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Capillary zone electrophoresis (CZE) is a powerful tool that is progressively being applied for the separation of monoclonal antibody (mAb) charge variants. Mass spectrometry (MS) is the desired detection method concerning identification of mAb variants. In biopharmaceutical applications, there exist optimized and validated electrolyte systems for mAb variant quantification. However, these electrolytes interfere greatly with the electrospray ionization (ESI) process. Here, a heart-cut CZE–CZE–MS setup with an implemented mechanical four-port valve interface was developed that used a generic ε-aminocaproic acid based background electrolyte in the first dimension and acetic acid in the second dimension. Interference-free, highly precise mass data (deviation less than 1 Da) of charge variants of trastuzumab, acting as model mAb system, were achieved. The mass accuracy obtained (low parts per million range) is discussed regarding both measured and calculated masses. Deamidation was detected for the intact model antibody, and related mass differences were significantly confirmed on the deglycosylated level. The CZE–CZE–MS setup is expected to be applicable to a variety of antibodies and electrolyte systems. Thus, it has the potential to become a compelling tool for MS characterization of antibody variants separated in ESI-interfering electrolytes. Graphical Abstract Two-dimensional capillary zone electrophoresis mass spectrometry for the characterization of intact monoclonal antibody (mAb) charge variants. A generic, but highly electrospray-interfering electrolyte system was used as first dimension for mAb charge variant separation and coupled to a volatile electrolyte system as second dimension via a four-port nanoliter valve. In this way, interference-free and precise mass spectrometric data of separated mAb charge variants, including deamidation products, were obtained

PCR-ribotyping has been adopted in many laboratories as the method of choice for C. difficile typing and surveillance. However, issues with the conventional agarose gel-based technique, including inter-laboratory variation and interpretation of banding patterns have impeded progress. The method has recently been adapted to incorporate high-resolution capillary gel-based electrophoresis (CE-ribotyping), so improving discrimination, accuracy and reproducibility. However, reports to date have all represented single-centre studies and inter-laboratory variability has not been formally measured or assessed. Here, we achieved in a multi-centre setting a high level of reproducibility, accuracy and portability associated with a consensus CE-ribotyping protocol. Local databases were built at four participating laboratories using a distributed set of 70 known PCR-ribotypes. A panel of 50 isolates and 60 electronic profiles (blinded and randomized) were distributed to each testing centre for PCR-ribotype identification based on local databases generated using the standard set of 70 PCR-ribotypes, and the performance of the consensus protocol assessed. A maximum standard deviation of only ±3.8bp was recorded in individual fragment sizes, and PCR-ribotypes from 98.2% of anonymised strains were successfully discriminated across four ribotyping centres spanning Europe and North America (98.8% after analysing discrepancies). Consensus CE-ribotyping increases comparability of typing data between centres and thereby facilitates the rapid and accurate transfer of standardized typing data to support future national and international C. difficile surveillance programs.

A standardized data workflow is described for large-scale serum metabolomic studies using multisegment injection-capillary electrophoresis-mass spectrometry. Multiplexed separations increase throughput (<4 min/sample) for quantitative determination of 66 polar/ionic metabolites in serum filtrates consistently detected (coefficient of variance (CV) <30%) with high frequency (>75%) from a multi-ethnic cohort of pregnant women ( n = 1,004). We outline a validated protocol implemented in four batches over a 7-month period that includes details on preventive maintenance, sample workup, data preprocessing and metabolite authentication. We achieve stringent quality control (QC) and robust batch correction of long-term signal drift with good mutual agreement for a wide range of metabolites, including serum glucose as compared to a clinical chemistry analyzer (mean bias = 11%, n = 668). Control charts for a recovery standard (mean CV = 12%, n = 2,412) and serum metabolites in QC samples (median CV = 13%, n = 202) demonstrate acceptable intermediate precision with a median intraclass coefficient of 0.87. We also report reference intervals for 53 serum metabolites from a diverse population of women in their second trimester of pregnancy. A standardized protocol and data workflow for high-throughput analysis of the maternal serum metabolome is outlined. It uses multisegment injection–capillary electrophoresis–mass spectrometry and is applied to a multi-ethnic cohort of pregnant women.

