Analysis of microcystins by capillary zone electrophoresis coupling with electrospray ionization mass spectrometry

In this paper, a rapid and effective method based on capillary zone electrophoresis (CZE) coupled with electrospray ionization mass spectrometry (ESI-MS) was established for the trace analysis of microcystin (MC) isomers in crude algae sample. The experimental conditions including the composition, acidity and concentration of buffer, separation voltage, injection time, and MS detection parameters were investigated in detail. A capillary separation system was as follows: a uncoated fused-silica capillary tube (50 μm i.d. × 90 cm), 40 mmol L⁻¹ ammonium acetate solution (pH 9.86) as running buffer, 25 kV as separation voltage, 20 kV × 3 s water first and 20 kV × 20 s for sample injection. Mass analysis was performed in ESI source, with sheath gas temperature 150 °C, sheath gas pressure 10 psi, and sheath gas flow 6 L min⁻¹. And sheath liquid was 7.5 mmol L⁻¹ acetic acid in 50% isopropanol-water (3 μL min⁻¹). Protonation and ammonium adduct molecular ions m/z 506.9 (MC-LR) and 532.0 (MC-YR) were used for the quantification of MCs. Under these conditions, two MCs were baseline separated within 9 min, the calibration curves were obtained in the range of 0.11-10.0 μg mL⁻¹ and 0.16-10.5 μg mL⁻¹ for MC-LR and MC-YR, respectively. Meanwhile, limits of detection were 0.05 and 0.08 μg mL⁻¹ for MC-LR and MC-YR, respectively. The recoveries for the two MCs were in the range of 95.8-108%. The developed approach had been successfully applied to the analysis of MCs in crude algae samples.     

Enzyme inhibitor screening by electrospray mass spectrometry with immobilized enzyme on magnetic silica microspheres

In this study, a novel technique for screening inhibitors by electrospray mass spectrometry (ESI-MS) with immobilized enzyme on magnetic microspheres has been demonstrated. First, the model enzyme acetylcholinesterase (AChE) is immobilized onto the 3-glycidoxypropyltrimethoxysilane (GLYMO)-modified magnetic silica microspheres. AChE activity was monitored by biochemical assay that is based on mixing of AChE immobilized microspheres and model substrate acetylcholine, separating and detecting the product through ESI-MS. Stability of the enzyme-immobilized microspheres was investigated. No apparent loss of enzyme activity was observed after fivefold reuse of AChE-immobilized microspheres. The enzyme-immobilized bioassay was used to effectively identify AChE inhibitors among two standard samples, huperzine A and huperzine B, and their source herbal Huperzia serrata, all of which were spiked into the substrate. The inhibition was determined by measuring a decrease of product formation using ESI-MS.     

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.     

Capillary electrophoresis inductively coupled plasma mass spectrometry

Chemical speciation (extraction of elemental information and identification of molecular environment for an analyte in a complex sample) has been a long sought after goal for analytical chemists. Recently, because of successful developments in more sensitive element-specific detectors and gentle separation schemes, which preserve the true chemical information in a real sample, routine speciation experiments are becoming a common occurrence in the scientific literature. For many reasons, the combination of capillary electrophoresis (for separation of different chemical species) with inductively coupled plasma mass spectrometry (for element and isotope specific detection) has emerged as the method of choice for these analyses. In this article the basic principles of capillary electrophoresis inductively coupled plasma mass spectrometry are discussed. Design consideration for instrument interface, anticipated difficulties with speciation experiments and applications for specific matrices and analytes are also presented in this article.     

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.     

Identification of hyaluronic acid oligosaccharides by direct coupling of capillary electrophoresis with electrospray ion trap mass spectrometry

A new method for the identification of oligosaccharides obtained by enzymatic digestion of hyaluronic acid (HA) with bacterial hyaluronidase (HA lyase, E.C. 4.2.2.1, from Streptococcus agalactiae) using online capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS) is presented. A fused-silica capillary coated with polyacrylamide was used with a 40 mM ammonium acetate buffer at pH 9.0 and a separation voltage of +30 kV applied to the inlet. Separation was achieved for oligosaccharides containing 4-16 monomers. The migration behavior follows the chain length of the oligomers, regardless of charge state. However, no linear relationship was found for the relation between mobility and chain length. Using an ion trap mass analyzer, complementary structural information was obtained by MS/MS and MS(n) experiments.     

