Determination of glyphosate as cross-contaminant in a commercial herbicide by capillary electrophoresis-electrospray ionization-mass spectrometry

Capillary electrophoresis combined with mass spectrometry (CE-MS) was used for the rapid determination of the negatively charged herbicide, Glyphosate, in a selective dried granule (DG) formulation. The CE-MS method was required to ensure product safety from the risk of cross contamination of a selective herbicide formulation. Glyphosate separation was achieved by using a bare fused-silica capillary column, operated in the reversed-polarity mode, using ammonium formate buffer, pH 2.5. The total CE-MS analysis time was under 10 min and the limit of detection was 10 ng/mL. The CE-MS analysis of Glyphosate was simple, rapid, and selective. The method involved minimal sample handling and was proven to be ideal for cross-contamination investigations in manufacturing samples.     

Positional and geometrical anionic isomer separations by capillary electrophoresis-electrospray ionization-mass spectrometry

Capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) was applied to the analysis of polar positional and geometrical anionic isomers. Since the investigated positional and geometrical anionic isomers have different pK(a) values, they could be separated by CE-ESI-MS under simple analytical conditions using a bare fused-silica capillary and volatile ammonium acetate buffer after optimizing buffer pH. Ortho-, meta-, para-hydroxybenzoate positional isomers were completely separated on a fused-silica capillary with 20 mM ammonium acetate buffer at pH 10.0, and cis-, trans-cyclohexane dicarboxylate geometrical isomers could be also separated with 20 mM ammonium acetate buffer at pH 4.0. Several analytical parameters affecting ESI-MS sensitivity were also investigated. It was found that both running buffer pH and sheath liquid pH had significant effects on the selectivity and the sensitivity on CE-ESI-MS analysis while sheath flow rate and other parameters had little influence. Under optimized conditions, linearity, detection limit, and repeatability of the analysis of hydroxybenzoate isomers were examined, and good results were obtained. It was found that the method presented in this paper is a simple, robust, and cost-effective method for simultaneous analysis of positional and geometrical anionic isomers as well as of other small anionic compounds.     

Twenty years of interface development for capillary electrophoresis-electrospray ionization-mass spectrometry

Capillary electrophoresis-electrospray ionization-mass spectrometry has the potential to become a preferred tool for the analysis of biological mixtures and other complex samples. The development of improved interfaces in the past twenty years has been critical in demonstrating the feasibility of this technique. However, a compromise still exists between interfaces that give optimal performance and those that are practical for commercial applications. The first section of this review focuses on the technological advances in CE-ESI-MS as they relate to the key interface features for both sheath-flow and sheathless systems: delivery of the sheath liquid, shaping of the emitter tip, formation of electrical contact, and practicality in terms of ease of use and lifetime. In the second section, we review the fundamental processes that affect interface performance. Because of the complex natures of both capillary electrophoresis and electrospray ionization, flow rate, arrangement of the electrical circuit, electrochemistry, tip geometry and location of electrical contact must all be carefully managed in the design of a successful interface.     

Qualitative and quantitative analysis of amino acids by capillary electrophoresis-electrospray ionization-tandem mass spectrometry

We describe a method to identify and quantify amino acids using capillary electrophoresis-electrospray ionization-triple-quadrupole tandem mass spectrometry (CE-ESI-MS/MS). Amino acids, including physiological amino acids, were first separated by CE under acidic pH conditions and then detected by MS/MS. To efficiently introduce the whole sample into the capillary, no electrical potential was applied to the electrospray probe until running electrophoresis. The position of the electrosprayer with respect to the MS capillary entrance drastically affected sensitivity and generation of cluster ions. MS/MS with multiple reaction monitoring (MRM) detection was performed to obtain sufficient selectivity and sensitivity. Under optimized CE-MS/MS conditions, the minimum detectable levels for 32 free amino acids normally found in proteins and other physiological amino acids were between 0.1 and 14 micromol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. For most amino acids, this constitutes a severalfold increase in sensitivity compared to CE-MS. The relative standard deviations (% RSD) for all amino acids were better than 0.4% for migration times and between 1.4% and 8.6% for peak areas (n = 10). Since amino acids exhibited characteristic MS/MS spectra, this approach is useful for the simultaneous, selective, quantitative, and reproducible analysis of amino acids in physiological and biological samples that contain various kinds of matrices. The power of the method was demonstrated by analyzing amino acids in human urine.     

