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。     

Profiling of polar ionogenic metabolites in Polish wines by capillary electrophoresis-mass spectrometry

The composition of wine is determined by a complex interaction between environmental factors, genetic factors (i.e., grape varieties), and winemaking practices (including technology and storage). Metabolomics using NMR spectroscopy, GC-MS, and/or LC-MS has shown to be a useful approach for assessing the origin, authenticity, and quality of various wines. Nonetheless, the use of additional analytical techniques with complementary separation mechanisms may aid in the deeper understanding of wine's metabolic processes. In this study, we demonstrate that CE-MS is a very suitable approach for the efficient profiling of polar ionogenic metabolites in wines. Without using any sample preparation or derivatization, wine was analyzed using a 10-min CE-MS workflow with interday RSD values for 31 polar and charged metabolites below 3.8% and 23% for migration times and peak areas, respectively. The utility of this workflow for the global profiling of polar ionogenic metabolites in wine was evaluated by analyzing different cool-climate Polish wine samples.     

Analysis of naphthenic acids in produced water samples by capillary electrophoresis-mass spectrometry

A capillary electrophoresis-mass spectrometry method was used to analyze naphthenic acids in produced water samples. It was possible to detect cyclopentanecarboxylic, benzoic, cyclohexanebutyric, 1-naphthoic, decanoic, 3,5-dimethyladamantane-1-carboxylic, 9-anthracenecarboxylic, and pentadecanoic acids within ca. 13 min using a buffer composed of 40 mmol/L ammonium hydroxide, 32 mmol/L acetic acid and 20% v/v isopropyl alcohol, pH 8.6. The proposed method showed good repeatability, with relative standard deviation (RSD) values of 6.6% for the sum of the peak areas and less than 2% for the analysis time. In the interday analysis, the RSD values for the sum of the peak areas and migration time were 10.3% and 10%, respectively. The developed method demonstrated linear behavior in the concentration range between 5 and 50 mg/L for benzoic, decanoic, 3,5-dimethyladamantane-1-carboxylic and 9-anthracenecarboxylic acids, and between 10 and 50 mg/L for cyclopentanecarboxylic, cyclohexanebutyric, 1- naphthoic, and pentadecanoic acids. The detection limits values ranged from 0.31 to 1.64 mg/L. Six produced water samples were analyzed and it was possible to identify and quantify cyclopentanecarboxylic, benzoic, cyclohexanebutyric, and decanoic acids. The concentrations varied between 4.8 and 98.9 mg/L, proving effective in the application of complex samples.     

毛细管电泳-质谱联用技术及其在蛋白质组学中的应用

蛋白质组学研究在生物学、精准医学等方面发挥着重要的作用。然而研究面临的巨大挑战来自生物样品的复杂性,因此在质谱（MS）鉴定技术不断革新的同时,发展分离技术以降低样品复杂度尤为重要。毛细管电泳（CE）技术具有上样体积小、分离效率高、分离速度快等优势,其与质谱的联用在蛋白质组学研究中越来越受到关注。低流速鞘流液和无鞘流液接口的发展及商品化推动了CE-MS技术的发展。目前毛细管区带电泳（CZE）、毛细管等电聚焦（CIEF）、毛细管电色谱（CEC）等分离模式已与质谱联用,其中CZE-MS应用最广泛。目前被广泛采用的蛋白质组学研究策略主要是基于酶解肽段分离鉴定的"自下而上（bottom-up）"策略。首先,CE-MS技术对酶解肽段的检测灵敏度高达1 zmol,已成功应用于单细胞蛋白质组学;其次,毛细管电泳技术与反相液相色谱互补,为疏水性质相近的肽段（尤其是翻译后修饰肽段）的分离鉴定提供了新的途径。基于整体蛋白质分离鉴定的自上而下"top-down"策略可以直接获得更精准、更完整的蛋白质信息。CE技术在蛋白质大分子的分离方面具有分离效率高、回收率高的优势,其与质谱的联用提高了整体蛋白质的鉴定灵敏度和覆盖度。非变性质谱（native MS）是一种在近生理条件下从完整蛋白质复合物水平上进行分析的质谱技术。CE与非变性质谱联用已被尝试用于蛋白质复合体的分离鉴定。该文引用了与CE-MS和蛋白质组学应用相关的93篇文献,综述了以上介绍的CE-MS的研究进展以及在蛋白质组学分析中的应用优势,并总结和展望了其应用前景。     

