Determination of amino acids by capillary electrophoresis-electrospray ionization-mass spectrometry: An evaluation of different protein hydrolysis procedures

In this work, a CE equipment, online hyphenated to an IT MS analyzer by a linear sheath liquid interface promoting ESI, was used to develop a method for quantitative determination of amino acids. Under appropriate conditions (BGE composition, 0.8% HCOOH, 20% CH(3)OH; sheath liquid composition, 0.8% HCOOH, 60% methanol; V(ESI), +4.50 W), analytical curves of all amino acids from 3 to 80 mg/L were recorded presenting acceptable linearity (r > 0.99). LODs in the range of 16-172 mu mol/L were obtained. BSA, a model protein, was submitted to different hydrolysis procedures (classical acid and basic, and catalyzed by the H(+) form of a cation exchanger resin) and its amino acid profiles determined. In general, the resin-mediated hydrolysis yields were overall similar or better than those obtained by classical acid or basic hydrolysis. The resulting experimental-to-theoretical BSA concentration ratios served as correction factors for the quantitation of amino acids in Brazil nut resin generated hydrolysates.

Application of capillary electrophoresis-electrospray ionization mass spectometry in the determination of molecular diversity.

By means of capillary electrophoresis coupled online to electrospray ionization MS, a library of theoretically 171 disubstituted xanthene derivatives was analyzed. The method allowed the purity and makeup of the library to be determined: 160 of the expected compounds were found to be present, and 12 side-products were also detected in the mixture. Due to the ability of capillary electrophoresis to separate analytes on the basis of charge, most of the xanthene derivatives could be resolved by simple capillary electrophoresis-MS procedures even though 124 of the 171 theoretical compounds were isobaric with at least one other molecule in the mixture. Any remaining unresolved peaks were resolved by MS/MS experiments. The method shows promise for the analysis of small combinatorial libraries with fewer than 1000 components.

Determination of fluoxetine and norfluoxetine enantiomers in human plasma by polypyrrole-coated capillary in-tube solid-phase microextraction coupled with liquid chromatography-fluorescence detection

A sensitive, selective, and reproducible in-tube polypyrrole-coated capillary (PPY) solid-phase microextraction and liquid chromatographic method for fluoxetine and norfluoxetine enantiomers analysis in plasma samples has been developed, validated, and further applied to the analysis of plasma samples from elderly patients undergoing therapy with antidepressants. Important factors in the optimization of in-tube SPME efficiency are discussed, including the sample draw/eject volume, draw/eject cycle number, draw/eject flow-rate, sample pH, and influence of plasma proteins. Separation of the analytes was achieved with a Chiralcel OD-R column and a mobile phase consisting of potassium hexafluorophosphate 7.5 mM and sodium phosphate 0.25 M solution, pH 3.0, and acetonitrile (75:25, v/v) in the isocratic mode, at a flow rate of 1.0 mL/min. Detection was carried out by fluorescence absorbance at Ex/Em 230/290 nm. The multifunctional porous surface structure of the PPY-coated film provided high precision and accuracy for enantiomers. Compared with other commercial capillaries, PPY-coated capillary showed better extraction efficiency for all the analytes. The quantification limits of the proposed method were 10 ng/mL for R- and S-fluoxetine, and 15 ng/mL for R- and S-norfluoxetine, with a coefficient of variation lower than 13%. The response of the method for enantiomers is linear over a dynamic range, from the limit of quantification to 700ng/mL, with correlation coefficients higher than 0.9940. The in-tube SPME/LC method can therefore be successfully used to analyze plasma samples from ageing patients undergoing therapy with fluoxetine. (C) 2009 Elsevier B.V. All rights reserved.

