutical effluents.

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. Usually, during breakthrough, the retained peak displays a wide array of deformations and it is not rare that multiple retained peaks appear for a given injected analyte. However, under certain conditions, it has been demonstrated that these deleterious effects could fully disappear, leaving only one breakthrough peak and one symmetrical peak on the chromatogram. This so-called "total breakthrough" phenomenon was recently highlighted in the specific context of the 2D-LC separation of peptides but has yet to be explained. In the present paper, we describe the results of a comprehensive study aiming to better understand and define the conditions of emergence of both breakthrough and total breakthrough phenomena in liquid chromatography. The effects of a broad range of parameters, including the nature of the solute, the retention mechanism, the injection and elution conditions, the column temperature, and the injected sample concentration on the occurrence of both phenomena were investigated. While breakthrough was found to occur for all studied compounds, it appears that the presence of positive charges on the molecule is a prerequisite for observing a total breakthrough phenomenon. Among all the parameters investigated in this work, only the injection conditions and the analyte retention were found to be impactful on the onset of both phenomena. This finding allowed us to suggest one necessary and sufficient condition, relying on the injection of critical volumes to observe each respective phenomenon. These critical volumes only depend on the column dead volume and the retention factor of the analyte in the injection solvent.

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. Specifically, three instrument types, namely Agilent®, Shimadzu®, and Waters®, were included through 21 laboratories (n = 7 for each instrument). First, method transfer was performed to assess the separation quality and to set up the specific instrument parameters of Agilent® and Shimadzu® instruments. Second, the inter-laboratory study was performed following a protocol defined by the sending lab. Analytical results were examined regarding consistencies within- and between-laboratories criteria. Afterwards, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. Reproducibility variance was larger than that observed during the first study involving only one single type of instrumentation. Indeed, we clearly observed an 'instrument type' effect. Moreover, the reproducibility variance was larger when considering all instruments than each type separately which can be attributed to the variability induced by the instrument configuration. Nevertheless, repeatability and reproducibility variances were found to be similar than those described for LC methods; i.e. reproducibility as %RSD was around 15 %. These results highlighted the robustness and the power of modern analytical SFC technologies to deliver accurate results for pharmaceutical quality control analysis.

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. An efficient analytical method was needed to characterize these neutral positional isomers and identify the mixtures to reject. Two samples were provided by the pharmaceutical company: Sample A was conform, Sample B was not. 8 RPLC columns were used with different gradients to screen Sample A. Next, the best RPLC separation was used as the second dimension fast analysis in a comprehensive 2D-RPLC systems. Two columns were used as first dimension: a fluorinated one and a zirconium based one. An order of magnitude was gained in peak capacity, but a better sample characterization was still needed. An off-line RPLC x SFC x Q-TOF/MS analysis was performed collecting 96 RPLC fractions and analyzing them by SFC with Q-TOF/MS detection. A home-made software associated the 96 SFC MS chromatograms to produce either base peak (BPC) or extract ion (EIC) contour plots that allowed for a satisfying characterization of the samples. Subtracting the EIC of expected m/z compounds from the Sample B BPC contour plot produced a unique new contour plot clearly pointing out unexpected compounds explaining the failure of the synthesis and possibly allowing improving the synthesis process.

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 (2D) 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. The effect of the volume of the column, the volume and the shape of the sample band, the retention properties of the analyte and the band broadening experienced by the analytes and the sample solvent are investigated. A good agreement between the simulations and the experimental observations with caffeine and methylparaben injected in acetonitrile/water (ACN/H2O) mobile phase with different injection volumes is obtained. Save the difference in dwell volume, key features of experimental and simulated chromatograms agree within a few %. The simulations are also validated against a number of simple mathematical rules of thumb that can be established to predict the occurrence of a breakthrough fraction and estimate the amount of breakthrough.