Determination of caffeine and its metabolites in urine by capillary electrophoresis-mass spectrometry

The caffeine content of foods and beverages varies considerably, interfering with our ability to obtain valid interpretations in many human studies with regard to the mechanism of action(s) of caffeine and/or its metabolites. The rate of metabolism of caffeine and other xanthine drugs also varies greatly from one individual to another. Therefore, it is extremely important to develop accurate, reliable analytical methods to quantify caffeine and its metabolites in simple and complex matrixes. A simple method is described for the separation and characterization of caffeine and its major metabolites employing capillary electrophoresis (CE) coupled to ultraviolet-absorption and mass spectrometry (MS) detection. After optimization of the electrophoresis separation conditions, a reliable separation of caffeine and 11 of its major metabolites was achieved in 50 mM ammonium carbonate buffer, pH 11.0. The volatile aqueous electrolyte system used with a normal electroosmotic flow polarity also provided an optimal separation condition for the characterization of the analytes by MS. The CE method achieved baseline resolution for all 12 compounds in less than 30 min. The CE-MS method is suitable for use as a routine procedure for the rapid separation and characterization of caffeine and its metabolites. The usefulness of this method was demonstrated by the extraction, separation, and identification of caffeine and its 11 metabolites from normal urine samples. The urine specimens were first acidified to obtain optimum binding efficiency to the sorbents of the off-line, solid-phase extraction procedure employed here, and an acidified eluent solvent was employed for the desorption step to maximize the recovery of the bound analytes.     

Chiral capillary electrophoresis-mass spectrometry of tetrahydroisoquinoline-derived neurotoxins: observation of complex stereoisomerism

Previous studies have shown that certain 1,2,3,4-tetrahydroisoquinoline derivatives (TIQs) are neurotoxins inducing Parkinsonism. Further, individual enantiomers of these toxins such as (R/S)-N-methylsalsolinol ((R/S)-NMSal) possess distinct neurotoxicological properties. In this work, a chiral capillary electrophoresis (CE) method with electrospray ionization-tandem mass spectrometric (ESI-MS/MS) detection was developed for the quantification of TIQ enantiomers. Enantioseparation was achieved with sulfated β-cyclodextrin (sulfated β-CD) as chiral selector. To avoid any potential contamination of MS ionization source by the non-volatile chiral selector, partial filling technique was deployed in the CE separation. TIQ derivatives, including (R/S)-6,7-dihydroxy-1-methy-TIQ (salsolinol, Sal), (R/S)-1-benzyl-TIQ (BTIQ), and (R/S)-NMSal, were base-line resolved with resolution values (R) ranging from 3 (for Sal) to 4.5 (for BTIQ), which were much better than those reported previously by HPLC methods. ESI-MS/MS detection of the resolved TIQ enantiomers was specific and sensitive (LOD=1.2 μM for Sal enantiomers). The proposed chiral CE-MS/MS method was used to study in vitro formation of (R/S)-NMSal. It was found that NMSal was formed from the incubation of epinine (a dopamine metabolite) with acetaldehyde (a metabolite of alcohol). More interestingly, four isomers of NMSal were separated and detected in the incubation solution. They were identified as (R)-e.e-NMSal, (R)-e.a-NMSal, (S)-e.e-NMSal, and (S)-e.a-NMSal. This was the first lab evidence that this Parkinsonian neurotoxin exists in multiple isomeric forms.     

Determination of urinary androgen glucuronides by capillary electrophoresis with electrospray tandem mass spectrometry

Capillary electrophoresis–electrospray tandem mass spectrometry (CE‐ESI/MS/MS) is a simple and highly sensitive method for quantifying seven urinary androgen glucuronides. The urine samples were diluted and filtered through a membrane filter, and the filtrate was injected into a CE‐MS/MS system without further sample preparation steps such as extraction and derivatization. The calibration ranges were 0.01–5 µg/mL for glucuronides of androsterone and 11β‐OHAn‐3G, and 5–500 ng/mL for glucuronides of 11‐ketoAn, DHEA, testosterone, epitestosterone and DHT. The linearity of the method was 0.992–0.998, and the limits‐of‐detection at a signal‐to‐noise ratio of 3 were 5–10 ng/mL. The coefficients of variation were in the range of 4.0–9.0% for intra‐day assay and 4.1–9.8% for inter‐day assay. The proposed method may be applicable to metabolic profiling in both quantitative and qualitative analysis. Copyright © 2008 John Wiley & Sons, Ltd.     