New insights in carbohydrate-deficient transferrin analysis with capillary electrophoresis-mass spectrometry

Capillary zone electrophoresis (CZE) with UV detection has been widely used for the determination of carbohydrate-deficient transferrin (CDT), an indirect marker of the chronic alcohol consumption (≥60-80g/day). A commercially available method (CEofix? CDT kit), containing a bilayer anionic coating, allows for the analysis of CDT with a high resolution between transferrin (Tf) glycoforms with reduced protein adsorption onto the capillary wall. Although widely used in routine analysis, this procedure presents some limitations in terms of selectivity and sensitivity which may be overcome with mass spectrometry (MS). However, the available method is not MS-compatible due to the non-volatile coating as well as the phosphate and borate buffers present in the background electrolyte (BGE). This study firstly consisted in developing MS-compatible separation conditions, i.e., coating and BGE compositions. Numerous cationic, neutral, and anionic coatings were evaluated in combination with BGEs covering a broad range of pH values. A bilayer coating composed of a cationic layer of 10% polybrene (m/v) and an anionic layer of 10% dextran sulfate (m/v) combined with a BGE composed of 20mM ammonium acetate at pH 8.5 provided the best results in terms of glycoforms' resolution, efficiency, adsorption reduction, migration times' repeatability, and coating stability. The method was then transferred to CZE-MS after investigations of the electrospray ionization (ESI) source, equipped with a sheath-flow interface, and the time-of-flight (TOF/MS) parameters. A successful MS detection of tetrasialo-Tf was obtained during infusion, while the experiments highlighted the challenges and issues encountered with intact glycoprotein analysis by CZE-ESI-MS.

Optimization of a method for determination of phenolic acids in exotic fruits by capillary electrophoresis

In this work, the separation of nine phenolic acids (benzoic, caffeic, chlorogenic, p-coumaric, ferulic, gallic, protocatechuic, syringic, and vanillic acid) was approached by a 32 factorial design in electrolytes consisting of sodium tetraborate buffer(STB) in the concentration range of 10-50 mmol L(-1) and methanol in the volume percentage of 5-20%. Derringer`s desirability functions combined globally were tested as response functions. An optimal electrolyte composed by 50 mmol L(-1) tetraborate buffer at pH 9.2, and 7.5% (v/v) methanol allowed baseline resolution of all phenolic acids under investigation in less than 15 min. In order to promote sample clean up, to preconcentrate the phenolic fraction and to release esterified phenolic acids from the fruit matrix, elaborate liquid-liquid extraction procedures followed by alkaline hydrolysis were performed. The proposed methodology was fully validated (linearity from 10.0 to 100 mu g mL(-1), R(2) > 0.999: LOD and LOQ from 1.32 to 3.80 mu g mL(-1) and from 4.01 to 11.5 mu g mL(-1), respectively; intra-day precision better than 2.8% CV for migration time and 5.4% CV for peak area; inter-day precision better than 4.8% CV for migration time and 4.8-11% CV for peak area: recoveries from 81% to 115%) and applied successfully to the evaluation of phenolic contents of abiu-roxo (Chrysophyllum caimito), wild mulberry growing in Brazil (Morus nigra L.) and tree tomato (Cyphomandra betacea). Values in the range of 1.50-47.3 mu g g(-1) were found, with smaller amounts occurring as free phenolic acids. (C) 2009 Elsevier B.V. All rights reserved.

Simultaneous analysis of five antidepressant drugs using direct injection of biofluids in a capillary restricted-access media-liquid chromatography-tandem mass spectrometry system

Direct analysis, with minimal sample pretreatment, of antidepressant drugs, fluoxetine, imipramine, desipramine, amitriptyline, and nortriptyline in biofluids was developed with a total run time of 8 min. The setup consists of two HPLC pumps, injection valve, capillary RAM-ADS-C18 pre-column and a capillary analytical C 18 column connected by means of a six-port valve in backflush mode. Detection was performed with ESI-MS/MS and only 1 mu m of sample was injected. Validation was adequately carried out using FLU-d(5) as internal standard. Calibration curves were constructed under a linear range of 1-250 ng mL(-1) in plasma, being the limit of quantification (LOQ), determined as 1 ng mL(-1), for all the analytes. With the described approach it was possible to reach a quantified mass sensitivity of 0.3 pg for each analyte (equivalent to 1.1-1.3 fmol), translating to a lower sample consumption (in the order of 103 less sample than using conventional methods). (C) 2008 Elsevier B.V. All rights reserved.