Analysis of the lipophilic peptaibol alamethicin by nonaqueous capillary electrophoresis-electrospray ionization-mass spectrometry

The microheterogeneous peptaibol alamethicin F30 isolated from the culture broth of Trichoderma viride was analyzed by nonaqueous CE-electrospray-MS using an IT and a TOF mass analyzer. Compared to aqueous buffers, higher separation selectivity was observed for methanolic BGE allowing the detection of more minor components. The low electrophoretic mobility observed for neutral analytes under nonaqueous conditions may be explained by ion-dipole interactions between the peptide analytes and electrolyte ions. The amino acid sequences of the individual components were derived from MS(n) using the doubly or triply charged pseudomolecular ions as well as characteristic fragments as precursor ions. The exchange of Ala by alpha-aminoisobutyric acid (Aib) which is frequently observed for peptaibols was detected for several components. Additional variations included the exchange of Gln to Glu, and the loss of the C-terminal amino alcohol or of the first six amino acids from the N-terminus with concomitant formation of pyroglutamyl residues. In most cases comigration of the Aib peptaibols with the respective Ala component was observed as the mass difference of 14 Da as the result of the amino acid exchange was not sufficient to translate into an electrophoretic separation under the conditions applied. However, proper selection of the precursor ions allowed the unequivocal analysis of the components. Additional TOF-MS measurements were performed in order to resolve the ammonium adducts from comigrating compounds (i.e., Aib-Ala exchange) and to confirm the amino acid composition of the individual components. Except for neutral compounds migrating close to the EOF the mass accuracy was better than 4 ppm for the doubly charged pseudomolecular ions and better than 2 ppm for triply charged ions.     

Determination of tobacco-specific N-nitrosamines in rabbit serum by capillary zone electrophoresis and capillary electrophoresis-electrospray ionization-mass spectrometry with solid-phase extraction

In this paper, we propose a new strategy for separation and determination of tobacco-specific N-nitrosamines (TSNAs), a group of strong carcinogens found only in tobacco products, by using CZE and CE-MS associated with SPE. Six TSNAs: N'-nitrosonornicotine, N'-nitrosoanatabine, N'-nitrosoanabasine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, and 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol were simultaneously separated by either of two CZE methods, one of which worked with ammonium formate buffer (pH 2.5) and another with citrate buffer (pH 2.4), as well as a CE-MS method. The CZE conditions including pH and concentration of running buffer, capillary length, applied voltage, and capillary temperature were systematically optimized. For CE-MS method, an optimized sheath liquid consisted of methanol-water was used at a flow rate of 10 muL/min. With SPE procedure, our proposed CE-MS method was successfully applied to determine TSNAs after 15 min metabolism in rabbits. A comparison study between CZE and CE-MS methods for quantitative purposes was carried out, showing that both methods provided similar separation efficiency, selectivity, repeatability, linearity, and recovery. However, CE-MS method was better suited for the analysis of TSNAs in complicated biological samples for its sensitivity and extra information on molecular structure. Having good accordance with our previous work by using LC-MS, the new CE-MS method is expected to be an alternative to the LC-MS method and applied to study the metabolism of TSNAs.     

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 some acidic drugs in surface and sewage treatment plant waters by capillary electrophoresis-electrospray ionization-mass spectrometry

We describe an analytical method involving solid-phase extraction (SPE) and capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) for determining some pharmaceutical compounds - naproxen, clofibric acid and bezafibrate - in real water samples. The electrospray parameters were optimized to maximize sensitivity. When a mixed aqueous-organic solvent and CZE-ESI-MS were used to analyze these drugs in water samples, the capillary was coated with hexadimethrin bromide (HDB) to permanently reverse the EOF. The method was developed from off-line SPE-CZE-MS and was validated with surface water. The detection limits were 100 ng.L(-1) for all analytes. The method was applied to analyze water samples from the influent and effluent of a sewage treatment plant. A liquid-liquid extraction step was required before SPE, and the compounds studied were found, some of them between detection and quantification limits.     