Recent years showed a boost in knowledge about the presence and fate of micropollutants in the environment. Instrumental and methodological developments mainly in liquid chromatography coupled to mass spectrometry hold a large share in this success story. These techniques soon complemented gas chromatography and enabled the analysis of more polar compounds including pesticides but also household chemicals, food additives, and pharmaceuticals often present as traces in surface waters. In parallel, sample preparation techniques evolved to extract and enrich these compounds from biota and water samples. This review article looks at very polar and ionic compounds using the criterion log P ≤ 1. Considering about 240 compounds, we show that (simulated) log D values are often even lower than the corresponding log P values due to ionization of the compounds at our reference pH of 7.4. High polarity and charge are still challenging characteristics in the analysis of micropollutants and these compounds are hardly covered in current monitoring strategies of water samples. The situation is even more challenging in biota analysis given the large number of matrix constituents with similar properties. Currently, a large number of sample preparation and separation approaches are developed to meet the challenges of the analysis of very polar and ionic compounds. In addition to reviewing them, we discuss some trends: for sample preparation, preconcentration and purification efforts by SPE will continue, possibly using upcoming mixed-mode stationary phases and mixed beds in order to increase comprehensiveness in monitoring applications. For biota analysis, miniaturization and parallelization are aspects of future research. For ionic or ionizable compounds, we see electromembrane extraction as a method of choice with a high potential to increase throughput by automation. For separation, predominantly coupled to mass spectrometry, hydrophilic interaction liquid chromatography applications will increase as the polarity range ideally complements reversed phase liquid chromatography, and instrumentation and expertise are available in most laboratories. Two-dimensional applications have not yet reached maturity in liquid-phase separations to be applied in higher throughput. Possibly, the development and commercial availability of mixed-mode stationary phases make 2D applications obsolete in semi-targeted applications. An interesting alternative will enter routine analysis soon: supercritical fluid chromatography demonstrated an impressive analyte coverage but also the possibility to tailor selectivity for targeted approaches. For ionic and ionizable micropollutants, ion chromatography and capillary electrophoresis are amenable but may be used only for specialized applications such as the analysis of halogenated acids when aspects like desalting and preconcentration are solved and the key advantages are fully elaborated by further research.

The analysis of myo -inositol phosphates (InsPs) and myo -inositol pyrophosphates (PP-InsPs) is a daunting challenge due to the large number of possible isomers, the absence of a chromophore, the high charge density, the low abundance, and the instability of the esters and anhydrides. Given their importance in biology, an analytical approach to follow and understand this complex signaling hub is desirable. Here, capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESI-MS) is implemented to analyze complex mixtures of InsPs and PP-InsPs with high sensitivity. Stable isotope labeled (SIL) internal standards allow for matrix-independent quantitative assignment. The method is validated in wild-type and knockout mammalian cell lines and in model organisms. SIL-CE-ESI-MS enables the accurate monitoring of InsPs and PP-InsPs arising from compartmentalized cellular synthesis pathways, by feeding cells with either [ 13 C 6 ]- myo -inositol or [ 13 C 6 ]- D -glucose. In doing so, we provide evidence for the existence of unknown inositol synthesis pathways in mammals, highlighting the potential of this method to dissect inositol phosphate metabolism and signalling. Myo -Inositol phosphates (InsPs) and pyrophosphates (PP-InsPs) are important second messengers but their analysis remains challenging. Here, the authors develop a capillary electrophoresis-mass spectrometry method for the identification and quantitation of InsP and PP-InsP isomers in cells and tissues.

The NISTmAb is a monoclonal antibody Reference Material from the National Institute of Standards and Technology; it is a class-representative IgG1κ intended serve as a pre-competitive platform for harmonization and technology development in the biopharmaceutical industry. The publication series of which this paper is a part describes NIST’s overall control strategy to ensure NISTmAb quality and availability over its lifecycle. In this paper, the development and qualification of methods for monitoring NISTmAb charge heterogeneity are described. Capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF) assays were optimized and evaluated as candidate assays for NISTmAb quality control. CIEF was found to be suitable as a structural characterization assay yielding information on the apparent pI of the NISTmAb. CZE was found to be better suited for routine monitoring of NISTmAb charge heterogeneity and was qualified for this purpose. This paper is intended to provide relevant details of NIST’s charge heterogeneity control strategy to facilitate implementation of the NISTmAb as a test molecule in the end user’s laboratory.Graphical AbstractRepresentative capillary zone electropherogram of the NIST monoclonal antibody (NISTmAb). The NISTmAb is a publicly available research tool intended to facilitate advancement of biopharmaceutical analytics.

Both capillary electrophoresis (CE) and atomic absorption spectrometry (AAS) are analytical techniques of high performance given their efficient separation and their sensitive detection, respectively. They are predominantly adduced as standalone techniques, also widely used in pharmaceutical sciences. However, with CE, only indirect detection or direct detection of metal complexes using additives to the background electrolyte is feasible, while AAS is not capable of separating different compounds of the same metal. Combining these two techniques would compensate for these limitations by complementing each other. Surprisingly, the hyphenation of CE and AAS represents rather a rare liaison. Therefore, the aim of the current perspective was to shed light on examples where the hybrid technique of CE-AAS was successfully applied thus far. In this context, particular attention was paid to the extent to which the studies and the analytes under investigation might also be relevant from a pharmaceutical point of view. It revealed that the combination of CE and AAS has great potential for research in pharmaceutical sciences. With respect to online hyphenation, this would be even better if interfaces were more widely available. Nevertheless, this overview demonstrates that CE-AAS is a valuable combination. Its potential just needs to be exploited.

