Capillary electrophoresis-mass spectrometry for impurity profiling of basic pharmaceuticals using non-volatile background electrolytes

A generic approach has been developed for coupling capillary electrophoresis (CE) using non-volatile background electrolytes (BGEs) with mass spectrometry (MS) using a sheath liquid interface. CE-MS has been applied for basic and bi-functional compounds using a BGE consisting of 100 mM of TRIS adjusted to pH 2.5 using phosphoric acid. A liquid sheath effect is observed which may influence the CZE separation and hence may complicate the correlation between CE-UV and CE-MS methods. The influence of the liquid sheath effect on the migration behavior of basic pharmaceuticals has been studied by simulation experiments, in which the BGE outlet vial is replaced by sheath liquid in a CE-UV experiment. As a consequence of the liquid sheath effect, phosphate based BGEs can be used without significant loss of MS sensitivity compared to volatile BGEs. The use of buffer constituents such as TRIS can lead to lower detection limits as loss of MS sensitivity can be compensated by better CE performance. TRIS based BGEs permit relatively high injection amounts of about 100 pmol while maintaining high resolution. The ESI-MS parameters were optimized for a generic method with maximum sensitivity and stable operation, in which the composition of the sheath liquid and the position of the capillary were found to be important. Furthermore, the nebulizing pressure strongly influenced the separation efficiency. The system showed stable performance for several days and a reproducibility of about 15% RSD in peak area has been obtained. Nearly all test compounds used in this study could be analyzed with an MS detection limit of 0.05% measured in scan mode using extracted ion chromatograms. As a result, CE-MS was found to be a valuable analytical tool for pharmaceutical impurity profiling.     

Metabolome analysis by capillary electrophoresis-mass spectrometry

Capillary electrophoresis (CE)-mass spectrometry (MS), as an analytical platform, has made significant contributions in advancing metabolomics research, if still limited up to this time. This review, covering reports published between 1998 and 2006, describes how CE-MS has been used thus far in this field, with the majority of the works dealing with targeted metabolite analyses and only a small fraction using it in the comprehensive context. It also discusses how some of the key features of CE-MS were exploited in selected metabolomic applications.     

Nonaqueous capillary electrophoresis-mass spectrometry

Nonaqueous background electrolytes broaden the application of capillary electrophoresis displaying altered separation selectivity and interactions between analytes and buffer additives compared to aqueous background electrolytes. In addition, nonaqueous capillary electrophoresis (NACE) appears to be ideally suited for online coupling with mass spectrometry due to the high volatility and low surface tension of many organic solvents. Despite these advantages and an increasing use of nonaqueous background electrolytes in CE, coupling of NACE to mass spectrometry has not yet been applied very often to date. The present review summarizes the applications of online NACE-MS with regard to the analysis of drugs, stereoisomers, peptides, alkaloids, polymers and others. A brief discussion of solvent effects in NACE and pH of nonaqueous background electrolyte systems is also presented.     

Quantitation in capillary electrophoresis-mass spectrometry

CE-MS has evolved into a strong alternative to LC-MS. Most of CE-MS applications deal with characterization and identification. However, quantitative aspects have gained importance in, e.g., pharmaceutical and biotechnological applications. Here we summarize and evaluate various methodological aspects in order to achieve sensitive and reproducible results. Similar to LC-MS, aspects of matrix influence on the electrospray process need to be carefully addressed when quantitative results are intended by CE-MS. Due to a more complicated coupling special emphasis needs to be put on the CE-MS interface. Generally linearity over more than three orders of magnitude can be achieved by CE-ESI-MS. Furthermore, a literature survey has been performed in order to give an overview over quantitative measurements performed by CE-MS. The precision can be doubled when changing from a structural related to an isotopically labeled internal standard. Thus a level of precision better than 5% RSD can be achieved.     