Capillary electrophoresis using diol-bonded fused-silica capillaries

In this paper, 3-glycidoxypropyltrimethoxysilane was used to produce diol-bonded capillaries at room temperature for capillary electrophoresis (CE). A variety of standard reference compounds and authentic biological samples including ribonucleotides, peptides and proteins were used to test the columns. It was found that greatly suppressed electroosmotic flow was measured over a pH range of 3–10. Lower than 1.6% relative standard deviation (>10 runs) in migration time was observed for the analysis of test proteins. For real samples of ribonucleotides in tumor cell extracts, 1 million theoretical plates and excellent peak shapes were obtained. The high column efficiency and symmetrical peaks allowed the separation of samples with only 0.6% maximum difference in migration times. The diol-bonded fused-silica capillary columns were stable when used in a pH range of 2–8 under typical CE conditions. The column preparation method involved a simple dynamic coating procedure at room temperature, greatly simplifying the more typical static coating methods that require vacuum pumps and ovens.     

Capillary electrophoresis off-line matrix-assisted laser desorption/ionisation mass spectrometry of intact and digested proteins using cationic-coated capillaries

Capillary electrophoresis (CE) was coupled off-line with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the analysis of proteins and peptides. CE fractions were collected directly on a matrix-coated MALDI target, using a sheath-flow interface. Protein adsorption during CE separations was prevented by coating the capillaries with the physically adsorbed, cationic polymer PolyE-323. The CE/MALDI-MS system was used for the analysis of model proteins and peptides at physiological pH as well as analysis of proteins in tear fluid. Moreover, tryptic on-target digestion of the collected protein fractions, with subsequent MALDI-MS and MS/MS peptide analysis, was demonstrated.     

Subatmospheric electrospray interface for coupling of microcolumn separations with mass spectrometry

A modular subatmospheric electrospray interface with fiber optic UV detection close to the electrospray tip was developed for coupling of microcolumn separation techniques with mass spectrometry. The interface was based on a liquid junction with a removable microelectrospray tip. The electrospray tip was enclosed in a subatmospheric chamber attached in front of the sampling orifice of the mass spectrometer. The inlet of the liquid junction was maintained at atmospheric pressure, and thus no pressure drop developed across the separation column. The flow rate of the electrosprayed liquid from the liquid junction reservoir was adjusted by the pressure in the electrospray chamber. In this approach, a continuous and stable electrospray could be achieved without the use of an external pump. Since the electrospray did not depend on fluid delivery from the separation column, coated capillaries without electroosmotic flow as well as capillaries with electroosmotic flow could be used for capillary electrophoresis. In addition, the interface was found to be effective with capillary liquid chromatography. The use of a fiber optic UV detector placed close to the exit of the separation column provided additional detection information and a simple means of troubleshooting. The interface did not significantly influence the quality of the separation, even with columns generating several hundred thousand theoretical plates. Peptide samples in the submicromolar concentration range were detected, corresponding to a limit of detection in the attomole range.     

Interface for coupling nonaqueous wide-bore capillary electrophoresis with mass spectrometry

A liquid-junction-type interface where a thin spraying capillary is inserted inside the separation capillary was constructed for coupling nonaqueous wide-bore capillary electrophoresis (CE) to mass spectrometry (MS). The robust structure of the interface provided fairly easy capillary handling. The study was carried out with uncoated CE capillaries of 200 and 320 microm inner diameter (ID). 1-Propanol-acetonitrile (80:20 v/v) with acetate electrolyte provided a low conducting medium for CE and good spraying conditions for electrospray ionization (ESI) without sheath-flow and drying gas. Methamphetamine, alprenolol, and levorphanol served as model compounds. Approximate detection limits with the 200 microm ID capillary were 35-265 ng/mL.     