Metabolic fingerprinting using direct flow injection electrospray ionization tandem mass spectrometry for the characterization of proanthocyanidins from the barks of Hancornia speciosa

Direct flow injection electrospray ionization ion trap tandem mass spectrometry (ESI-IT-MS/MS) was used to investigate the polyphenolic compounds present in an infusion from the barks of Hancornia speciosa Gom. (Apocynaceae), a native Brazilian plant popularly known as 'mangabeira', used as a source of nutrition and against gastric disorders. After a simple sample filtration pretreatment the characteristic fingerprint of the infusion was performed in negative ion ESI mode in a few minutes. At low capillary-voltage activation, the deprotonated molecules ([M-H](-)) were observed and using collision-induced dissociation the product ion spectra showed the presence of a homologous series of B-type proanthocyanidins, as well as another series containing their respective C-glycosylated derivatives, with a degree of polymerization from 1 up to 6 units of interlinked catechins. Therefore, direct flow injection allowed us to identify the key compounds without preparative isolation of the components. Copyright (C) 2007 John Wiley & Sons, Ltd.

From Sample Processing to Quantification: A Full Electrochemical Approach for Neutral Analyte Derivatization, Capillary Electrophoresis Separation, and Contactless Conductivity Detection

A thin-layer electrochemical flow cell coupled to capillary electrophoresis with contactless conductivity detection (EC-CE-(CD)-D-4) was applied for the first time to the derivatization and quantification of neutral species using aliphatic alcohols as model compounds. The simultaneous electrooxidation of four alcohols (ethanol, 1-propanol, 1-butanol, and 1-pentanol) to the corresponding carboxylates was carried out on a platinum working electrode in acid medium. The derivatization step required 1 min at 1.6 V vs. Ag/AgCl under stopped flow conditions, which was preceded by a 10 s activation at 0 V. The solution close to the electrode surface was then hydrodynamically injected into the capillary, and a 2.5 min electrophoretic separation was carried out. The fully automated flow system operated at a frequency of 12 analyses per hour. Simultaneous determination of the four alcohols presented detection limits of about 5 x 10(-5) mol As a practical application with a complex matrix, ethanol concentrations were determined in diluted pale lager beer and in nonalcoholic beer. No statistically significant difference was observed between the EC-CE-(CD)-D-4 and gas chromatography with flame ionization detection (GC-FID) results for these samples. The derivatization efficiency remained constant over several hours of continuous operation with lager beer samples (n = 40).

Development of Nanoinjector Devices for Electrospray Ionization - Tandem Mass Spectrometry (ESI-MSn)

In mass spectrometric (MS) systems with electrospray ionization (ESI), the sample can be analyzed coupled to separation systems (such as liquid chromatography or capillary electrophoresis) or simply by direct infusion. The greatest benefit of the type of injection is the possibility of continuous use of small amounts of samples over a long period of time. This extended analysis time allows a complete study of fragmentation by mass spectrometry, which is critical for structure elucidation of new compounds, or when using an ion trap mass analyzer. The injector filled with the sample is placed at the ESI source inlet creating an electric field suitable for the continuous formation of a spray (solvent and sample) and consequently, the gradual and even release of the sample. For the formation of the spray, is necessary that the injector end is metalized. The formation of a bilayer of titanium and gold provided an excellent attachment of the film, resulting in a nanoinjector for ionization/spray formation in the system for MS. The nanoinjectors showed high repeatability and stability over 100 min by continuous sampling with 10 mu L of sample.

