▸ The ESI process is deeply explained in the context of CE. ▸ The electrochemical processes involved in CE–ESI-MS are presented. ▸ CE–ESI-MS interfaces are classified based on their operating flow ...rates. ▸ Electrospray and nanospray interfaces and their applications are overviewed.
Capillary electrophoresis (CE) hyphenated to electrospray ionization (ESI) mass spectrometry (MS) is a powerful tool for analyzing a wide variety of analytes in different matrices. The major issue with CE–ESI-MS lies in finding a suitable and versatile interface to ensure the best CE and ESI operations. Thus, the development and improvement of CE–ESI-MS interfaces have been the subjects of much research. The first part of the present review focuses on the fundamental aspects of the three steps of the ESI process, i.e., spray formation, droplet evolution, and the production of gas-phase ions. In the second part of the review, the electrochemical reactions involved in the ESI and CE processes and their influences on the sensitivity and performance are discussed in detail. Then, the existing interfaces are divided into two major classes according to their operating flow rate (electrospray vs. nanospray regime). The particular characteristics of these two regimes are discussed by considering their practical impacts on ionization and the MS response. Finally, the current CE–ESI-MS interfaces are summarized, including their major advantages, drawbacks, and fields of application.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
•A CE-MS two-step analytical strategy was implemented in bioanalysis.•CE-ESI-TOF/MS was implemented for the screening step with on-line preconcentration.•CE-ESI-MS/MS with QqQ was used for ...quantitation and was validated for COC and MTD.•QqQ was equipped with a new sprayer and a new ESI source.•The two-step workflow was applied to an analysis of toxicological cases.
The combination of capillary electrophoresis (CE) and mass spectrometry (MS) is particularly well adapted to bioanalysis due to its high separation efficiency, selectivity, and sensitivity; its short analytical time; and its low solvent and sample consumption. For clinical and forensic toxicology, a two-step analysis is usually performed: first, a screening step for compound identification, and second, confirmation and/or accurate quantitation in cases of presumed positive results. In this study, a fast and sensitive CE-MS workflow was developed for the screening and quantitation of drugs of abuse in urine samples. A CE with a time-of-flight MS (CE-TOF/MS) screening method was developed using a simple urine dilution and on-line sample preconcentration with pH-mediated stacking. The sample stacking allowed for a high loading capacity (20.5% of the capillary length), leading to limits of detection as low as 2ngmL−1 for drugs of abuse. Compound quantitation of positive samples was performed by CE-MS/MS with a triple quadrupole MS equipped with an adapted triple-tube sprayer and an electrospray ionization (ESI) source. The CE-ESI-MS/MS method was validated for two model compounds, cocaine (COC) and methadone (MTD), according to the Guidance of the Food and Drug Administration. The quantitative performance was evaluated for selectivity, response function, the lower limit of quantitation, trueness, precision, and accuracy. COC and MTD detection in urine samples was determined to be accurate over the range of 10–1000ngmL−1 and 21–1000ngmL−1, respectively.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
► Monoclonal antibodies (mAbs) are essential drugs for treatment of various diseases. ► The heterogeneity of mAbs is significant and should be well characterized. ► Reversed-phase liquid ...chromatography is a powerful strategy for mAbs analysis. ► Capillary-zone electrophoresis is a good option, because of its MS compatibility. ► Adsorption of mAbs is a major concern with both electrophoresis and chromatography.
Recombinant monoclonal antibodies (mAbs) have become particularly relevant for the treatment of autoimmune diseases or cancers. Because of their inherent complexity and for safety reasons, there is a need to develop powerful analytical methods to provide detailed characterizations of mAbs.
The aim of the present review is to detail the state-of-the-art of analytical strategies for mAb characterization. It focuses on the most important separation techniques used in this field, specifically, the chromatographic and electrophoretic approaches and their combination with mass spectrometry (MS). Thanks to recent improvements in separation science and MS devices, mAbs can be analyzed more easily. However, there is still a need to find new approaches that avoid adsorption issues.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
In recent years, a growing number of biopharmaceutical proteins have been produced and are already available, or will be soon available, in the market. These molecules are more complex to analyze ...than conventional low molecular weight drugs, and thus need powerful analytical approaches for the entire development and delivery process. This review summarizes the analytical techniques available for intact protein determination and the main development steps in which they are applicable. A strong emphasis has been put on separation techniques, liquid chromatography and electrophoretic techniques, but mass spectrometry and spectroscopic approaches are also mentioned. Overall, we highlight how several analytical strategies are necessary to obtain global information.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Among the various biological matrices used in metabolomics, urine is a biofluid of major interest because of its non-invasive collection and its availability in large quantities. However, significant ...sources of variability in urine metabolomics based on UHPLC-MS are related to the analytical drift and variation of the sample concentration, thus requiring normalization. A sequential normalization strategy was developed to remove these detrimental effects, including: (i) pre-acquisition sample normalization by individual dilution factors to narrow the concentration range and to standardize the analytical conditions, (ii) post-acquisition data normalization by quality control–based robust LOESS signal correction (QC-RLSC) to correct for potential analytical drift, and (iii) post-acquisition data normalization by MS total useful signal (MSTUS) or probabilistic quotient normalization (PQN) to prevent the impact of concentration variability. This generic strategy was performed with urine samples from healthy individuals and was further implemented in the context of a clinical study to detect alterations in urine metabolomic profiles due to kidney failure. In the case of kidney failure, the relation between creatinine/osmolality and the sample concentration is modified, and relying only on these measurements for normalization could be highly detrimental. The sequential normalization strategy was demonstrated to significantly improve patient stratification by decreasing the unwanted variability and thus enhancing data quality.
