CE‐ESI‐MS coupled with dynamic pH junction online concentration for analysis of peptides in human urine samples

In this article, an approach has been developed for the analysis of some small peptides with similar pI values by CE‐ESI‐MS based on the online concentration strategy of dynamic pH junction. The factors affected on the separation, detection and online enrichment, such as BGE, injection pressure, sheath flow liquid and separation voltage have been investigated in detail. Under the optimum conditions, i.e. using 0.5 mol/L formic acid (pH 2.15) as the BGE, preparing the sample in 50 mM ammonium acetate solution (pH 7.5), 50 mbar of injection pressure for 300 s, using 7.5 mM of acetic acid in methanol–water (80% v/v) solution as the sheath flow liquid and 20 kV as the separation voltage, four peptides with similar pI values, such as L ‐Ala‐L ‐Ala (pI=5.57), L ‐Leu‐D ‐Leu (pI=5.52), Gly‐D ‐Phe (pI=5.52) and Gly‐Gly‐L ‐Leu (pI=5.52) achieved baseline separation within 18.3 min with detection limits in the range of 0.2–2.0 nmol/L. RSDs of peak migration time and peak area were in the range of 1.45–3.57 and 4.93–6.32%, respectively. This method has been applied to the analysis of the four peptides in the spiked urine sample with satisfactory results.     

CE‐MS method development for peptides analysis,especially hepcidin,an iron metabolism marker

A method for the resolution of a peptides mixture including hepcidin‐25, an iron metabolism marker, was developed by CE‐ESI‐MS. Several strategies were tested to optimize peptide separation, such as the addition of cyclodextrins or organic solvents in the BGE or the use of coated capillaries. Best results in terms of resolution, symmetry and efficiency were obtained with a BGE made of 500 mM ammonium acetate pH 4.5/ACN 70:30 v/v. Using the methodology of experimental design, BGE concentration, sheath liquid composition and MS‐coupling parameters were then optimized in order to obtain the best signal intensity for hepcidin. Finally, a 225 mM BGE and a sheath liquid composed of isopropanol/water 80:20 v/v containing 0.5% v/v formic acid were selected as it constitutes the best compromise for selectivity, peak shape and sensitivity.     

Optimization of capillary electrophoretic-electrospray ionization-mass spectrometric analysis of catecholamines

The capillary electrophoretic-mass spectrometric analysis (CE-MS) of catecholamines was optimized with coaxial sheath flow interface and electrospray ionization (ESI). The parameters studied included the sheath liquid composition and its flow rate, separation conditions in ammonium acetate buffer together with the ESI and cone voltages as mass spectrometric parameters. In addition, the effect of ESI voltage on injection as well as the siphoning effect were considered. The optimized conditions were a sheath liquid composition of methanol-water (80:20 v/v) with 0.5% acetic acid, with a flow rate of 6 microL/min. The capillary electrophoretic separation parameters were optimized with 50 mM ammonium acetate buffer, pH 4.0, to +25 kV separation voltage together with a pressure of 0.1 psi. The most intensive signals were obtained with an ESI voltage of +4.0 kV and a cone voltage of +20 V. The nonactive ESI voltage during injection as well as avoidance of the siphoning effect increased the sensitivity of the MS detection considerably. The use of ammonium hydroxide as the CE capillary conditioning solution instead of sodium hydroxide did not affect the CE-MS performance, but allowed the conditioning of the capillary between analyses to be performed in the MS without contaminating the ion source.     

Capillary electrophoresis-mass spectrometry for the analysis of quaternary ammonium herbicides

Conditions for the simultaneous determination of the three herbicides paraquat, diquat and difenzoquat and the two plant growth regulators chlormequat and mepiquat by pressure-assisted capillary electrophoresis coupled to mass spectrometry (ion-trap) using electrospray as ionisation source have been established. A 200 mM formic acid-ammonium formate buffer solution at pH 3.0 with 50% of methanol was used as carrier electrolyte. Some capillary electrophoresis-mass spectrometry parameters such as sheath liquid and sheath gas flow-rates, sheath liquid composition, electrospray voltage andthe CE capillary position were optimised. The MS and MS-MS spectra of positive ions were studied in order to obtain structural information for the confirmation of the identity. The use of labelled standards allowed to confirm fragment ions assignation. The detection limits, based on a signal-to-noise ratio of 3:1, were between 0.5 and 2.5 mg l(-1) with hydrodynamic injection (10 s) and between 1 and 10 microg l(-1) with elecrokinetic injection (20 s, 10 kV) using standards in ultrapure water. Quality parameters such as linearity and run-to-run precision (n=6) were established. Quantitation was carried out using labelled standards. The method has been applied to the analysis of contaminated irrigation water and spiked mineral water samples.     

