毛细管电泳-无鞘流电喷雾质谱用于药物分析

毛细管电泳-质谱联用技术具有分离效率高、检测灵敏度高、样品消耗量少,可同时提供样品的结构信息等优点,成为复杂样品分离分析的强有力工具。但是,毛细管电泳与质谱联用的接口技术依然未能很好的解决。为了拓展我们发展的金箔包裹的毛细管电泳分离柱尖端直接作为喷雾电极和无鞘流质谱接口的应用,本文报道了用无鞘流接口毛细管电泳-电喷雾质谱联用（CE-ESI-MS）分析5种酪氨酸激酶抑制剂（舒尼替尼、甲磺酸伊马替尼、吉非替尼、达沙替尼、埃罗替尼）的研究结果。这种接口集分离与电喷雾离子化于一根毛细管中,制作简单,成本低廉,且可批量制作。实验发现采用非水毛细管电泳分离模式不仅可以对5种酪氨酸激酶抑制剂实现基线分离,而且可以获得稳定的质谱信号。考察了电解质溶液组成对分离效果的影响,得到优化的背景电解质组成,即含2%（v/v）乙酸及5 mmol/L乙酸铵的乙腈-甲醇（80∶20, v/v）混合溶剂。在优化的条件下,5种激酶抑制剂可以得到基线分离,无鞘接口也可以长时间保持稳定的电喷雾,分析物的保留时间日内、日间重复性（RSD值）分别小于0.5%和0.8%,接口批次间的RSD值小于2.6%。与水相分离条件下的CE-MS对比,非水相条件下的5种酪氨酸激酶抑制剂的分离柱效更高,检测灵敏度更高,绝对检出限达到amol级。此外,采用无鞘流CE-MS分析了各类有机酸（千层纸素A、丹酚酸C和迷迭香酸）和脂溶性的大环内酯类抗生素（阿奇霉素、红霉素和环孢素A）,均可以获得良好的分离效果和质谱检测结果。     

Open-tubular capillary electrochromatography-mass spectrometry with sheathless nanoflow electrospray ionization for analysis of amino acids and peptides

A novel, rugged sheathless capillary electrochromatography-electrospray ionization (CEC-ESI) device, in which an open-tubular separation capillary and an electrospray tip are integrated with a Nafion tubing junction, is coupled to mass spectrometry (MS) for the analysis of amino acids and peptides. A stable electrospray was generated at nanoflow rates by applying a positive electrical potential at the Nafion membrane junction. To sustain the stable spray, an electroosmotic flow (EOF) to the spray was supported by coating the fused silica capillary with Lupamin, a high-molecular-weight linear positively charged polyvinylamine (PVAm) polymer, which also minimizes analyte adsorption. Electrochromatographic separation of amino acids and peptides was further enhanced by the chromatographic selectivity of Lupamin stationary phase for these molecules. The device was very reliable and reproducible for CEC-ESI-MS analyses of amino acids and peptides for over a hundred injections. The separation and detection behaviors of amino acids and peptides under different conditions including pH, concentration, and composition of mobile phases on Lupamin-coated and uncoated capillaries have been investigated. The relationship between nano electrospray stability and EOF is discussed.     

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.     

A novel sheathless interface for capillary electrophoresis/electrospray ionization mass spectrometry using an in-capillary electrode

A simple and rugged sheathless interface for capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was designed using common laboratory tools and chemicals. The interface uses a small platinum (Pt) wire that is inserted into the CE capillary through a small hole near the terminus. The position of the wire inside the CE capillary and within the buffer solution is analogous to standard CE separation operations where the terminus of the CE capillary is placed inside a buffer reservoir along with a grounded platinum electrode. By combining the use of the in-capillary electrode interface with sharpening of the fused silica tip of the CE capillary outlet, a stable electrospray current was maintained for an extended period of time. The design was successfully applied to CE/ESI-MS separations and analysis of mixtures of peptides and proteins. A detection limit of approximately 4 femtomole (S/N = 3) was achieved for detection of myoglobin utilizing a 75-µm-i.d. aminopropylsilane treated CE column and using a wide scan range of 550–1300 Da. The advantages of this new design include (1) a stable CE and ESI current, (2) durability, (3) a reduced risk of sparking between the capillary tip and the inlet of the mass spectrometer, (4) lack of any dead volume, and (5) facile fabrication with common tools and chemicals.     

