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.     

Analyses of gibberellins by capillary electrophoresis-mass spectrometry combined with solid-phase extraction

A new, simple and rapid capillary electrophoresis-mass spectrometry (CE-MS) method, using a cationic polymer-coated capillary to reverse electroosmotic flow, is proposed and validated for the separation and simultaneous quantification of 11 gibberellins (GAs). Under optimum conditions, a baseline separation of 11 GAs, including GA1, GA3, GA4, GA5, GA6, GA7, GA13, GA19, GA20, GA24 and GA53 was accomplished within 25 min using 70 mM ammonium formate/formic acid buffer (pH 3.8) and 2% (v/v) acetonitrile with -25 kV as the separation voltage. Satisfactory results were obtained in terms of linearity (R2 between 0.984 and 0.995), precision (RSD of migration time below 0.8%) and sensitivity (LOD between 0.31 and 1.02 microM). Furthermore, a novel solid-phase extraction (SPE) procedure was developed for the pre-concentration and purification of GAs using Oasis MAX cartridges. The combination of SPE and CE-MS approach was applied to screen for endogenous GAs present in coconut (Cocos nucifera L.) water sample. To illustrate the applicability of the method, GA1 and GA3 were successfully detected and quantified in coconut water. Finally, the GA1 and GA3 identities were further unequivocally confirmed by CE-tandem MS experiments operating in the multiple reaction monitoring mode.     

Determination of caffeine and its metabolites in urine by capillary electrophoresis-mass spectrometry

The caffeine content of foods and beverages varies considerably, interfering with our ability to obtain valid interpretations in many human studies with regard to the mechanism of action(s) of caffeine and/or its metabolites. The rate of metabolism of caffeine and other xanthine drugs also varies greatly from one individual to another. Therefore, it is extremely important to develop accurate, reliable analytical methods to quantify caffeine and its metabolites in simple and complex matrixes. A simple method is described for the separation and characterization of caffeine and its major metabolites employing capillary electrophoresis (CE) coupled to ultraviolet-absorption and mass spectrometry (MS) detection. After optimization of the electrophoresis separation conditions, a reliable separation of caffeine and 11 of its major metabolites was achieved in 50 mM ammonium carbonate buffer, pH 11.0. The volatile aqueous electrolyte system used with a normal electroosmotic flow polarity also provided an optimal separation condition for the characterization of the analytes by MS. The CE method achieved baseline resolution for all 12 compounds in less than 30 min. The CE-MS method is suitable for use as a routine procedure for the rapid separation and characterization of caffeine and its metabolites. The usefulness of this method was demonstrated by the extraction, separation, and identification of caffeine and its 11 metabolites from normal urine samples. The urine specimens were first acidified to obtain optimum binding efficiency to the sorbents of the off-line, solid-phase extraction procedure employed here, and an acidified eluent solvent was employed for the desorption step to maximize the recovery of the bound analytes.     

Metabolome analysis by capillary electrophoresis-mass spectrometry

Capillary electrophoresis (CE)-mass spectrometry (MS), as an analytical platform, has made significant contributions in advancing metabolomics research, if still limited up to this time. This review, covering reports published between 1998 and 2006, describes how CE-MS has been used thus far in this field, with the majority of the works dealing with targeted metabolite analyses and only a small fraction using it in the comprehensive context. It also discusses how some of the key features of CE-MS were exploited in selected metabolomic applications.     

Nonaqueous capillary electrophoresis-mass spectrometry

Nonaqueous background electrolytes broaden the application of capillary electrophoresis displaying altered separation selectivity and interactions between analytes and buffer additives compared to aqueous background electrolytes. In addition, nonaqueous capillary electrophoresis (NACE) appears to be ideally suited for online coupling with mass spectrometry due to the high volatility and low surface tension of many organic solvents. Despite these advantages and an increasing use of nonaqueous background electrolytes in CE, coupling of NACE to mass spectrometry has not yet been applied very often to date. The present review summarizes the applications of online NACE-MS with regard to the analysis of drugs, stereoisomers, peptides, alkaloids, polymers and others. A brief discussion of solvent effects in NACE and pH of nonaqueous background electrolyte systems is also presented.     

Quantitation in capillary electrophoresis-mass spectrometry

CE-MS has evolved into a strong alternative to LC-MS. Most of CE-MS applications deal with characterization and identification. However, quantitative aspects have gained importance in, e.g., pharmaceutical and biotechnological applications. Here we summarize and evaluate various methodological aspects in order to achieve sensitive and reproducible results. Similar to LC-MS, aspects of matrix influence on the electrospray process need to be carefully addressed when quantitative results are intended by CE-MS. Due to a more complicated coupling special emphasis needs to be put on the CE-MS interface. Generally linearity over more than three orders of magnitude can be achieved by CE-ESI-MS. Furthermore, a literature survey has been performed in order to give an overview over quantitative measurements performed by CE-MS. The precision can be doubled when changing from a structural related to an isotopically labeled internal standard. Thus a level of precision better than 5% RSD can be achieved.     