Influence of solvent properties on separation and detection performance in non-aqueous capillary electrophoresis-mass spectrometry of basic analytes

The versatility of non-aqueous capillary electrophoresis (NACE) results mainly from the variety of physico-chemical properties of the different solvents. They provide solubility for a wide range of analytes, enable to control electrophoretic selectivity, but affect in some cases UV absorbance detection. The coupling of NACE to electrospray mass spectrometry (ESI-MS) allows to cope with the high UV cut-off of some CE relevant solvents (e.g., formamides). In this paper the pure organic solvents methanol, acetonitrile, dimethylsulfoxide, formamide, N-methylformamide and N,N-dimethylformamide are evaluated against water for the preparation of ammonium acetate electrolytes to separate the basic model substances 2-aminobenzimidazole, procaine, propranolol and quinine with NACE-MS. MS coupling is assisted with the sheath liquid water-isopropanol (1:4, v/v) with 0.1% formic acid. The goal of the paper is to assess the influence of the solvent on selectivity, separation speed, and peak efficiency for a given set of model compounds on a simple empirical basis. It should give the user an idea how the separation quality is changed when nothing but the running solvent is altered. The obtained efficiency results were discussed with respect to physico-chemical models described in literature (assuming longitudinal diffusion as the only source of band broadening), but no satisfying correlations with solvent properties could be traced. The feasibility of all six organic solvents for MS coupling was demonstrated and the influence of the separation solvent on the MS detection performance was compared. In the seven different solvents, the shortest run time was obtained with acetonitrile, the best peak resolution with the amphiprotic solvents (especially methanol) best peak efficiency with methanol and formamide, and the most sensitive ESI-MS detection with acetonitrile and methanol, but with only slight advantage to water.     

二维毛细管区带电泳/胶束电动毛细管色谱分离尿样中的药物及其对映体

建立了毛细管区带电泳(CZE)/胶束电动毛细管色谱(MEKC)二维毛细管电泳分离平台,CZE毛细管和MEKC毛细管通过一段带微孔的聚四氟乙烯(polytetrafluoroethylene, PTFE)套管固定。样品在CZE毛细管中分离后进入MEKC毛细管进一步分离,在二维转换过程中采用动态pH连接-胶束扫集法避免第一维分离区带在接口处扩散。将该方法成功用于鼠尿样品中4种药物及其对映体的分离,各组分的理论塔板数为(2.8～4.3)×104/m,检出限为0.015～0.052 mg/L,实际样品中峰面积和迁移时间的相对标准偏差(n=7)分别为1.7%～3.8%和1.3%～4.6%。方法重现性好、灵敏度和分离度高、峰容量大,适用于尿样中多种药物组分及其对映体的同时分离检测。     

Concepts and recent advances in microchip electrophoresis coupled to mass spectrometry: Technologies and applications

The online coupling of microchip electrophoresis (ME) as a fast, highly efficient, and low-cost miniaturized separation technique to mass spectrometry (MS) as an information-rich and sensitive characterization technique results in ME–MS an attractive tool for various applications. In this paper, we review the basic concepts and latest advances in technology for ME coupled to MS during the period of 2016–2021, covering microchip materials, structures, fabrication techniques, and interfacing to electrospray ionization (ESI)–MS and matrix-assisted laser desorption/ionization–MS. Two critical issues in coupling ME and ESI–MS include the electrical connection used to define the electrophoretic field strength along the separation channel and the generation of the electrospray for MS detection, as well as, a miniaturized ESI-tip. The recent commercialization of ME–MS in zone electrophoresis and isoelectric focusing modes has led to the widespread application of these techniques in academia and industry. Here we summarize recent applications of ME–MS for the separation and detection of antibodies, proteins, peptides, carbohydrates, metabolites, and so on. Throughout the paper these applications are discussed in the context of benefits and limitations of ME–MS in comparison to alternative techniques.     