Amino acid analysis of dried blood spots for diagnosis of phenylketonuria using capillary electrophoresis-mass spectrometry equipped with a sheathless electrospray ionization interface

We describe a capillary electrophoresis-mass spectrometry (CE-MS) method for newborn screening of a representative amino acid metabolic disease, namely, phenylketonuria (PKU). Underivatized phenylalanine and tyrosine in a dried blood spot (DBS) were simultaneously determined by CE-MS equipped with an ionophore membrane-packed sheathless electrospray ionization interface, which was developed by our group. The method was optimized for rapid determination of the underivatized amino acids, phenylalanine and tyrosine extracted from a DBS. Under the optimized conditions, the limit of detection of phenylalanine and tyrosine (signal-to-noise ratio, 3) was 0.03 and 0.07 mg/L in DBS, respectively, with a CE run time of less than 3 min. For repeated runs of a sample, coefficients of variation (CVs) for migration time were less than 3.7 %, whereas CVs for the area ratio under the curve were 2.1 and 2.9 % for 20 consecutive runs of 49.5 mg/kg Phe and 36.2 mg/kg Tyr, respectively. However, the relative standard deviations of intra- and interday assays for DBS samples were <6.2 and <5.8 %, respectively, which were substantially due to sample extraction from DBS. The analytical method was applied to real clinical samples of Korean neonates, and results were compared with those of conventional methods for PKU diagnosis, which required reference analytical methods such as isotope dilution CE-MS or high-performance liquid chromatography-mass spectrometry for quality assurance of the conventional kit-based assays. The distinct advantages of high sensitivity and extremely low sample volume, as well as a simple, easy, and economic sample pretreatment, were demonstrated for the proposed method.     

Performance of a sheathless porous tip sprayer for capillary electrophoresis-electrospray ionization-mass spectrometry of intact proteins

The performance of a prototype porous tip sprayer for sheathless capillary electrophoresis-mass spectrometry (CE-MS) of intact proteins was studied. Capillaries with a porous tip were inserted in a stainless steel needle filled with static conductive liquid and installed in a conventional electrospray ionization (ESI) source. Using a BGE of 100 mM acetic acid (pH 3.1) and a positively charged capillary coating, a highly reproducible and efficient separation of four model proteins (insulin, carbonic anhydrase II, ribonuclease A and lysozyme) was obtained. The protein mass spectra were of good quality allowing reliable mass determination of the proteins and some of their impurities. Sheath-liquid CE-MS using the same porous tip capillary and an isopropanol-water-acetic acid sheath liquid showed slightly lower to similar analyte responses. However, as noise levels increased with sheath-liquid CE-MS, detection limits were improved by a factor 6.5-20 with sheathless CE-MS. The analyte response in sheathless CE-MS could be enhanced using a nanoESI source and adding 5% isopropanol to the BGE, leading to improved detection limits by 50-fold to 140-fold as compared to sheath liquid interfacing using the same capillary - equivalent to sub-nM detection limits for three out of four proteins. Clearly, the sheathless porous tip sprayer provides high sensitivity CE-MS of intact proteins.     

Design and performance of a sheathless capillary electrophoresis/mass spectrometry interface by combining fused-silica capillaries with gold-coated nanoelectrospray tips

A simple sheathless capillary electrophoresis (CE)/mass spectrometry (MS) interface was constructed by combining widely used nanospray needles with fused-silica capillaries and it was successfully applied for the separation of peptides. The end of the CE capillary was pulled to a taper, etched and then fitted into the metal-coated nanospray borosilicate capillary. The nanospray needle can be used for several CE runs, but it can be easily and rapidly changed in the case of accidental breakage or evaporation of the coating. A fast capillary electrochromatographic method was also developed for MS analysis of peptides containing numerous basic amino acids.     