Analysis of nucleotides by pressure-assisted capillary electrophoresis-mass spectrometry using silanol mask technique

A method for the determination of nucleotides based on pressure-assisted capillary electrophoresis-electrospray ionization mass spectrometry (PACE-MS) is described. To prevent multi-phosphorylated species from adsorbing onto the fused-silica capillary, silanol groups were masked with phosphate ions by preconditioning the capillary with the background electrolyte containing phosphate. During preconditioning, nebulizer gas was turned off to avoid contamination of MS detector with phosphate ions. To detect nucleotides using the CE positive mode at a pH 7.5, it was necessary to apply air pressure to the inlet capillary during electrophoresis to supplement the electroosmotic flow (EOF) toward the cathode. Moreover, we exchanged the running electrolyte every analysis using the buffer replenishment system to obtain the required reproducibility. Under the optimized conditions, 14 phosphorylated species such as nucleotides, nicotinamide-adenine dinucleotides and coenzyme A (CoA) compounds were well determined in less than 20 min. The relative standard deviations (n=6) of the method were better than 0.9% for migration times and between 1.7% and 8.1% for peak areas. The detection limits for these species were between 0.5 and 1.7 micromol/L with pressure injection of 50 mbar for 30 s (30 nL) at a signal-to-noise ratio of 3. This approach is robust and quantitative compared to the previous method, and its utility is demonstrated by the analysis of intracellular nucleotides and CoA compounds extracted from Escherichia coli wild type, pfkA and pfkB knockout mutants. The methodology was used to suggest that pfkA is the main functional enzyme.     

毛细管电泳-质谱联用技术的新进展

毛细管电泳-质谱(CE-MS)联用技术综合了CE的高效分离能力、广泛的样品适应性和MS的高灵敏度、可提供结构信息等优势,已发展成为一种重要的分离分析手段。本文对近几年来CE-MS联用接口技术的发展作一简单介绍,并对CE-MS在生命分析、食品药品分析等领域的一些应用进展予以综述。     

Sheathless capillary electrophoresis-mass spectrometry using a pulsed electrospray ionization source

A sheathless interface has been developed for coupling CE with electrospray IT mass spectrometer. This interface utilized a pulsed ESI source. The use of a pulsed electrospray source allows the use of a sprayer with larger orifice, and thus alleviates the problem of column clogging during conductive coating and CE analysis. A pulsed ESI source operated at 20 Hz and 20% duty cycle was found to produce the optimal signals. For better signals, the maximum ion injection time in the IT mass spectrometer has to be set to a value close to the actual spraying time (10 ms). Using a sprayer with 50 microm od, more stable and enhanced signals were obtained in comparison with continuous CE-ESI-MS under the same flow rate (150 nL/min). The utility of this design is demonstrated with the analysis of synthetic drugs by CE-MS.     

Urine profiling using capillary electrophoresis-mass spectrometry and multivariate data analysis

This work presents the development of a general and fast method for metabolic profiling of urine, using capillary electrophoresis-electrospray ionisation mass spectrometry (CE-ESIMS) and multivariate data analysis (DA). Human urine samples collected before and after ingestion of paracetamol were analysed at acidic and basic CE conditions, using both positive and negative ESI-MS detection. Analysis of the entire resulting data set, with no prior knowledge of the target compounds, using pair-wise 'fuzzy' correlation and eigenvalue analysis enabled the samples to be discriminated between on the basis of blank urine and urine collected after drug intake. By generating two-dimensional loadings plots, it was also possible to identify the m/z values of the substances responsible for the differentiation between control and dosed samples.     

Sensitivity enhancement in capillary electrophoresis-mass spectrometry of anionic metabolites using a triethylamine-containing background electrolyte and sheath liquid