Microfabricated liquid junction hybrid capillary electrophoresis‐mass spectrometry interface for fully automated operation

One of the challenging instrumental aspects in coupling an automated CE instrument with ESI mass spectrometry (CE‐MS) is finding the balance between the stability, reproducibility and sensitivity of the analysis and compatibility with the standard CE instrumentation. Here, we present a development of a new liquid junction based electrospray interface for automated CE‐MS, with a focus on the technical design followed by computer modeling of transport conditions as well as characterization of basic performance of the interface. This hybrid arrangement designed as a microfabricated unit attachable to the automated CE instrument allows using of a wide range of separation capillaries with respect to their diameter, length or internal coating (e.g., for suppressed electroosmotic flow). Different compositions of the ESI liquid and background electrolyte solutions can be used if needed. The microfabricated part, prepared by laser machining from polyimide, includes a self‐aligning liquid junction, a short transport channel, and a pointed sprayer for the electrospray ionization. This microfabricated part is positioned in a plastic connection block securing the separation capillary and flushing ports. Transport conditions were modelled using computer simulation and the real life performance of the interface was compared to that of a commercial sheath liquid interface. The basic performance of the interface was demonstrated by separations of peptides, proteins, and oligosaccharides.     

A sheath-flow nanospray interface for capillary electrophoresis/mass spectrometry

We have developed a novel sheath-flow interface for low-flow electrospray ionization mass spectrometry (ESI-MS) and capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS). The interface is composed of two capillaries. One is a tapered fused-silica ESI emitter suitable for microliter and nanoliter flow rate electrospray and the other is a tail-end gold-coated CE separation column that is inserted into the emitter. A sheath liquid is supplied between the column and the emitter capillaries. The gold coating and the sheath liquid are used as the conducting media for ESI and the CE circuit. This novel design was initially evaluated by an infusion ESI-MS analysis of the most common antiretroviral dideoxynucleosides, followed by CE/MS coupling analysis of several antidepressant drugs. With infusion studies, the effects of the sheath liquid and the sample flow rates on detection sensitivity and signal stability were investigated. For an emitter with an internal diameter of 30 microm, the optimum flow rates for the sheath and the sample were 200 and 300 nL/min, respectively. The main improvement of this approach in comparison with conventional sheath liquid approaches using an ionspray interface is the gain in sensitivity. Sensitivities were three times better for dideoxynucleosides analyzed by infusion and 12 times higher for antidepressant drugs analyzed by CE/MS with this interface compared with ionspray. The emitter is durable, disposable, and simple to fabricate.     

Optimization of CE-ESI-MS Parameters for the Analysis of Ecstasy and Derivatives in Urine

Capillary electrophoresis (CE) was interfaced with a quadrupole mass spectrometer, using pneumatically-assisted electrospray ionization (ESI) in order to analyze Ecstasy and derivatives in urine. The influence of the sheath liquid composition, including the type and the percentage of the organic solvent, as well as the nature of the conductive acid modifier, were examined in order to find optimal coupling conditions. A fractional factorial design was also used to optimize the electrospray experimental parameters, such as the nebulizing gas pressure, the electrospray voltage, the drying gas flow rate, the drying gas temperature, the skimmer voltage, and the sheath liquid flow rate. The separation conditions were optimized in terms of temperature, electrolyte concentration, percentage of organic modifier, as well as capillary type. Finally, the optimal CE-ESI-MS conditions were applied to the analysis of Ecstasy and other related amphetamines in urine samples, following a liquid-liquid extraction procedure.     