Recent developments and applications of capillary and microchip electrophoresis in proteomics and peptidomics (2015–mid 2018)

This review summarizes recent developments and applications of capillary and microchip electroseparation methods in proteomic and peptidomic analyses since the year 2015 to ca. mid 2018. Sample preparation procedures for the removal of interfering components or for pre‐fractionation and preconcentration of proteins and peptides of interest are discussed. The innovations in coupling of capillary or microchip electroseparation methods with different modes of mass spectrometry detection are covered. In addition, significant recent applications of capillary electromigration methods in both bottom‐up and top‐down proteomics as well as in determinations of post‐translational modifications of proteins are presented. Moreover, several examples of the utilization of capillary electromigration methods coupled with mass spectrometry detection for clinical proteomics and peptidomics are described.     

毛细管电泳指纹图谱及毛细管电泳-质谱联用在中药质量控制中的作用

以作者多年从事毛细管电泳、毛细管电泳-质谱联用及其指纹图谱对中药的研究成果为基础,介绍了毛细管电泳实验条件的优化方法、毛细管电泳指纹图谱的研究方法与评价方法及其在中药质量控制中的作用。     

Profiling of sulfoconjugates in urine by using precursor ion and neutral loss scans in tandem mass spectrometry. Application to the investigation of heavy metal toxicity in rats

This paper reports a liquid chromatographic/electrospray ionization mass spectrometric (LC/ESI-MS) method for profiling a wide range of structurally different sulfoconjugated compounds in urine and its application to the characterization of biomarkers for heavy metal toxicity in rat urine. Sulfoconjugates were first isolated by solid-phase extraction and the LC separation was performed on a reversed-phase column. Sulfoconjugates were detected in a triple-quadrupole mass spectrometer by simultaneously monitoring constant losses of 80 u (or 80 Th for doubly charged ions), precursors of m/z 80 (SO(3) (-*)) and precursors of m/z 97 (HSO4-). The ESI-MS detection conditions were optimized on dehydroepiandrosterone sulfate and estradiol sulfate and tested on other sulfoconjugates. The analysis of urine samples from humans and rats by using the developed method allowed the detection of about 15 peaks in each mode of detection. It was then applied to the investigation of heavy metal toxicity in rats. Comparative analysis of the chromatographic fingerprints of urine from control and uranium- and cadmium-treated rats showed several variations in the chromatographic pattern of the sulfoconjugates. Diagnostic m/z ratios were confirmed by analyzing individual urine samples and one of the observed variations seemed to be specific to uranium toxicity. The ion responsible for this variation has been identified as 4-ethylphenol sulfate by comparison of its chromatographic retention time and collision-induced dissociation mass spectra (MS(2) and MS(3) performed on a quadrupole ion trap instrument) with those of the synthesized compound.     

Analysis of therapeutic peptides in human urine by combination of capillary zone electrophoresis-electrospray mass spectrometry with preparative capillary isotachophoresis sample pretreatment

The presented study deals with the off-line coupling of preparative isotachophoresis (pITP) with on-line combination of capillary zone electrophoresis with electrospray mass spectrometric detection (CZE-ESI-MS) used for the analysis of therapeutic peptides (anserine, carnosine, and buserelin) in complex matrix (urine). Preparative capillary isotachophoresis, operating in a discontinuous fractionation mode in column-coupling configuration, served as a sample pretreatment technique to separation, and fractionation of mixture of therapeutic peptides present in urine at low concentration level. The fractions isolated by pITP procedure were subsequently analyzed by capillary zone electrophoresis with electrospray mass spectrometric detection. Acetic acid at 200 mmol L(-1) concentration served as background electrolyte in CZE stage and it is compatible with MS detection in positive ionization mode. In pITP fractionation procedure, sodium cation (10 mmol L(-1) concentration) as leading ion and beta-alanine as terminating ion (20 mmol L(-1) concentration) were used. While using CZE-ESI-MS, the limits of detection were 0.18 μg mL(-1) for carnosine, 0.17 μg mL(-1) for anserine and 0.64 μg mL(-1) for buserelin in water and 0.19 μg mL(-1) for carnosine, 0.50 μg mL(-1) for anserine and 0.74 μg mL(-1) for buserelin in 10 times diluted urine, respectively. The cleaning power of pITP sample pretreatment was proved as the peptides provided the higher MS signals at lower concentration levels resulting from the minimized matrix effects. The quality of obtained MS/MS spectra was very good so that they can provide information about the structure of analytes, and they were used for verification of the analytes identities. The pITP pretreatment improved the detection limits of the analyzed therapeutic peptides at least 25 times compared to the CZE-ESI-MS itself.     