Arsenic speciation by coupling capillary zone electrophoresis with mass spectrometry

Summary Capillary zone electrophoresis (CZE) has been coupled with mass spectrometry to enable the identification of mineral and organometallic compounds of arsenic in speciation studies. The electrophoretic effluent was introduced through a concentric interface into the mass spectrometer. Make-up liquid was added to enable electric contact at the outlet of the separation capillary and to assist the electronebulization process. After ionization, the ions were analyzed and quantified with an ion-trap detector. Optimization of the coupling conditions (geometry of the concentric interface, composition and flow rate of the sheath liquid, electronebulization and detection conditions) is described. The results show that the geometry of the concentric interface and the positioning of the outlet of the separation capillary have a critical effect on stability and sensitivity. Programming the electronebulization and detection conditions throughout the analysis enabled identification and quantification of the seven arsenic compounds of interest (neutral, and positively or negatively charged species) in less than 20 min at the ppm level. Limits of detection ranged from 0.5 to 3.3 mg L⁻¹, corresponding to amounts injected ranging from 15 to 60 pg. The linear dependence of mass spectrometric response on arsenic concentration was verified for concentrations ranging from 5 to 200 mgL⁻¹. For the two positively charged species, arsenobetaine and arsenocholine, an on-line preconcentration technique (field-amplified sample injection) enabled reduction of the detection limits by approximately one order of magnitude to 110 and 160 μgL⁻¹, respectively.     

Capillary electrophoresis time-of-flight mass spectrometry of an opium powder

Summary Intensive work has been invested in recent years to evaluate the performance of capillary electrophoresis (CE) in forensic analysis. Tremendous progress has also been achieved in interfacing CE to sensitive and specific detection systems such as the mass spectrometer (MS). We have recently developed an electrospray time-of-flight mass spectrometer (ESI-TOFMS) for use as a detector for fast and efficient liquid phase separations. In the present paper we investigated ESI-TOFMS for the analysis of an opium powder. Both continuous infusion and CE were studied for direct sample introduction into the TOFMS and mixture separation, respectively. CEMS analysis of the opium was performed in a citrate buffer, using aqueous or mixed aqueous/organic eluents. Low fmol detection was achieved.     

Capillary electrophoresis coupled to mass spectrometry from a polymer modified poly(dimethylsiloxane) microchip with an integrated graphite electrospray tip

Hybrid capillary-poly(dimethysiloxane)(PDMS) microchips with integrated electrospray ionization (ESI) tips were directly fabricated by casting PDMS in a mould. The shapes of the emitter tips were drilled into the mould, which produced highly reproducible three-dimensional tips. Due to the fabrication method of the microfluidic devices, no sealing was necessary and it was possible to produce a perfect channel modified by PolyE-323, an aliphatic polyamine coating agent. A variety of different coating procedures were also evaluated for the outside of the emitter tip. Dusting graphite on a thin unpolymerised PDMS layer followed by polymerisation was proven to be the most suitable procedure. The emitter tips showed excellent electrochemical properties and durabilities. The coating of the emitter was eventually passivated, but not lost, and could be regenerated by electrochemical means. The excellent electrochemical stability was further confirmed in long term electrospray experiments, in which the emitter sprayed continuously for more than 180 h. The PolyE-323 was found suitable for systems that integrate rigid fused silica and soft PDMS technology, since it simply could be applied successfully to both materials. The spray stability was confirmed from the recording of a total ion chromatogram in which the electrospray current exhibited a relative standard deviation of 3.9% for a 30 min run. CE-ESI-MS separations of peptides were carried out within 2 min using the hybrid PDMS chip resulting in similar efficiencies as for fused silica capillaries of the same length and thus with no measurable band broadening effects, originating from the PDMS emitter.     

Capillary electrophoresis coupled to mass spectrometry for clinical diagnostic purposes

The introduction of fast, sensitive, and robust techniques for proteomic analysis into clinical practice represents a major step toward a new diagnostic approach of body fluids. In addition, proteomics emerges as a key technology for the discovery of disease biomarkers in various body fluids. However, even in relatively protein-deprived body fluids such as urine, the complexity and wide dynamic range of protein expression pose a considerable challenge to both separation and identification technologies. In the present review we discuss from a clinical point-of-view recent advances of the use of proteomics in clinical diagnosis as well as therapy evaluation. We focus on capillary electrophoresis coupled to mass spectrometry (CE-MS) and discuss CE-MS from an application point of view evaluating its merits and vices with regard to biomarker discovery. This review further presents examples of clinical applications of CE-MS for detection and identification of biomarkers in urine.     