Coupling of capillary electrophoresis with electrospray ionization multiplexing ion mobility spectrometry

In this work, ion mobility spectrometry (IMS) function as a detector and another dimension of separation was coupled with CE to achieve two‐dimensional separation. To improve the performance of hyphenated CE‐IMS instrument, electrospray ionization correlation ion mobility spectrometry is evaluated and compared with traditional signal averaging data acquisition method using tetraalkylammonium bromide compounds. The effect of various parameters on the separation including sample introduction, sheath fluid of CE and drift gas, data acquisition method of IMS were investigated. The experimental result shows that the optimal conditions are as follows: hydrodynamic sample injection method, the electrophoresis voltage is 10 kilo volts, 5 mmol/L ammonium acetate buffer solution containing 80% acetonitrile as both the background electrolyte and the electrospray ionization sheath fluid, the ESI liquid flow rate is 4.5 μL/min, the drift voltage is 10.5 kilo volts, the drift gas temperature is 383 K and the drift gas flow rate is 300 mL/min. Under the above conditions, the mixture standards of seven tetraalkylammoniums can be completely separated within 10 min both by CE and IMS. The linear range was 5–250 μg/mL, with LOD of 0.152, 0.204, 0.277, 0.382, 0.466, 0.623 and 0.892 μg/mL, respectively. Compared with traditional capillary electrophoresis detection methods, the developed CE‐ESI‐IMS method not only provide two sets of qualitative parameters including electrophoresis migration time and ion drift time, ion mobility spectrometer can also provide an additional dimension of separation and could apply to the detection ultra‐violet transparent compounds or none fluorescent compounds.     

Nonaqueous capillary electrophoresis-electrospray ionization-ion trap-mass spectrometry analysis of pyrrolo- and pyrido[1,2-a]azepine alkaloids in Stemona

Stemona alkaloids represent an outstanding class of natural compounds due to their pharmacological profile and their complex and unusual molecular structures. The aim of this study was the development of the first CE method for the separation, identification and quantification of these pyrrolo- and pyrido[1,2-a]azepine derivatives in three Stemona species. The best results were obtained with a NACE-ESI-IT-MS method, utilizing an electrolyte of 50 mM ammonium acetate, 1 M acetic acid and 10% methanol in ACN and a separation voltage of 30 kV. Samples were injected voltage-assisted with 20 kV for 1 s. Isopropanol:water (1:1) was used as ESI sheath liquid at a flow rate of 3 microL/min. The assay was applied for the qualitative profiling of Stemona alkaloids in S. curtisii, S. collinsae and S. tuberosa. For unambiguous peak assignment of more than forty unidentified alkaloids MS/MS experiments were performed and fragmentation patterns studied. Subsequently the method was validated for the quantitative determination of four selected derivatives (RSD inter- and intraday <6%, LODs <7.5 microg/mL, LOQs <25.0 microg/mL, for all analytes, recovery rates >98.9%) in several Stemona sp. extracts.     

CE hydrogen deuterium exchange-MS in peptide analysis

CE and hydrogen-deuterium (H/D) exchange MS are useful tools in the analysis and characterisation of peptides. This study reports the facile coupling of these tools in the H/D exchange CE-MS analysis of model and pharmaceutically important peptides, using a sheath flow interface. The peptides varied in mass from 556 (leucine enkephalin) to 1620 Da (bombesin), and in charge state from 0.33 (leucine enkephalin) to 3.0 (substance P). The application of a BGE composed of ammonium formate buffer (25 mM, pD 3.5 in D(2)O (>98% D atom)), a sheath liquid composed of formic acid (0.25% v/v in D(2)O) and ACN (30:70 v/v), and dissolving the samples in a mixture of ACN/D(2)O (50:50 v/v) facilitates complete H/D exchange. Because of complete H/D exchange the ESI mass spectra produced are easy to interpret and comparable to those obtained from LC-MS analysis. The CE-H/D-MS approach has the advantage of requiring lower volumes of deuterated solvents. The b- and y-series fragments produced by using in-source decomposition correspond to those predicted. With the peptides studied, the complete exchange H/D exchange observed with both the molecular and fragment ions helps to confirm both amino acid composition and sequence.     