Negative mode sheathless capillary electrophoresis electrospray ionization-mass spectrometry for metabolite analysis of prokaryotes

Capillary electrophoresis (CE) was coupled to negative mode electrospray ionisation-mass spectrometry (MS) for separation and detection of phosphorylated and acidic metabolites in extracts of prokaryotes. Unlike previous CE-MS systems for metabolite analysis, a sheathless interface was used to improve sensitivity. To accomplish this, the separation capillary was modified by creating a porous junction near the outlet where the electrospray voltage and cathodic voltage for CE were applied. The outlet of the capillary was pulled to a 5 microm inner diameter to form an electrospray emitter and had a frit fabricated near the exit to prevent clogging. During analysis pressure was applied at the inlet of the separation column to create sufficient flow towards the detector. Limits of detection for 19 metabolites in full scan mode ranged from 20 nM for ADP ribose to 2.5 microM for alpha-ketoglutarate for 40 nL injections. Extracts of Escherichia coli, strain DH5-alpha, were analyzed using this system. In full scan mode, 118 different metabolites were detected. Tandem mass spectrometry was also employed to attempt identification. Reproducible fragmentation of 19 parent peaks was found and 10 of these produced spectra that were consistent with identification obtained from matching to compounds in the MetaCyc database. These results demonstrate the utility of a sensitive CE-MS system for large scale metabolite detection in biological samples.     

Copper-coated microsprayer interface for on-line sheathless capillary electrophoresis electrospray mass spectrometry of carbohydrates

A sturdy home-built sheathless CE/ESI-QTOF-MS system was developed and optimized for carbohydrate analysis. The interface and employed methodology provided a simple analytical solution to laborious CE/MS interfacing methods and to problems in characterization of complex carbohydrate mixtures that require high-resolution separation of the components. The CE/ESI interface, feasible in any MS laboratory, consists of a one-piece CE column having the CE terminus in-laboratory shaped as a microsprayer and coated with copper. The CE microsprayer was inserted into an in-house made stainless steel clenching device and the whole assembly was mounted onto a quadrupole TOF mass spectrometer. The analytical potential of the interface in terms of suitability, microsprayer performance, copper coat durability, ionization efficiency, spray stability, and sensitivity was tested first on a simple mixture of standard saccharides, which were separated, resolved, and detected with high separation efficiency. The approach was next assessed for the screening of a biological sample, a complex mixture of O-glycosylated sialylated amino acids from urine of a patient suffering from Schindler disease. Preliminary data allow this method to be considered as one of general applicability in structural glycobiology and glycomics and easy to be implemented for proteomic surveys as well.     

Capillary electrophoresis mass spectrometry with sheathless electrospray ionization for high sensitivity analysis of underivatized amino acids

A high durability sheathless electrospray ionization interface of CE-MS is applied for the sensitive analysis of underivatized amino acids. The sheathless interface was realized using an ionophore membrane-packed electro-conduction channel. The interface functioned well with a volatile alkaline background electrolyte (BGE) and uncoated fused-silica capillaries for CE-MS analysis of underivatized amino acids. High electroosmotic flow with alkaline BGE facilitated high separation efficiency (>100,000 theoretical plates) and short analysis time (<15 min). Both the short-term stability and long-term durability are particularly suited for routine applications. Using electrokinetic injection and the multiple reaction monitoring (MRM) mode with a triple-quadrupole analyzer, high sensitivity was achieved, which yielded detection limits of 0.05-0.81 μM. For the quantitation of underivatized amino acids, quantification precisions (RSDs) for intra- and inter-day analyses were less than 3%. Recoveries from serum were 96.3-101.8% for isotope dilution mass spectrometry (IDMS). When compared with HPLC-IDMS for human serum samples, highly agreeable (96.9-102.0%) results were obtained with the proposed CE-IDMS method.     