A beveled tip sheath liquid interface for capillary electrophoresis-electrospray ionization-mass spectrometry

A simple and durable sheath liquid interface for capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) has been developed. This interface utilized a beveled tip emitter and was found to be more sensitive than the conventional sheath liquid interface. The use of a beveled tip reduces the optimal flow rate and therefore decreases sample dilution. The interface utilized a 380 microm inner diameter and 400 microm outer diameter beveled tapered tip. Because of the large inner diameter and outer diameter of the tip, the interface is robust and can be easily implemented. The performance of this interface for CZE-ESI-MS and micelle electrokinetic capillary electrophoresis-electrospray-mass spectrometry, as demonstrated by the analysis of synthetic drugs and triazine mixtures, was significantly better than results obtained using a conventional sheath liquid interface.     

Characterization of the methanolic extract of hops using capillary electrophoresis-electrospray ionization-mass spectrometry

Hops are used almost exclusively for bitterness and flavor by brewers. We propose the first analytical application of CZE coupled to ESI-MS for the separation and structural elucidation of organic compounds in the methanolic extracts of hops, and different extraction procedures of the plant material have been carried out. The proposed method permits the identification of hop polyphenols (flavonoids glycosides and chalcones), bitter acids (alpha-acids and beta-acids), and their oxidation products. The optimization of CZE parameters (pH, concentration, and type of buffer) and ESI-MS parameters (nature and flow rate of the sheath liquid, nebulizer pressure, drying gas flow rate, temperature, and compound stability) have permitted the development of a rapid, simple, direct, and straightforward CZE-ESI-MS method for the identification of components of methanolic extracts from different hops used in the brewing process.     

A polyamine coating for enhanced capillary electrophoresis-electrospray ionization-mass spectrometry of proteins and peptides

A procedure for enhanced capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) of proteins is presented. The use of a newly presented capillary coating, PolyE-323, provided fast separations of typically a few minutes with high efficiency, good deactivation, and no bleeding into the mass spectrometer. Capillaries coated with PolyE-323 showed high stability over a range of pH 2-10, and tolerance towards methanol and acetonitrile, two modifiers commonly used in CE-ESI-MS. Due to the speed and simplicity of the coating procedure, the polymeric surface could, if necessary, easily be regenerated. This capability is especially valuable when working with samples of complex matrix, where a capillary surface cleaning step might be desired in order to eliminate possible memory effects. The potential of PolyE-323-coated capillaries in bioanalysis using CE-ESI-MS was demonstrated by analyzing peptides and proteins up to 66 kDa using time of flight (TOF)-MS. Due to the stable, anodal electroosmotic flow generated by the coating, the use of a sheathless ESI interface was enabled, demonstrated in peptide analysis with attomole sensitivity. The fast on-line CE-ESI-TOF system using PolyE-323-coated capillaries provided efficient separation and detection of a large number of peaks in a short time, exemplified by the analysis of a tryptic digest of bovine serum albumin (BSA). The capability of the developed capillary surface coating was demonstrated by the separation of human plasma and cerebrospinal fluid (CSF).     

Experimental designs to investigate capillary electrophoresis-electrospray ionization-mass spectrometry enantioseparation with the partial-filling technique