Development of nanoinjector devices for electrospray ionization - tandem mass spectrometry (ESI-MSn)

In mass spectrometric (MS) systems with electrospray ionization (ESI), the sample can be analyzed coupled to separation systems (such as liquid chromatography or capillary electrophoresis) or simply by direct infusion. The greatest benefit of the type of injection is the possibility of continuous use of small amounts of samples over a long period of time. This extended analysis time allows a complete study of fragmentation by mass spectrometry, which is critical for structure elucidation of new compounds, or when using an ion trap mass analyzer. The injector filled with the sample is placed at the ESI source inlet creating an electric field suitable for the continuous formation of a spray (solvent and sample) and consequently, the gradual and even release of the sample. For the formation of the spray, is necessary that the injector end is metalized. The formation of a bilayer of titanium and gold provided an excellent attachment of the film, resulting in a nanoinjector for ionization/spray formation in the system for MS. The nanoinjectors showed high repeatability and stability over 100 min by continuous sampling with 10 µL of sample.

Development, characterization, and application of flowing atmospheric-pressure afterglow ionization for mass spectrometric analysis of ambient organic aerosols

Addressing current limitations of state-of-the-art instrumentation in aerosol research, the aim of this work was to explore and assess the applicability of a novel soft ionization technique, namely flowing atmospheric-pressure afterglow (FAPA), for the mass spectrometric analysis of airborne particulate organic matter. Among other soft ionization methods, the FAPA ionization technique was developed in the last decade during the advent of ambient desorption/ionization mass spectrometry (ADI–MS). Based on a helium glow discharge plasma at atmospheric-pressure, excited helium species and primary reagent ions are generated which exit the discharge region through a capillary electrode, forming the so-called afterglow region where desorption and ionization of the analytes occurs. Commonly, fragmentation of the analytes during ionization is reported to occur only to a minimum extent, predominantly resulting in the formation of quasimolecular ions, i.e. [M+H]+ and [M–H]– in the positive and the negative ion mode, respectively. Thus, identification and detection of signals and their corresponding compounds is facilitated in the acquired mass spectra. The focus of the first part of this study lies on the application, characterization and assessment of FAPA–MS in the offline mode, i.e. desorption and ionization of the analytes from surfaces. Experiments in both positive and negative ion mode revealed ionization patterns for a variety of compound classes comprising alkanes, alcohols, aldehydes, ketones, carboxylic acids, organic peroxides, and alkaloids. Besides the always emphasized detection of quasimolecular ions, a broad range of signals for adducts and losses was found. Additionally, the capabilities and limitations of the technique were studied in three proof-of-principle applications. In general, the method showed to be best suited for polar analytes with high volatilities and low molecular weights, ideally containing nitrogen- and/or oxygen functionalities. However, for compounds with low vapor pressures, containing long carbon chains and/or high molecular weights, desorption and ionization is in direct competition with oxidation of the analytes, leading to the formation of adducts and oxidation products which impede a clear signal assignment in the acquired mass spectra. Nonetheless, FAPA–MS showed to be capable of detecting and identifying common limonene oxidation products in secondary OA (SOA) particles on a filter sample and, thus, is considered a suitable method for offline analysis of OA particles. In the second as well as the subsequent parts, FAPA–MS was applied online, i.e. for real time analysis of OA particles suspended in air. Therefore, the acronym AeroFAPA–MS (i.e. Aerosol FAPA–MS) was chosen to refer to this method. After optimization and characterization, the method was used to measure a range of model compounds and to evaluate typical ionization patterns in the positive and the negative ion mode. In addition, results from laboratory studies as well as from a field campaign in Central Europe (F–BEACh 2014) are presented and discussed. During the F–BEACh campaign AeroFAPA–MS was used in combination with complementary MS techniques, giving a comprehensive characterization of the sampled OA particles. For example, several common SOA marker compounds were identified in real time by MSn experiments, indicating that photochemically aged SOA particles were present during the campaign period. Moreover, AeroFAPA–MS was capable of detecting highly oxidized sulfur-containing compounds in the particle phase, presenting the first real-time measurements of this compound class. Further comparisons with data from other aerosol and gas-phase measurements suggest that both particulate sulfate as well as highly oxidized peroxyradicals in the gas phase might play a role during formation of these species. Besides applying AeroFAPA–MS for the analysis of aerosol particles, desorption processes of particles in the afterglow region were investigated in order to gain a more detailed understanding of the method. While during the previous measurements aerosol particles were pre-evaporated prior to AeroFAPA–MS analysis, in this part no external heat source was applied. Particle size distribution measurements before and after the AeroFAPA source revealed that only an interfacial layer of OA particles is desorbed and, thus, chemically characterized. For particles with initial diameters of 112 nm, desorption radii of 2.5–36.6 nm were found at discharge currents of 15–55 mA from these measurements. In addition, the method was applied for the analysis of laboratory-generated core-shell particles in a proof-of-principle study. As expected, predominantly compounds residing in the shell of the particles were desorbed and ionized with increasing probing depths, suggesting that AeroFAPA–MS might represent a promising technique for depth profiling of OA particles in future studies.