Nonaqueous capillary electrophoresis method for the analysis of gleevec and its main metabolite in human urine

The viability of nonaqueous capillary electrophoresis (NACE) was investigated for determination of gleevec and its main metabolite in human urine using a fused-silica capillary. Baseline separation of the studied solutes was obtained using a nonaqueous solution composed of 12 mM ammonium acetate and 87.6 mM acetic acid in methanol-acetonitrile (ACN) (80:20, v:v) providing analysis time shorter than 3 min. Different aspects including stability of the solutions, linearity, accuracy and precision were studied in order to validate the method in the urine matrix. Detection limits of 24 microg L(-1) for gleevec and its metabolite were obtained. A robustness test of the method was carried out using the Plackett-Burman fractional factorial model with a matrix of 15 experiments. The developed method is simple, rapid and sensitive and has been used to determine gleveec and its metabolite at clinically relevant levels in human urine. Before NACE determination, a solid-phase extraction (SPE) procedure with a C18 cartridge was necessary. Real determination of these analytes in two patient urines were done.     

Off-line capillary electrophoresis/fully automated nanoelectrospray chip quadrupole time-of-flight mass spectrometry and tandem mass spectrometry for glycoconjugate analysis

Implementation and optimization of an off-line capillary electrophoresis (CE)/(−)nanoESIchip-quadrupole time-of-flight (QTOF) mass spectrometric (MS) and tandem MS system for compositional mapping and structural investigation of components in complex carbohydrate mixtures is described. The approach was developed for glycoscreening and applied to O-glycosylated peptides from urine of a patient suffering from α-N-acetylhexosaminidase deficiency, known as Schindler's disease. The fundamental issue of sensitivity, previously representing a serious drawback of the off-line CE/MS analysis, could be positively addressed by the off-line conjunction of CE with automated chip-based ESI-QTOF-MS to provide flexibility for CE/chip MS coupling and enhance structural elucidation of single components in heterogeneous mixtures. Copyright © 2004 John Wiley & Sons, Ltd.     

A new interface used to couple capillary electrophoresis with an inductively coupled plasma mass spectrometry for speciation analysis

In this work, a novel and high-efficiency interface has been developed in coupling CE with inductively coupled plasma MS (ICPMS). The interface completely avoids laminar flow in CE capillary caused by the suction of nebulizer, and can be easily and stably operated at room temperature with high analyte transport efficiency to ICPMS. The new interface has a liquid dead volume smaller than 5 nL, which was much smaller than those (65-2500 microL) reported previously for other interfaces. All above features led to a higher sensitivity and a better electrophoretic resolution for CE-ICPMS coupled with this new interface. With the help of this new interface, we have successfully separated and determined five species of arsenic, As(III), As(V), monomethylarsonic acid, dimethylarsinic acid and p-aminophenylarsonic acid using CE-ICPMS within 11 min with a detection limit of 0.046-0.075 ng/mL and an RSD of 2-6% (n=6).     

毛细管电泳-电感耦合等离子体质谱法测定藻类中6种不同形态的砷化合物

建立了一种利用毛细管电泳与电感耦合等离子体质谱联用技术(CE-ICP-MS)分析检测6种不同形态砷化合物的方法。详细研究了缓冲溶液的种类、pH值和浓度,分离电压以及进样时间等因素对6种砷化合物的分离度、灵敏度和重现性等的影响。结果表明,在最佳条件下,三价砷(As3+)、一甲基砷(MMA)、二甲基砷(DMA)、五价砷(As5+)、砷胆碱(AsC)和砷甜菜碱(AsB)6种化合物在25 min内得到完全分离。6次平行测定中,6种砷化合物峰面积的相对标准偏差(RSD)为3%～5%,检出限(以As计)(3倍信噪比)为0.08～0.12 μg/L。应用该方法成功地对海带中6种砷化合物进行了分析,回收率为90%～103%。该方法具有耗时短、灵敏度高、样品消耗量少、稳定性好等优点,可用于藻类样品中不同形态砷化合物的分析。