CE-ESI-MS of biological anions in plastic capillaries at high pH

In this study, the potential of poly(methylmethacrylate) (PMMA, Plexiglas) and polyether ether ketone (PEEK) tubing for CE-ESI-MS separations of anions at high pH values was examined. A set of model compounds of biological interest was used to investigate the main operational parameters for CE-ESI-MS, such as the sheath-flow interface design, the polarity of the ionization voltage, the use of ammonia-based separation electrolytes, and the sheath liquid composition. Optimum separations and detection sensitivities in negative ESI mode were obtained using a running electrolyte of 75 mM of ammonia at pH 11 and a sheath liquid of 60:40 v/v or 75:25 v/v isopropanol/water with 0.5% v/v of ammonia. At these experimental conditions, PMMA and PEEK capillaries show good hydrolytic stabilities and lower EOF values than fused-silica columns. Better separation resolutions were obtained with PMMA capillary, but this plastic rapidly swelled and bled because of its limited chemical resistance to the sheath liquid. PMMA columns equipped with a fused-silica tip were used for a safer exposure to the sheath liquid, but the inner surface of the fused-silica tips had limited stability at pH 11. On the other hand, good separations and reproducibility on migration times and peak areas were obtained using PEEK capillaries without capillary column deterioration.     

Metabolite profiling of human urine by CE-ESI-MS using separation electrolytes at low pH

We investigated the potential of CE coupled to electrospray MS (CE-ESI-MS) in metabolite profiling of human urine without any sample prefractionation step. A heterogeneous mixture of biologically relevant compounds covering a broad range of physicochemical properties was used to optimize separation conditions in fused-silica capillaries. A running electrolyte containing 50 mM of acetic acid and 50 mM of formic acid at pH 2.5 was used for the CE separations. A sheath-flow electrospray interface was employed for CE-ESI-MS analysis. Sheath liquids containing 80:20 v/v methanol/water with 0.1% v/v of acetic acid or 60:40 v/v isopropanol/water with 0.5% v/v of ammonia were selected for optimum detection in positive and negative ESI modes, respectively. Reproducibility and sensitivity were studied, and strategies for identification of the separated urinary compounds are suggested. We report major advantages and disadvantages of CE-ESI-MS for metabolite profiling of human body fluids. This work may be regarded as a first step in the use of CE-ESI-MS for reliable differential analysis of body fluids from healthy and diseased individuals.     

Capillary electrophoresis-electrospray ion-trap mass spectrometry for the separation of chlorophenols

Eighteen positional isomers of chlorophenols were separated by capillary electrophoresis (CE) and detected on-line by electrospray ionization ion-trap mass spectrometry (MS). Conditions for the coupling of CE to MS, e.g., the concentration of carrier electrolyte, the sheath liquid composition and the sheath gas flow-rate were optimized. Diethylmalonic acid (5 mM) at pH 7.25 and isopropanol-250 mM dimethylamine (80:20) as sheath liquid were used. The activation parameters for ion-trap mass spectrometric analysis of chlorophenols were optimized. The mass spectra, obtained for all the analytes, revealed that the [M-H]- ion was the base peak for all chlorophenols. Moreover, conditions for CE-MS-MS detection were established and [M-H-HCl]- ions were detected.     

Development of a capillary zone electrophoresis-electrospray ionisation tandem mass spectrometry method for the analysis of fluoroquinolone antibiotics

The applicability of a capillary zone electrophoresis–electrospray ionisation tandem mass spectrometric (CZE–ESI-MS–MS) method for the separation of nine fluoroquinolones was investigated. Method optimisation involved systematic trouble-shooting starting with the type and duration of capillary pre-washing and conditioning, the choice of both the CE run buffer, MS sheath liquid, CE run potential, ESI spray voltage, sheath gas flow-rate, MS capillary voltage and CE capillary and MS capillary temperatures. Another extremely important factor was found to be the degree to which the CE capillary protrudes into the ESI chamber as well as whether or not sheath gas and spray voltage are employed during the CE injection or not. The importance of the latter has, to our knowledge, not been addressed elsewhere. Nine fluoroquinolones have been separated and detected in a single run by this technique.     