Mass spectrometric and capillary electrophoretic investigation of the enzymatic degradation of heparin-like glycosaminoglycans

Difficulties in determining composition and sequence of glycosaminoglycans, such as those related to heparin, have limited the investigation of these biologically important molecules. Here, we report methodology, based on matrix-assisted laser desorption ionization MS and capillary electrophoresis, to follow the time course of the enzymatic degradation of heparin-like glycosaminoglycans through the intermediate stages to the end products. MS allows the determination of the molecular weights of the sulfated carbohydrate intermediates and their approximate relative abundances at different time points of the experiment. Capillary electrophoresis subsequently is used to follow more accurately the abundance of the components and also to measure sulfated disaccharides for which MS is not well applicable. For those substrates that produce identical or isomeric intermediates, the reducing end of the carbohydrate chain was converted to the semicarbazone. This conversion increases the molecular weight of all products retaining the reducing terminus by the “mass tag” (in this case 56 Da) and thus distinguishes them from other products. A few picomoles of heparin-derived, sulfated hexa- to decasaccharides of known structure were subjected to heparinase I digestion and analyzed. The results indicate that the enzyme acts primarily exolytically and in a processive mode. The methodology described should be equally useful for other enzymes, including those modified by site-directed mutagenesis, and may lead to the development of an approach to the sequencing of complex glycosaminoglycans.

Development of advanced capillary electrophoresis techniques with UV and mass spectrometry detection for forensic, pharmaceutical and environmental applications

Capillary electrophoresis (CE) is a modern analytical technique, which is electrokinetic separation generated by high voltage and taken place inside the small capillaries. In this dissertation, several advanced capillary electrophoresis methods are presented using different approaches of CE and UV and mass spectrometry are utilized as the detection methods. ^ Capillary electrochromatography (CEC), as one of the CE modes, is a recent developed technique which is a hybrid of capillary electrophoresis and high performance liquid chromatography (HPLC). Capillary electrochromatography exhibits advantages of both techniques. In Chapter 2, monolithic capillary column are fabricated using in situ photoinitiation polymerization method. The column was then applied for the separation of six antidepressant compounds. ^ Meanwhile, a simple chiral separation method is developed and presented in Chapter 3. Beta cycodextrin was utilized to achieve the goal of chiral separation. Not only twelve cathinone analytes were separated, but also isomers of several analytes were enantiomerically separated. To better understand the molecular information on the analytes, the TOF-MS system was coupled with the CE. A sheath liquid and a partial filling technique (PFT) were employed to reduce the contamination of MS ionization source. Accurate molecular information was obtained. ^ It is necessary to propose, develop, and optimize new techniques that are suitable for trace-level analysis of samples in forensic, pharmaceutical, and environmental applications. Capillary electrophoresis (CE) was selected for this task, as it requires lower amounts of samples, it simplifies sample preparation, and it has the flexibility to perform separations of neutral and charged molecules as well as enantiomers. ^ Overall, the study demonstrates the versatility of capillary electrophoresis methods in forensic, pharmaceutical, and environmental applications.^