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.     

Capillary electrophoresis/electrospray ion trap mass spectrometry for the analysis of negatively charged derivatized and underivatized glycans.

The increasing interest in the development of glycoproteins for therapeutic purposes has created a greater demand for methods to characterize the sugar moieties bound to them. Traditionally, released carbohydrates are derivatized using such methods as permethylation or fluorescent tagging prior to analysis by high performance liquid chromatography (HPLC), capillary electrophoresis (CE), or direct infusion mass spectrometry. However, little research has been performed using CE with on-line mass spectrometry (MS) detection. The CE separation of neutral oligosaccharides requires the covalent attachment of a charged species for electrophoretic migration. Among charged labels which have shown promise in assisting CE and HPLC separation is the fluorophore 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). This report describes the qualitative profiling of charged ANTS-derivatized and underivatized complex glycans by CE with on-line electrospray ion trap mass spectrometry. Several neutral standard glycans including a maltooligosaccharide ladder were derivatized with ANTS and subjected to CE/UV and CE/MS using low pH buffers consisting of citric and 6-aminocaproic acid salts. The ANTS-derivatized species were detected as negative ions, and multiple stage MS analysis provided valuable structural information. Fragment ions were easily identified, showing promise for the identification of unknowns. N-Linked glycans released from bovine fetuin were used to demonstrate the applicability of ANTS derivatization followed by CE/MS for the analysis of negatively charged glycans. Analyses were performed on both underivatized and ANTS-derivatized species, and sialylated glycans were separated and detected in both forms. The ability of the ion trap mass spectrometer to perform multiple stage analysis was exploited, with MS5 information obtained on selected glycans. This technique presents a complementary method to existing methodologies for the profiling of glycan mixtures.     

A snapshot of enzyme catalysis using electrospray ionization mass spectrometry

Insights into the early molecular events involving protein-ligand/substrate interactions such as protein signaling and enzyme catalysis can be obtained by examining these processes on a very short, millisecond time scale. We have used time-resolved electrospray mass spectrometry to delineate the catalytic mechanism of a key enzyme in bacterial lipopolysaccharide biosynthesis, 3-deoxy-d-manno-2-octulosonate-8-phosphate synthase (KDO8PS). Direct real-time monitoring of the catalytic reaction under single enzyme turnover conditions reveals a novel hemiketal phosphate intermediate bound to the enzyme in a noncovalent complex that establishes the reaction pathway. This study illustrates the successful application of mass spectrometry to reveal transient biochemical processes and opens a new time domain that can provide detailed structural information of short-lived protein-ligand complexes.     

Combination of MALDI-TOF mass spectrometry with immobilized enzyme microreactor for peptide mapping

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been combined with immobilized enzyme microreactor for the rapid, sensitive, and accurate tryptic mapping of protein and polypeptides. The technique utilizes the trypsin microreactor by immobilized enzyme on the glycidyl methacrylate (GMA)-modified cellulose membrane. The membrane micro-reactor was used for the tryptic mapping of cytochrome C and the results were compared with those obtained by using free trypsin. A significant increase in the overall sensitivity of the process was observed using the membrane microreactor, as well as the elimination of background signals due to the autolysis of the trypsin. Further, membrane microreactor digestions were found to be rapid and convenient.     

Capillary electrophoresis/mass spectrometry relevant to pharmaceutical and biotechnological applications

Advanced analytical techniques play a crucial role in the pharmaceutical and biotechnological field. In this context, capillary electrophoresis/mass spectrometry (CE/MS) has attracted attention due to efficient and selective separation in combination with powerful detection allowing identification and detailed characterization. Method developments and applications of CE/MS have been focused on questions not easily accessible by liquid chromatography/mass spectrometry (LC/MS) as the analysis of intact proteins, carbohydrates, and various small molecules, including peptides. Here, recent approaches and applications of CE/MS relevant to (bio)pharmaceuticals are reviewed and discussed to show actual developments and future prospects. Based on other reviews on related subjects covering large parts of previous works, the paper is focused on general ideas and contributions of the last 2 years; for the analysis of glycans, the period is extended back to 2006.