Analysis of glyphosate and glufosinate by capillary electrophoresis-mass spectrometry utilising a sheathless microelectrospray interface

The potential of capillary electrophoresis combined with mass spectrometry for the simultaneous determination of two herbicides (glyphosate and glufosinate) and their metabolites (aminomethylphosphonic acid and methylphosphinicopropionic acid) as the native species is demonstrated utilising a simple microelectrospray interface. The interface uses the voltage applied to the CE capillary to drive separation and generate the electrospray, avoiding sample dilution associated with the use of a sheath liquid interface. The chemistry of the internal walls of the capillary has a marked influence on peak shape, and appropriate choice is essential to successful operation of the interface. A linear polyacrylamide coated capillary, which has no electroosmotic flow, gave best reproducibility, with precision of migration time and peak area in the range 1-2 and 7-12% RSD, respectively, for the four analytes. Limits of detection, low-pg on-column, are substantially better than for previous methods and calibration curves over the range 1-100 microM have R2 values greater than 0.97. The observed concentration limit of detection for glyphosate in water is 1 microM and for a water-acetone extract of wheat is 2.5 microM, allowing the underivatised herbicide to be detected at 10% of the maximum residue limit in wheat.     

A new sheathless electrospray interface for coupling of capillary electrophoresis to ion-trap mass spectrometry

A simple laboratory-made sheathless electrospray interface for coupling of capillary electrophoresis to ion-trap mass spectrometry (CE/MS) was developed. The interface was machined in-house and it was designed to be freely interchangeable with the commercially available ionization sources for the mass spectrometer. Sharpened fused-silica capillaries were coated with nickel by a simple electrodeless plating procedure and were used as all-in-one columns/emitters. The electrodeless plating produced a 2-5- micro m thick smooth nickel layer that lasted for more than 8 h of continuous electrospraying. The performance of the CE/MS interface was examined by using four cationic imipramine derivatives as test substances. Relative detection limits were calculated on the basis of the extracted ion electrophorograms and were in the range 6-130 nmol/L, corresponding to absolute detection limits in the range of 20-400 amol. The system was applied for analysis of impurities in an impure imipramine N-oxide preparation, and two of the impurities could be identified on the basis of online-MS(MS) spectra recorded in scan-dependent mode.     

Ionic probe attachment ionization mass spectrometry

A new ionization method that uses metal-complex-based ionization probes containing the 2,6-bis(oxazolinyl)pyridine (pybox) ligand is presented. This method was proven to effectively ionize large complex molecules, including biomolecules. The preparation of the charged probes and their application to the ionization of biomolecules using cold-spray ionization mass spectrometry are shown.     

Photoelectron resonance capture ionization mass spectrometry: a soft ionization source for mass spectrometry of particle-phase organic compounds

Photoelectron resonance capture ionization (PERCI) is a soft and sensitive ionization method, based on the attachment of low-energy (<1 eV) photoelectrons to organic analyte molecules. PERCI has been developed in our laboratory for the real-time analysis of organic particles by mass spectrometry, and is employed here to monitor the heterogeneous reaction of ozone with oleic acid. Simplified identification of the reaction products is possible as a result of the soft nature of PERCI, giving predominantly the [M--H](-) ions. The major particle-phase products are identified as: 1-nonanal, nonanoic acid, 9-oxononanoic acid, and azelaic acid, consistent with proposed mechanisms. New insight into this well-studied heterogeneous reaction is gained as additional minor particle-phase products, consistent with the Criegee mechanism, are readily detected.     

An automated, sheathless capillary electrophoresis-mass spectrometry platform for discovery of biomarkers in human serum

A capillary electrophoresis-mass spectrometry (CE-MS) method has been developed to perform routine, automated analysis of low-molecular-weight peptides in human serum. The method incorporates transient isotachophoresis for in-line preconcentration and a sheathless electrospray interface. To evaluate the performance of the method and demonstrate the utility of the approach, an experiment was designed in which peptides were added to sera from individuals at each of two different concentrations, artificially creating two groups of samples. The CE-MS data from the serum samples were divided into separate training and test sets. A pattern-recognition/feature-selection algorithm based on support vector machines was used to select the mass-to-charge (m/z) values from the training set data that distinguished the two groups of samples from each other. The added peptides were identified correctly as the distinguishing features, and pattern recognition based on these peptides was used to assign each sample in the independent test set to its respective group. A twofold difference in peptide concentration could be detected with statistical significance (p-value < 0.0001). The accuracy of the assignment was 95%, demonstrating the utility of this technique for the discovery of patterns of biomarkers in serum.     