Applications of capillary electrophoresis and mass spectrometry for the analysis of thiosalts

Thiosalt species are unstable, partially oxidized sulfur oxyanions formed in sulfur-rich environments but also during the flotation and milling of sulfidic minerals especially those containing pyrite (FeS₂) and pyrrhotite (Fe₍₁₋ₓ₎S, x = 0 to 0.2). Detecting and quantifying the major thiosalt species such as sulfate (SO₄²⁻), thiosulfate (S₂O₃²⁻), trithionate (S₃O₆²⁻), tetrathionate (S₄O₆²⁻) and higher polythionates (SₓO₆²⁻, where 3 ≤ x ≤ 10) in the milling process and in the treated tailings is important to understand how thiosalts are generated and provides insight into potential treatment. As these species are unstable, a fast and reliable analytical technique is required for their analysis. Three capillary zone electrophoresis (CZE) methods using indirect UV-vis detection were developed for the simultaneous separation and determination of five thiosalt anions: SO₄²⁻, S₂O₃²⁻, S₃O₆²⁻, S₄O₆²⁻ and S₅O₆²⁻. Both univariate and multivariate experimental design approaches were used to optimize the most critical factors (background electrolyte (BGE) and instrumental conditions) to achieve fast separation and quantitative analysis of the thiosalt species. The mathematically predicted responses for the multivariate experiments were in good agreement with the experimental results. Limits of detection (LODs) (S/N = 3) for the methods were between 0.09 and 0.34 μg/mL without a sample stacking technique and nearly four-fold increase in LODs with the application of field-amplified sample stacking. As direct analysis of thiosalts by mass spectrometry (MS) is limited by their low m/z values and detection in negative mode electrospray ionization (ESI), which is typically less sensitive than positive ESI, imidazolium-based (IP-L-Imid and IP-T-Imid) and phosphonium-based (IP-T-Phos) tricationic ion-pairing reagents were used to form stable high mass ions non-covalent +1 ion-pairs with these species for ESI-MS analysis and the association constants (Kassoc) determined for these ion-pairs. Kassoc values were between 6.85 × 10² M⁻¹ and 3.56 × 10⁵ M⁻¹ with the linear IP-L-Imid; 1.89 ×10³ M⁻¹ and 1.05 × 10⁵ M⁻¹ with the trigonal IP-T-Imid ion-pairs; and 7.51×10² M⁻¹ and 4.91× 10⁴ M⁻¹ with the trigonal IP-T-Phos ion-pairs. The highest formation constants were obtained for S₃O₆²⁻ and the imidazolium-based linear ion-pairing reagent (IP-L-Imid), whereas the lowest were for IP-L-Imid: SO₄²⁻ ion-pair.

Quantitative Analysis and Determination of Microcystin in water by Capillary Electrophoresis Mass Spectrometry

The presence of harmful algal blooms (HAB) is a growing concern in aquatic environments. Among HAB organisms, cyanobacteria are of special concern because they have been reported worldwide to cause environmental and human health problem through contamination of drinking water. Although several analytical approaches have been applied to monitoring cyanobacteria toxins, conventional methods are costly and time-consuming so that analyses take weeks for field sampling and subsequent lab analysis. Capillary electrophoresis (CE) becomes a particularly suitable analytical separation method that can couple very small samples and rapid separations to a wide range of selective and sensitive detection techniques. This paper demonstrates a method for rapid separation and identification of four microcystin variants commonly found in aquatic environments. CE coupled to UV and electrospray ionization time-of-flight mass spectrometry (ESI-TOF) procedures were developed. All four analytes were separated within 6 minutes. The ESI-TOF experiment provides accurate molecular information, which further identifies analytes.