An experimental design approach is described to evaluate the main electrophoretic parameters involved in the enantioseparation of pharmaceuticals by capillary electrophoresis (CE) coupled to electrospray ionization-mass spectrometry (ESI-MS). For all experiments, the partial-filling technique was applied to avoid the chiral selector entering in the mass spectrometer ion source with a negative effect on the electrospray performance. To carry out enantioseparation, a volatile buffer constituted of 20 mM ammonium acetate at pH 4.0, and a polyvinyl alcohol-coated capillary were used. Methadone was employed as the model compound and three different cyclodextrins (CDs), namely sulfobutyl ether-beta-CD, carboxymethylated-beta-CD and hydroxypropyl-beta-CD, were selected in order to study the countercurrent process. Two different experimental designs were chosen: (i) a full-factorial design to examine the effects and significance of the investigated factors, and (ii) a central composite face-centered design to establish the mathematical model of the selected responses in function of experimental factors. The chiral selector concentration, percentage of the capillary filled with the chiral selector, and drying gas nebulization pressure were three relevant factors taken into consideration. For each CD, the methadone enantiomeric resolution, apparent selectivity, and migration time of the second enantiomer were established as responses. The latter were systematically related to experimental parameters with the help of multiple linear regression. It is noteworthy that the behaviour was different in function of the chiral selector charge. Results revealed that the nebulization pressure involved in the electrospray process and the CD concentration had a significant effect on the enantiomeric resolution, while the effect of the separation zone length was less pronounced. Finally, response surfaces were drawn from the mathematical model and experimental conditions were selected to allow a robust determination of methadone enantiomers by CE-MS.     

Validation of chiral capillary electrophoresis-electrospray ionization-mass spectrometry methods for ecstasy and methadone in plasma

Due to its selectivity and sensitivity, CE coupled to MS (CE-MS) has evolved as a useful analytical tool for determining drugs and metabolites in biological samples. A generic CE-ESI/MS method was developed for the enantioselective determination of basic compounds in plasma. The use of protein precipitation (PP) prior to a hydrodynamic injection (HD) was well adapted to high-concentration samples (>1 ppm) and allowed high throughput. In contrast, the combination of liquid-liquid extraction (LLE) and electrokinetic injection (EK) was time-consuming but did allow detection at the ppb level. Both approaches were fully validated according to ICH guidelines and SFSTP protocols for two pharmaceutical compounds (ecstasy and methadone (MTD)). Deuterated internal standards (IS) in the analytical procedures were used and good quantitative performance was obtained in terms of trueness and precision (repeatability and intermediate precision) since accuracy profiles were within the acceptance limits (30% for biological assay). Methods were linear over the concentration range of 0.50-175 ng/mL and 0.25-5 microg/mL for LLE-EK and PP-HD procedures, respectively. The LLE-EK methodology was finally successfully applied to quantitation of ecstasy and MTD in real cases obtained from toxicology.     

Development of capillary zone electrophoresis-electrospray ionization-mass spectrometry for the determination of lamotrigine in human plasma

A method of coupling capillary zone electrophoresis (CZE) with electrospray ionization-mass spectrometry (ESI-MS) detection has been developed for monitoring an antiepileptic drug, lamotrigine (LTG) in human plasma. The CZE-MS was developed in three stages: (i) CZE separation and ESI-MS detection of LTG and tyramine (TRM, internal standard) were simultaneously optimized by studying the influence of CZE background electrolyte (BGE) pH, BGE ionic strength, and nebulizer pressure of the MS sprayer; (ii) sheath liquid parameters, such as pH, ionic strength, organic modifier content, and flow rate of the sheath liquid, were systematically varied under optimum CZE-MS conditions developed in the first stage; (iii) MS sprayer chamber parameters (drying gas temperature and drying gas flow rate) were varied for the best MS detection of LTG. The developed assay was finally applied for the determination of LTG in plasma samples. The linear range of LTG in plasma sample assay was between 0.1-5.0 microg/mL with a limit of detection as low as 0.05 microg/mL and run time less than 6 min. Finally, the concentration-time profile of LTG in human plasma sample was found to correlate well when CZE-ESI-MS was compared to a more established method of high-performance liquid chromatography with ultraviolet detection.     

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.     

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.     