A biomolecular coating, or biocorona, forms on the surface of engineered nanomaterials (ENMs) immediately as they enter biological or environmental systems, defining their biological and environmental identity and influencing their fate and performance. This biomolecular layer includes proteins (the protein corona) and other biomolecules, such as nucleic acids and metabolites. To ensure a meaningful and reproducible analysis of the ENMs-associated biocorona, it is essential to streamline procedures for its preparation, separation, identification and characterization, so that studies in different labs can be easily compared, and the information collected can be used to predict the composition, dynamics and properties of biocoronas acquired by other ENMs. Most studies focus on the protein corona as proteins are easier to monitor and characterize than other biomolecules and play crucial roles in receptor engagement and signaling; however, metabolites play equally critical roles in signaling. Here we describe how to reproducibly prepare and characterize biomolecule-coated ENMs, noting especially the steps that need optimization for different types of ENMs. The structure and composition of the biocoronas are characterized using general methods (transmission electron microscopy, dynamic light scattering, capillary electrophoresis–mass spectrometry and liquid chromatography–mass spectrometry) as well as advanced techniques, such as transmission electron cryomicroscopy, synchrotron-based X-ray absorption near edge structure and circular dichroism. We also discuss how to use molecular dynamic simulation to study and predict the interaction between ENMs and biomolecules and the resulting biocorona composition. The application of this protocol can provide mechanistic insights into the formation, composition and evolution of the ENM biocorona, ultimately facilitating the biomedical and agricultural application of ENMs and a better understanding of their impact in the environment. Key points The authors provide a detailed workflow for the isolation and biophysical characterization of biomolecule corona (biocorona) components (proteins and metabolites) through mass spectrometry, advanced structural techniques (for example, transmission electron cryomicroscopy and synchrotron-based X-ray absorption near edge structure) and molecular dynamic simulations to model ENM–biocorona interactions. The designed pipeline normalizes the acquisition of data in different labs, increases their reproducibility, and facilitates their use for the prediction of the biocoronas acquired by less characterized ENMs. Engineered and anthropogenic nanoscale materials in the environment acquire a coating of biomolecules (biocorona) that modulates their properties, uptake and biodistribution. This protocol streamlines biocorona analysis to support the development of safe and sustainable nanotechnology.

The characteristic alkaloid component of the leaves of the catnip shrub (Catha edulis) is cathinone, and its synthetic analogs form a major group of recreational drugs. Cathinone derivatives are chiral compounds. In the literature, several chiral methods using cyclodextrins (CDs) have been achieved so far for diverse sets of analogs; however, a comprehensive investigation of the stability of their CD complexes has not been performed yet. To characterize the enantioselective complex formation, a systematic experimental design was developed in which a total number of 40 neutral, positively, and negatively charged CD derivatives were screened by affinity capillary electrophoresis and compared according to their cavity size, substituent type, and location. The functional groups responsible for the favorable interactions were identified in the case of para-substituted cathinone analog mephedrone, flephedrone, and 4-methylethcathinone (4-MEC) and in the case of 3,4-methylendioxy derivative butylone and methylenedioxypyrovalerone (MDPV). The succinylated-β-CD and subetadex exhibited the highest complex stabilities among the studied drugs. The complex stoichiometry was determined using the Job’s plot method, and the complex structures were further studied using ROESY NMR measurements. The results of our enantioselective complex formation study can facilitate chiral method development and may lead to evaluate potential CD-based antidotes for cathinone analogs.

Capillary electrophoresis (CE) is a potent method for analyzing chiral substances and is commonly used in the enantioseparation and chiral purity control of pharmaceuticals from different matrices. The adoption of Quality by Design (QbD) concepts in analytical method development, optimization and validation is a widespread trend observed in various analytical approaches including chiral CE. The application of Analytical QbD (AQbD) leads to the development of analytical methods based on sound science combined with risk management, and to a well understood process clarifying the influence of method parameters on the analytical output. The Design of Experiments (DoE) method employing chemometric tools is an essential part of QbD-based method development, allowing for the simultaneous evaluation of experimental parameters as well as their interaction. In 2022 the International Council for Harmonization (ICH) released two draft guidelines (ICH Q14 and ICH Q2(R2)) that are intended to encourage more robust analytical procedures. The ICH Q14 guideline intends to harmonize the scientific approaches for analytical procedures’ development, while the Q2(R2) document covers the validation principles for the use of analytical procedures including the recent applications that require multivariate statistical analyses. The aim of this review is to provide an overview of the new prospects for chiral CE method development applied for the enantiomeric purity control of pharmaceuticals using AQbD principles. The review also provides an overview of recent research (2012–2022) on the applicability of CE methods in chiral drug impurity profiling.