The design and synthesis of alanine-based indolicidin derivatives with identical physicochemical properties and their separation using capillary electrophoresis

Four novel alanine-based indolicidin peptide derivatives were designed containing one WPW motif and two alanine residues, resulting in peptides of similar sequence. The separation of these peptides with identical physicochemical properties including molar mass, charge, and secondary structure as characterized by circular dichroism spectroscopy is very difficult; and the separation of peptides with differing physicochemical properties has only previously been reported. Capillary electrophoresis parameters such as separation buffer concentration, separation buffer pH, capillary length, and separation voltage were investigated to optimize the analysis. Using optimized conditions of a background electrolyte containing 5 mM formic acid of pH 2.0, total capillary length of 51 cm and a voltage of 10 kV enabled a baseline separation of the four peptides. The relative standard deviation of the peak areas and migration times for method repeatability (n = 3) were found to be lower than 8% and 3%, respectively. In addition, reasoning for the separation of these peptides is proposed based on the acidity of the formic acid buffer and the hydrophobic grouping of the tryptophan residues in the peptide primary sequence.     

Evaluation of volatile eluents and electrolytes for high-performance liquid chromatography-electrospray ionization mass spectrometry and capillary electrophoresis-electrospray ionization mass spectrometry of proteins. II. Capillary electrophoresis.

Peptides and proteins were separated by capillary electrophoresis (CE) in fused-silica capillaries coated with an irreversibly adsorbed monolayer of derivatized polystyrene nanoparticles. Whereas phosphate buffer, pH 3.10, enabled the highly efficient separation of basic proteins with plate counts up to 1,400,000 m-1, volatile buffer components such as formic acid or acetic acid titrated with ammonia to the desired pH had to be used for the direct coupling of CE with electrospray ionization mass spectrometry (ESI-MS). Compared to 40 mM phosphoric acid-sodium hydroxide, pH 3.10, a background electrolyte containing 125 mM formic acid-ammonia, pH 4.00, was shown to yield equivalent separation efficiency. Investigation of the influence of buffered electrolytes on the ESI-MS signal of lysozyme at pH 2.70-4.00 showed that the charge state distribution shifted to lower charge states at higher pH with a concomitant five-fold decrease in signal intensity of the most abundant signal. The presence of trifluoroacetic acid in the background electrolyte greatly increased the level of baseline noise and completely inhibited the observation of any mass signals related to proteins. Full scan spectra could be acquired from 50-500 fmol amounts of proteins during coupled CE-ESI-MS utilizing 100-125 mM formic acid-ammonia, pH 3.10. However, compared to UV detection, considerable band broadening is observed with ESI-MS detection which is mainly attributed to column overloading, band spreading in the interface, and scanning data acquisition. Finally, the major whey proteins beta-lactoglobulin A, beta-lactoglobulin B, and alpha-lactalbumin were identified in a whey drink by comparison of molecular masses determined by CE-ESI-MS to molecular masses calculated from the amino acid sequence.     

Nonaqueous capillary electrophoresis-electrospray- mass spectrometry for the analysis of fluoxetine and its related compounds

The potential of nonaqueous capillary electrophoresis was investigated for the simultaneous separation of fluoxetine hydrochloride, its meta-isomer, and other related compounds. The resolution of these compounds was compared in aqueous and nonaqueous media. Baseline separation of the studied solutes required a buffer electrolyte solution composed of 25 mM ammonium acetate and 1 M acetic acid in acetonitrile, an applied voltage of 30 kV and a temperature of 20 degrees C. Selectivity was considerably affected by the nature of the solvent (water, methanol, and acetonitrile). Moreover, substituting acetate by formate in the background electrolyte resulted in migration time changes, which were attributed to an ion-pairing phenomenon. Finally, the method was successfully coupled on-line with electrospray ionization-mass spectrometry (ESI-MS) and allowed significant selectivity and sensitivity enhancement. The effect of ESI-MS parameters, such as nebulizing gas pressure, sheath liquid composition and flow rate, on resolution and method sensitivity was also discussed.     