Analyte responses in CE‐ESI‐MS using negative ionization are frequently relatively low, thereby limiting sensitivity in metabolomics applications. In order to enhance the ionization efficiency of anionic metabolites, BGEs and sheath liquids (SLs) of various compositions were evaluated. Pressure‐induced infusion and CE‐MS experiments showed that addition of triethylamine (TEA) to the BGE and SL enhanced analyte intensities. A BGE consisting of 25 mM TEA (pH 11.7) and an SL of water–methanol (1:1, v/v) containing 5 mM TEA was selected, providing separation and detection of ten representative test metabolites with good reproducibility (migration time RSDs<1%) and linearity (R²>0.99). This BGE yielded lower limits of detection (0.7–9.1 μM) for most test compounds when compared with common CE‐MS methods using a BGE and SL containing ammonium acetate (NH₄Ac) (25 and 5 mM, respectively). CE‐MS of human urine revealed an average amount of 231 molecular features in negative ionization mode when TEA was used in the BGE and SL, whereas 115 and 102 molecular features were found with an NH₄Ac‐containing BGE and SL, employing a bare fused‐silica (BFS) and Polybrene‐dextran sulfate‐Polybrene (PB‐DS‐PB)‐coated capillary, respectively. With the CE‐MS method using TEA, about 170 molecular features were observed that were not detected with the NH₄Ac‐based CE‐MS methods. For more than 82% of the molecular features that were detected with the TEA as well as the NH₄Ac‐containg BGEs (i.e. common features), the peak intensities were higher using TEA with gain factors up to 7. Overall, the results demonstrate that BGEs and SLs containing TEA are quite favorable for the analysis of anionic metabolites in CE‐MS.     

Chiral capillary electrophoresis-mass spectrometry: modes and applications

A review is presented to highlight several approaches for coupling capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS) for analysis of chiral compounds. A short discussion of commercially available CE-MS instruments and interface design is followed by a detail review on various modes of chiral CE-MS. In general, for each CE-MS mode, the capabilities, applications and limitations for chiral analysis have been pointed out. The first mode, chiral capillary zone electrophoresis-mass spectrometry (CZE-MS) in which neutral derivatized cyclodextrins (CDs) are used is possible using either column coupling with voltage switching or a partial-filling technique (PFT). However, some applications of direct coupling of CZE-MS mode are also reported. The second mode is a chiral electrokinetic chromatography-mass spectrometry (EKC-MS) in which a charged chiral selector such as a sulfated beta-CD or a vancomycin could be conveniently employed. This is because these chiral selectors have a significantly higher countercurrent electrophoretic mobility which prevents the entrance of these selectors into the mass spectrometer. The combination of counter-migration and PFT demonstrates that this synergism could be successfully applied to chiral analysis of a broader range of compounds. It is well-known that the on-line coupling of micellar electrokinetic chromatography to mass spectrometry (MEKC-MS) is problematic because the high surface activity and nonvolatile nature of conventional surfactant molecules lower the electrospray ionization efficiency. However, a recent report demonstrates that this hyphenation is now possible with the use of molecular micelles. Various MEKC-ESI-MS parameters that can be used to optimize both chiral resolution and ESI response are discussed. Finally, two recent examples that demonstrate the feasibility of using either open-tubular or packed chiral CEC with MS are reviewed. This survey will attempt to cover the state-of-the-art on various modes of CE-MS from 1998 up to 2002.     

Antioxidant compounds of propolis determined by capillary electrophoresis-mass spectrometry

Propolis is a resinous hive product rich in antioxidant compounds. Capillary electrophoresis coupled to mass spectrometric detection can provide selective information about the analytes present in complex extracts of propolis and has turned out to be an attractive alternative to HPLC methods. Therefore, a CE-ESI-MS method has been developed for the analysis of antioxidant compounds obtained from propolis. For this purpose, different electrophoretic parameters such as the nature, pH, and concentration of the separation buffer, as well as electrospray parameters (dry gas temperature and flow, nebulising gas pressure, and make-up flow) have been carefully optimised. Different phenolic compounds (e.g. pinobanksin 3-acetate, naringenin, pinocembrin, chrysin, daidzein, quercetin 3',7-dimethyl ether, apigenin, and kaempferid) could be detected. To confirm the identity of the phenolic compounds in propolis extracts, accurate mass data of the molecular ions were obtained by TOF MS. Limits of detection ranging from 6 mg/100 g of raw propolis for chrysin to 58 mg/ 100 g of raw propolis for luteolin, were obtained.     