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. In the present contribution, we compared four strategies designed to avoid this problem: (1) flow splitting, which consists in reducing the injection volume in the second dimension (2D), (2) on-line dilution with a make-up flow and (3) on-line dilution with Active Solvent Modulation (ASM), which both consist in reducing the solvent strength of the injected fractions, and (4) Total Breakthrough Strategy, which we recently proposed. Unlike the three preceding strategies, this latter one consists in injecting large volumes of strong solvent in 2D. The performance of each strategy was evaluated for sub-hour separations of a tryptic digest in on-line HILIC x RPLC. In this work, we considered the critical case for which the same column internal diameters (i.e. 2.1 mm here) are used in both dimensions. Peak capacity, peak shapes and peak intensities were considered for this evaluation. The highest peak capacity could be achieved with Total Breakthrough Strategy while the lowest one with on-line dilution using ASM. Peak intensities were usually higher with on-line dilution approaches (make-up flow and ASM). However, despite the presence of breakthrough, peak intensities were approximately 7-fold higher with Total Breakthrough Strategy than with flow splitting.

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. The model quality was assessed and was altered neither by the columns from different lots, nor by the complex matrices of environmental water samples. Then, the solute retention of any organic compound present in water samples is expected to be predicted within ± 14.3% acetonitrile by our model. Solute retention can therefore be used as a supplementary tool for the identification of environmental contaminants by UHPLC-HRMS, in addition to mass spectrometry data already used in the suspect screening approach.

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. In search for a high degree of orthogonality, two separation techniques involving two different retention mechanisms were evaluated: Size Exclusion Chromatography (SEC) and Reverse Phase Liquid Chromatography (RPLC). In SEC, the analytes are separated according to their molecular weight while according to their hydrophobicity in RPLC. The separation power of both individual separation techniques was first evaluated. Off-line and on-line LCxLC were compared on the basis of an optimization approach. It is shown that off-line SECxRPLC can provide, for the same analysis time of 150 min, a higher peak capacity (2600 vs 1700) than on-line RPLCxSEC while a similar dilution factor (close to 30) but also requires far fewer fractions to be analyzed (12 vs 400). Asphaltenes which constitute the heaviest fraction of crude oils (obtained from petroleum industry) were analyzed by the developed off-line SECxRPLC method. The resulting 2D-contour plots show that co-elutions could be removed leading, for the first time, to new information on high molecular weight species containing sulfur and vanadium.

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. Our focus was directed towards the reduction of band broadening and peak distortion. The conditions of the emergence of breakthrough could be investigated with high resolution mass spectrometry (HRMS) detection. The importance of both the injection volume and the difference in composition between injection and elution solvents was highlighted. Reported strategies to avoid bad peak shapes mostly rely either on flow splitting to limit the injection volume, or on on-line dilution. Here, we propose an alternative approach which consists in injecting large volumes in the second dimension. In this case, no flow-splitting nor dilution prior to the second dimension is required. Our results show that above a certain critical injected volume, depending on both the compound and the elution conditions, narrow and symmetrical peaks can be obtained, despite the persistence of breakthrough. As a result, the injected volume in the second dimension must be larger than the largest critical volume. This counter-intuitive approach was applied for the on-line HILIC x RPLC-UV-HRMS analysis of a complex tryptic digest sample. A peak capacity close to 1500 could be achieved in 30 min, which is two-fold higher than in RPLC x RPLC within the same analysis time.

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. Compared to conventional one-dimensional liquid chromatography techniques (1D-LC), LC × LC combines two different and complementary separation systems. In the context of ADC analysis, LC × LC has been proven to be a rapid and efficient analytical tool: (1) to provide a higher resolving power by increasing the overall peak capacity and thus allowing to gain more information within a single run and (2) to allow mass spectrometry (MS) coupling with some chromatographic techniques that are not MS-compatible and hence to facilitate the structural elucidation of ADCs. In this chapter, we present the coupling of different chromatographic techniques including hydrophobic interaction chromatography (HIC), reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC), ion exchange chromatography (IEX), and hydrophilic liquid chromatography (HILIC). The interest of HIC × SEC, SEC × SEC, HIC × RPLC, IEX × RPLC, RPLC × RPLC, and HILIC × RPLC, all hyphenated to high-resolution mass spectrometry (HRMS), is discussed in the context of the characterization of ADCs.

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. Both methods allow also the measurement of average DAR for both monomeric and multimeric species. In addition, the Fc fragments can be analyzed in the same run, providing a complete glycoprofile and the demonstration or absence of additional conjugation of this subdomain involved in FcRn and Fc-gammaR binding.