Feasibility of nonvolatile buffers in capillary electrophoresis-electrospray ionization-mass spectrometry of proteins

The combination of capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS) via a triaxial interface was studied as a potential means for the characterization of intact proteins. To evaluate the possibility to use a nonvolatile electrolyte for CE, the effect of sodium phosphate and ammonium borate on the MS signal of the proteins insulin, myoglobin, and bovine serum albumin (BSA) was investigated by employing infusion experiments, and compared to the effect of ammonium formate and formic acid. The study shows that with formic acid (50 mM, pH 2.4) the most intense protein signals were obtained, while the use of sodium phosphate buffer (5 and 10 mM, pH 7.5) almost completely diminished the MS response. Ammonium formate and ammonium borate (up to 100 mM, pH 8.5) also caused protein ion suppression, but especially with the borate buffer significant MS intensity remained. MS analysis of myoglobin revealed the loss of the heme group when an acidic CE electrolyte was used. Using a background electrolyte containing 25 mM ammonium borate (pH 8.5), it is demonstrated that a CE separation of a protein test mixture can be monitored with ESI-MS without degrading the MS performance allowing molecular weight determinations of the separated compounds. In the presence of borate, detection limits were estimated to be 5-10 microM (ca. 100 fmol injected). The usefulness of the CE-MS system employing a borate buffer is indicated by the analysis of a stored sample of BSA revealing several degradation products. A sample of placental alkaline phosphatase (PLAP), a potential therapeutic agent, was also analyzed by CE-MS indicating the presence of a protein impurity. Probably due to insufficient ionization of the PLAP (a complex glycoprotein), no MS signals of the intact protein were observed.     

Determination of heterocyclic aromatic amines by capillary electrophoresis coupled to mass spectrometry using in-line preconcentration

This paper shows the potentiality of capillary electrophoresis (CE) coupled to mass spectrometry (MS) for the analysis of heterocyclic aromatic amines obtaining good results in terms of sensitivity and precision. These compounds have a special interest since they can be carcinogenic for humans. The optimization of a CE-MS method was performed and the best conditions were obtained using a 16 mM formic acid/ammonium formate solution at pH 4.5 with 60% methanol as running electrolyte. For CE-MS coupling, a sheath liquid methanol/20 mM formic acid (75/25) solution at a flow rate of 3 microL/min and hydrodynamic injection of methanol mixtures for 10 s were used. Detection limits ranging from 18 ng/g to 360 ng/g and precisions up to 1.4% and 12% for migration time and concentration, respectively, were obtained. In order to improve sensitivity, field-amplified sample injection was applied as an in-line preconcentration method. Methanol/5 mM formic acid (50/50) as a sample solvent, 3 s hydrodynamic injection (0.5 psi) of a methanol plug, and 25 s of electrokinetic injection (10 kV) of the sample were found to be the optimum conditions. Detection limits up to 25 times lower and similar precisions than those reported for hydrodynamic injection were obtained.     

A new method for screening and determination of diuretics by on-line CE-ESI-MS

A rapid, high-resolution and effective new method for analyzing 12 diuretics by CE-ESI-MS was established in this paper. Ten diuretics (except two neutral compounds) could be fast separated by CE with a DAD at 214 nm with a 20 kV voltage within 6 min, using a 50 microm id and 48.5 cm effective length uncoated fused-silica capillary in a 40 mM ammonium formate buffer (pH 9.40). CE was coupled to the mass spectrometer applying an orthogonal electrospray interface with a triple-tube sheath liquid arrangement. The sheath liquid was composed of isopropanol-water (1:1 v/v) containing 30 mM acetic acid with a flow rate of 4 microL/min. Mass spectrum was employed in the positive mode and both full scan mode and SIM scan mode were utilized. All 12 diuretics could be detected and confirmed by MS in a single analysis. Under optimized conditions, LODs for the 12 diuretics were in the range of 0.13-2.7 micromol/L at an S/N of 3, and the correlation coefficients R(2 )were between 0.9921 and 0.9978. The RDSs (n = 5) of the method was 0.24-0.94 % for migration times and 1.6-8.8 % for peak areas. The recoveries of spiked samples of 12 diuretics were between 72.4% and 118%. The real urine samples were injected directly for analysis, with only simple filtration through a 0.22 microm membrane filter in order to remove solid particles, which may cause capillary blockage. Based on the migration times and characteristic ions, the diuretics in urine samples were detected successfully. This CE-ESI-MS method for analyzing diuretics will hopefully be applied to doping control.