On-line sheathless capillary electrophoresis/nanoelectrospray ionization-tandem mass spectrometry for the analysis of glycosaminoglycan oligosaccharides

A novel approach in glycosaminoglycomics, based on sheathless on-line capillary electrophoresis/nanoelectrospray ionization-quadrupole time of flight-mass spectrometry (CE/nanoESI-QTOF-MS) and tandem MS of extended chondroitin sulfate/dermatan (CS/DS) oligosaccharide chains is described. The methodology required the construction of a new sheathless CE/nanoESI-QTOF-MS configuration, its implementation and optimization for the high sensitivity analysis of CS/DS oligosaccharide mixtures from conditioned culture medium of decorin transfected human embryonic kidney (HEK) 293 cells. Under newly established sheathless on-line CE/(-)nanoESI conditions for glycosaminoglycan (GAG) ionization and MS detection, single CS/DS oligosaccharide components of extended chain length and increased sulfation degree were identified. Molecular ions corresponding to species carrying 5 and 6 negative charges could be generated for large GAG oligosaccharide species in the negative ion nanoESI-MS. The optimized on-line conditions enabled the detection of molecular ions assigned to oversulfated tetradeca-, octadeca-, and eicosasaccharide CS/DS molecules, which represent the category of largest sulfated GAG-derived oligosaccharides evidenced by CE/ESI-MS. By on-line CE/ESI tandem MS in data-dependent acquisition mode the oversulfated eicosasaccharide species could be sequenced and the localization of the additional sulfate group along the chain could be determined.     

Comparison of sheathless and sheath-flow electrospray interfaces for the capillary electrophoresis-electrospray ionization-mass spectrometry analysis of peptides

Capillary electrophoresis coupled to mass spectrometry via an electrospray interface provides a powerful system for separation and characterization of a high number of biomolecules. The present paper describes a home-made sheathless interface and compares it with a commercial sheath-flow interface, using a separation method based on a peptide hormone mixture of therapeutic interest. In a previous work, we optimized the parameters involved in a sheath-flow interface and obtained good results in sensitivity and reproducibility. The sheathless interface is performed with a graphite-coated electrospray ionisation (ESI) tip attached to the separation capillary. We demonstrate that electrolyte composition is the main parameter affecting signal sensitivity and separation resolution. The effect of the nature and concentration of the organic solvent added to the separation electrolyte is carefully studied. Furthermore, a general comparison of both interfaces is made in terms of separation, reproducibility, and sensitivity obtained under the optimized conditions described. Advantages and disadvantages of both coupling setups have been evaluated.     

Analysis of metallothioneins by means of capillary electrophoresis coupled to electrospray mass spectrometry with sheathless interfacing

Capillary electrophoresis (CE) coupled to electrospray mass spectrometry via sheathless interfacing has been applied to the analysis of mammalian metallothionein (MT) extracts. In a rabbit-liver extract, four (MT-2C, MT-2A, MT-2D and MT-2E) out of six known MT sub-isoforms were unambiguously identified under three CE-resolved peaks. A fourth peak was found to contain MT-1A and/or MT-2B, whose molecular masses differ by only 1 Da. Traces of non-N-acetylated MT-2D and MT-2E were observed in a fifth, minor peak. In a rat-liver extract, both MT-1 and MT-2 were resolved and identified. Non-N-acetylated MT-2 was also identified in a resolved, minor peak. Minimum detectable amounts of MTs have been estimated to be approximately 0.6 fmol per sub-isoform.