Separation of organic cations using novel background electrolytes by capillary electrophoresis

A background electrolyte for capillary electrophoresis containing tris(-hydroxymethyl) aminomethane (THAM) and ethanesulfonic acid (ESA) gives excellent efficiency for separation of drug cations with actual theoretical plate numbers as high as 300,000. However, the analyte cations often elute too quickly and consequently offer only a narrow window for separation. The best way to correct this is to induce a reverse electroosmotic flow (EOF) that will spread out the peaks by slowing their migration rates, but this has always been difficult to accomplish in a controlled manner. A new method for producing a variable EOF is described in which a low variable concentration of tributylammonium- or triethylammonium ESA is added to the BGE. The additive equilibrates with the capillary wall to give it a positive charge and thereby produce a controlled opposing EOF. Excellent separations of complex drug mixtures were obtained by this method.     

Sample treatments prior to capillary electrophoresis-mass spectrometry

Sample preparation is a crucial part of chemical analysis and in most cases can become the bottleneck of the whole analytical process. Its adequacy is a key factor in determining the success of the analysis and, therefore, careful selection and optimization of the parameters controlling sample treatment should be carried out. This work revises the different strategies that have been developed for sample preparation prior to capillary electrophoresis-mass spectrometry (CE-MS). Namely the present work presents an exhaustive and critical revision of the different samples treatments used together with on-line CE-MS including works published from January 2000 to July 2006.     

Glycosylation analysis of glycoproteins and proteoglycans using capillary electrophoresis-mass spectrometry strategies

This review highlights recent developments in glycosylation analysis by modern MS in combination with CE based preseparation. Focused on CE-MS strategies aimed for glycotyping, the review addresses the detailed glycoform analysis of glycoproteins, glycopeptides, and proteoglycans. Glycoform analysis in the context of modern glycoproteomics is briefly addressed, as well. CZE, CIEF, and frontal analysis CE approaches hyphenated to high-resolution multistage MS for the detailed analysis of protein-linked glycan structures are overviewed in a comprehensive way. Advantages and limitations of various methodological approaches and techniques as well as mass spectrometric instrumentation are discussed in the particular context of glycoanalysis.     

Simulation and optimization of peptide separation by capillary electrophoresis-mass spectrometry

The potential of capillary electrophoresis (CE) for the separation of peptides has been extensively demonstrated in the last decade. Their correct characterization and sequenciation is a difficult task that can be accomplished using CE-mass spectrometry (CE-MS). An important limitation of CE-MS is the buffer choice since it should provide an adequate CE separation without ruining the MS signal. In this work, a new strategy is used to help to solve this limitation based on the combination of two different methodologies. Namely, an ab initio semiempirical model that relates electrophoretic behavior of peptides to their sequence is first used to obtain in a fast and easy way adequate CE buffers compatible with MS analysis. Next, CE-MS is used to separate and characterize peptides via the determination of their relative molecular masses. The usefulness of this procedure is demonstrated analyzing in a single CE-MS run a group of 10 standard peptides of very different nature (i.e., relative molecular masses ranging from 132 to 1037 and isoelectric points ranging from 5.69 to 10.62). It is concluded that the use of this strategy can help to overcome the buffer limitation in CE-MS.     

毛细管电泳-质谱技术在手性化合物分离分析中的研究进展北大核心CSCD

目前使用的绝大多数药物为手性化合物,它们具有相似的物理和化学性质,但药理活性不同,且常以外消旋混合物的形式存在,因此对手性化合物的分离在生物、环境、食品和医药等领域一直备受关注。与广泛使用的液相色谱-质谱(LC-MS)相比,毛细管电泳-质谱(CE-MS)作为一种新型分离分析技术,具有分离效率高、样品和试剂消耗量低、选择性高和分离模式多样化等诸多优势,已经发展成为手性分析领域中有广阔应用前景的分析方法之一。CE-MS结合了CE的高分离效率和低样品消耗以及MS的高灵敏度和强结构解析能力,在蛋白质组学和代谢组学等领域发挥了重要作用。CE杰出的手性拆分能力与MS优势的结合,亦使CE-MS成为实现手性化合物高效分离分析的完美组合。在过去的十几年里,基于不同CE-MS分离模式的高性能手性分析体系层出不穷,如电动色谱-质谱(EKC-MS)、胶束电动色谱-质谱(MEKC-MS)和毛细管电色谱-质谱(CEC-MS)等,并成功应用于医药、生物、食品和环境科学等领域的手性化合物分析。该文主要综述了2011~2021年,CE-MS在手性化合物分析领域的技术、手性选择剂(如改性环糊精和聚合物表面活性剂等)的使用以及在医药等领域应用方面的研究进展,并讨论了不同手性分析模式的局限性,为未来的CE-MS手性分离分析技术发展及应用提供借鉴。     