Characterization of Atropa belladonna L. compounds by capillary electrophoresis-electrospray ionization-time of flight-mass spectrometry and capillary electrophoresis-electrospray ionization-ion trap-mass spectrometry

This short communication describes the characterization of seven tropane alkaloid compounds in Atropa belladonna L. Thus a rapid and easy CE-electrospray interface (ESI)-TOF-MS procedure is developed to analyze these compounds in a pharmaceutical preparations of A. belladonna L. leaf extract. Optimum electrophoretic separation is obtained using an alkaline solution of 60 mM ammonium acetate at pH 8.5 containing 5% isopropanol. Under the optimum CE-ESI-TOF-MS conditions several important compounds such as tropine, belladonnine, norhyoscyamine, apoatropine, hyoscyamine, 6beta-hydroxyhyoscyamine, and scopolamine have been simultaneously identified from A. belladonna L. CE-ESI-IT-MS has been used to discriminate the putative presence of littorine. The sensitivity, together with mass accuracy and true isotopic pattern of the TOF-MS, allowed the identification of a broad series of tropane alkaloid compounds present in pharmaceutical preparations of A. belladonna L. leaf extract.     

Analysis of low-molecular-mass organic acids using capillary zone electrophoresis-electrospray ionization mass spectrometry

A capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) method was developed to facilitate identification and determination of eleven low-molecular-mass (LMW) organic acids (i.e. oxalic, lactic, malonic, maleic, citric, tartaric, adipic, glutaric, gluconic, isosaccharinic and succinic acid) in different sample matrices. This CZE method was adapted to suit MS conditions. Sheath liquid, sheath flow and MS parameters were optimized to achieve high mass spectrometric sensitivity. The CZE-ESI-MS procedure showed good sensitivity (limit of detection of < 0.05-0.7 mg/l for all acids), linearity (r2 = 0.9925-0.9998) and reproducibility (2.09-5.34% RSD). The applicability of the CZE-ESI-MS was demonstrated on LMW organic acids in an ale sample. In addition the (here presented) method also provided quantification of fumaric, galacturonic and 2-ketoglutaric acid with high sensitivity.     

Determination of soil amino acids by high performance liquid chromatography-electro spray ionization-mass spectrometry derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate

Determination of amino acids by mass spectrometry (MS) is an important technique to investigate soil nitrogen transformation and cycling as amino acids being the major nitrogen-containing compounds in soil organic matter. However, researchers have long faced a critical problem in coupling an efficient separation technique to a sensitive MS detection system simultaneously. In this context, we established a new method of liquid chromatography coupled to mass spectrometry based on the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization method for convenient and accurate quantification of amino acids in soil samples. Baseline separation of 17 amino acid AQC-derivatives was achieved on an XTerra^R MS C₁₈ column using ammonium formate as a mobile phase modifier. The concentration of ammonium formate and the pH of the mobile phase were optimized in order to obtain sensitive MS signals. The response curves were linear over the range of 50-800 μmol L⁻¹ amino acids. The detection limits were 0.20-0.60 pmol μL⁻¹ on column and 0.07-0.24 μg g⁻¹ soil under the optimized conditions. The method has been applied successfully for the first time to determine amino acids in 4 types of soil samples, in which 15 amino acids were quantified by MS detector but methionine and cystine were below the detection limits. Both the recovery and the precision were satisfactory. Hence, this proposed technique shows a potential for the identification of amino acids in soil as well as tracing the transformation of soil amino acids with isotope dilution technique in nitrogen cycling investigation.     

Determination at ppb level of an anti-human immunodeficiency virus nucleoside drug by capillary electrophoresis-electrospray ionization tandem mass spectrometry

Capillary electrophoresis coupled with tandem mass spectrometry was used to indirectly separate and quantify the active metabolite of the anti-human immunodeficiency virus (anti-HIV) didanosine drug. The influence of several parameters (pH and ionic strength of volatile formic acid-ammonia buffer) upon electroosmotic flow, electrophoretic mobility and peak efficiency of several nucleosides (A, dA, ddA, C) has been studied. This paper illustrates the current importance in CE-MS technique as a complementary or substituted method to the known HPLC-radioimmunoassay or HPLC-UV method to measure levels of anti-HIV drugs. The limit of detection for 2',3'-dideoxyadenosine by this method is 2 microg 1(-1) in a formic acid-ammonia buffer (pH 2.5, 10 mM ionic strength).This methodology could be used to perform simultaneous detection of two or more anti-HIV nucleosides, such as stavudine or didanosine in combination therapy.