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•Sequential normalization strategy for urine metabolomics by UHPLC-QTOF-MS is proposed.•Pre-acquisition sample normalization enhanced the analytical conditions.•Post-acquisition data normalization corrected the unwanted variance in the dataset.•Sequential normalization significantly improved kidney failure patient stratification.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Sialylation is a glycosylation feature that occurs in different linkages at the non-reducing end of a glycan moiety, the linkage isomers are often differentially associated with various biological ...processes. Due to very similar physico-chemical properties, the separation of isomeric sialylated glycopeptides remains challenging but of utmost importance in the biomedicine and biotechnology, including biomarker discovery, glyco-engineering and biopharmaceutical characterization. This study presents the implementation of a high-resolution separation platform based on capillary electrophoresis - mass spectrometry (CE-MS) allowing for the selective analysis of α2,3- and α2,6-sialylated glycopeptides. These differentially linked glycopeptides showed an identical fragmentation pattern (collision induced dissociation) but different electrophoretic mobilities, allowing for baseline separation of the different linkages without the need for an extensive sample preparation. The different migration behavior between the two moieties was found to correlate with differences in pK
values. Using a novel methodology adapted from the so-called internal standard CE approach, a relative difference of 3.4·10
in pK
unit was determined. This approach was applied for the analysis of tryptic glycopeptides of prostate specific antigen, which shows highly complex and heterogeneous glycosylation. The developed platform therefore appears attractive for the identification of differentially linked sialic acids that may be related to pathological conditions.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Procedures for estimating the measurement uncertainty (MU) of the concentration of a given analyte in a sample are of major concern for analytical chemists. Unfortunately, it is still unclear how and ...why MU should be assessed. While several possibilities exist, an appropriate approach consists in using method validation data for the evaluation of MU. This was demonstrated by a validation study achieved in the framework of a clinical study related to caffeine in sports medicine, where the results were used for the evaluation of MU. After validation of the method developed using ultra-high pressure liquid chromatography–mass spectrometry for caffeine and its three main metabolites, accuracy profiles were built for each analyte. The first important conclusion is that the developed method was valid for all compounds and met the given specifications for the application (fit for purpose). Relevant estimates of combined standard uncertainty were computed to obtain uncertainty functions, which allow obtaining values of MU as a function of the concentration of the analyte. The great advantage of both uncertainty function and uncertainty profile is the development of a continuous model that enables easy calculation of the standard, expanded and relative expanded uncertainty at any concentration within the validation domain. In fact, the expanded uncertainty interval is assumed to contain 95% of all possible measurements, regardless of the concentration. Finally, the uncertainty function enables the determination of the lowest limit of quantification by selecting adequate acceptance limits, with the limit of quantification being defined as the point where the relative uncertainty equals the acceptance limit threshold. It has to be noted that further discussions remain mandatory to establish which criteria should be applied to define an adequate decision threshold, and the proposal afforded in this work may open new avenues in this direction.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
This review discusses the fundamental principles of TOF analyzers and covers the great progress that has been made in this area in recent years (i.e. orthogonal acceleration, reflectron). This paper ...also gives an overview of applications performed by CE coupled to TOF/MS detection. The main domains of interest include the analysis of biomolecules and natural compounds.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
This paper describes an efficient CE-UV-ESI-TOF/MS method for the determination and quantitation of intact insulin (INS) in a pharmaceutical formulation. The CE conditions were optimized to avoid the ...adsorption of proteins onto the capillary wall. Particular attention was paid regarding the choice of the internal standard (IS). A strategy based on multiple injections was selected and the methodology was validated according to international guidelines. The optimized method was applied with success to the analysis of INS formulations obtained from regular and parallel markets.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Drug–plasma protein interactions have a significant impact on both pharmacokinetics (i.e., absorption, distribution, metabolism, and excretion) and pharmacodynamics (pharmacological effects). ...Therefore, it is of high interest to evaluate this binding during the drug development process. Capillary electrophoresis (CE) is an interesting analytical tool for drug–protein binding characterization because it consumes a relatively low amount of reagents and enables assays that can be carried out under near-physiological conditions. The most interesting mode of CE for the study of biomolecular interactions is CE/frontal analysis (CE/FA). However, some confusion in how to conduct CE/FA experiments has emerged in the literature. The present study examines, using research into drug–albumin interactions as an example, the most important steps to take into consideration when building up new CE/FA binding assays. These include the following: choosing the buffer and applied voltage; evaluating protein adsorption onto the capillary wall; choosing the injection volume; choosing the drug and protein concentrations; and, finally, verifying the co-migration of the protein and drug–protein complex. The experimental part of the present report can serve as a checklist for developing the key parameters that need to be addressed for successful and reliable interaction studies. In a second time, short-end injection was used to enhance throughput. The strengths of the binding constants (
K
a) for nine selected drugs (basic, neutral, and acidic substances) to albumin, which is the most important plasma protein, were from log
K
a 2.9 to 5.4. These values were compared to those obtained with validated methods and good agreement was achieved.
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