Analysis of glycans derived from glycoconjugates by capillary electrophoresis-mass spectrometry

The high structural variation of glycan derived from glycoconjugates, which substantially increases with the molecular size of a protein, contributes to the complexity of glycosylation patterns commonly associated with glycoconjugates. In the case of glycoproteins, such variation originates from the multiple glycosylation sites of proteins and the number of glycan structures associated with each site (microheterogeneity). The ability to comprehensively characterize highly complex mixture of glycans has been analytically stimulating and challenging. Although the most powerful MS and MS/MS techniques are capable of providing a wealth of structural information, they are still not able to readily identify isomeric glycan structures without high-order MS/MS (MS(n) ). The analysis of isomeric glycan structures has been attained using several separation methods, including high-pH anion-exchange chromatography, hydrophilic interaction chromatography and GC. However, CE and microfluidics CE (MCE) offer high separation efficiency and resolutions, allowing the separation of closely related glycan structures. Therefore, interfacing CE and MCE to MS is a powerful analytical approach, allowing potentially comprehensive and sensitive analysis of complex glycan samples. This review describes and discusses the utility of different CE and MCE approaches in the structural characterization of glycoproteins and the feasibility of interfacing these approaches to MS.     

Analysis of nucleic acid constituents by on-line capillary electrophoresis-mass spectrometry

This review is focused on the capillary electrophoresis-mass spectrometric (CE-MS) analysis of nucleic acid constituents in the broadest sense, going from nucleotides and adducted nucleotides over nucleoside analogues to oligonucleotides. These nucleic acid constituents play an important role in a variety of biochemical processes. Hence, their isolation, identification, and quantification will undoubtedly help reveal the process of life and disease mechanisms, such as carcinogenesis, and can also be useful for antitumor and antiviral drug research to provide valuable information about mechanism of action, pharmacokinetics, pharmacodynamics, toxicity, therapeutic drug level monitoring, and quality control related to this substance class. Fundamental investigations into their structure, the search for modifications, the occurrence and biochemical impact of structural variation amongst others, are therefore of great value. In view of the related bioanalytical procedures, the coupling of CE to MS has emerged as a powerful tool for the analysis of the complex mixtures of nucleic acid constituents: CE confers rapid analysis and efficient resolution, while MS provides high selectivity and sensitivity with structural characterization of minute amounts of compound. After an introduction about the biochemical and analytical perspectives on the nucleic acid constituents, the different modes of CE used in this field of research as well as the relevant CE-MS interfaces and the difficulties associated with quantitative CE-MS are briefly discussed. A large section is finally devoted to field-oriented applications.     

Ionization of dichlorophenols for their analysis by capillary electrophoresis-mass spectrometry

The qualitative and quantitative betalain pigment content of two cultivars of prickly pear (Opuntia ficus-indica) fruits grown in southeastern Spain was evaluated. After methanolic extraction of crushed fruits, reversed-phase high-performance liquid chromatography and photodiode array detection were applied simultaneously for the separation, identification and quantification of these pigments. Two main pigments were obtained, which were identified as indicaxanthin (lambda(max) 484 nm) and betanin (lambda(max) 535 nm). Spectrophotometric evaluation of both pigments showed a yield of around 20-30 mg per 100 g of fresh pulp. When the influence of temperature (25 to 90 degrees C) on betacyanin pigment stability was investigated, the results revealed a substantial degree of thermodegradation at temperatures higher than 70 degrees C.