Highly sensitive capillary electrophoresis-mass spectrometry for rapid screening and accurate quantitation of drugs of abuse in urine

The combination of capillary electrophoresis (CE) and mass spectrometry (MS) is particularly well adapted to bioanalysis due to its high separation efficiency, selectivity, and sensitivity; its short analytical time; and its low solvent and sample consumption. For clinical and forensic toxicology, a two-step analysis is usually performed: first, a screening step for compound identification, and second, confirmation and/or accurate quantitation in cases of presumed positive results. In this study, a fast and sensitive CE-MS workflow was developed for the screening and quantitation of drugs of abuse in urine samples. A CE with a time-of-flight MS (CE-TOF/MS) screening method was developed using a simple urine dilution and on-line sample preconcentration with pH-mediated stacking. The sample stacking allowed for a high loading capacity (20.5% of the capillary length), leading to limits of detection as low as 2 ng mL⁻¹ for drugs of abuse. Compound quantitation of positive samples was performed by CE-MS/MS with a triple quadrupole MS equipped with an adapted triple-tube sprayer and an electrospray ionization (ESI) source. The CE-ESI-MS/MS method was validated for two model compounds, cocaine (COC) and methadone (MTD), according to the Guidance of the Food and Drug Administration. The quantitative performance was evaluated for selectivity, response function, the lower limit of quantitation, trueness, precision, and accuracy. COC and MTD detection in urine samples was determined to be accurate over the range of 10–1000 ng mL⁻¹ and 21–1000 ng mL⁻¹, respectively.     

Simultaneous chiral determination of methamphetamine and its metabolites in urine by capillary electrophoresis-mass spectrometry

A capillary electrophoresis-mass spectrometry method for the simultaneous chiral determination of enantiomers of methamphetamine (MA), amphetamine (AP), dimethylamphetamine (DMA) and p-hydroxymethamphetamine (pOHMA), in urine has been developed. The internal standards used were 2-phenylethylamine and 1-amino4-phenylbutane. The electrolyte was 1 M formic acid (pH 2.2). The chiral selector, which was added to the electrolyte, was a mixture of 3 mM beta-cyclodextrin and 10 mM heptakis(2,6-di-O-methyl)-beta-cyclodextrin. The detection limits were 0.03 microg ml(-1) for the enantiomers of MA and AP and 0.05 microg ml(-1) for the enantiomers of pOHMA using selected ion monitoring. In the analysis of healthy adult urine samples spiked with MA, AP and pOHMA, the precision of within-run assays (n = 4) for the migration time after correction with two internal standards were under 0.04%, and the detection yields utilizing solid phase extraction were 95-105%. This method was applicable to the analysis of urine samples of MA addicts and DMA addicts.     

Detection of singly- and doubly-charged quaternary ammonium drugs in equine urine by liquid chromatography/tandem mass spectrometry

Quaternary ammonium drugs (QADs) are anticholinergic agents some of which are known to have been abused or misused in equine sports. A recent review of literature shows that the screening methods reported thus far for QADs mainly cover singly-charged QADs. Doubly-charged QADs are extremely polar substances which are difficult to be extracted and poorly retained on reversed-phase columns. It would be ideal if a comprehensive method can be developed which can detect both singly- and doubly-charged QADs. This paper describes an efficient liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous detection and confirmation of 38 singly- and doubly-charged QADs at sub-parts-per-billion (ppb) to low-ppb levels in equine urine after solid-phase extraction.     

Determination of non-steroidal anti-inflammatory drugs in urine by combining an immobilized carboxylated carbon nanotubes minicolumn for solid-phase extraction with capillary electrophoresis-mass spectrometry

Last years, the usefulness of the use of carbon nanotubes (CNTs) as sorbent material have been demonstrated for a wide variety of compounds. In this work, it has been demonstrated for first time that immobilized carboxylated single-walled carbon nanotubes (c-SWNTs) offer clear advantages over the use of CNTs. The higher adsorption capacity has been attributed to the special orientation of c-SWNTs molecules on the glass surface. The potential of this new sorbent was evaluated for the preconcentration of non-steroidal anti-inflammatory drugs (NSAIDs) from urine samples. Purified samples were analysed by capillary electrophoresis-mass spectrometry detection allowing the determination of 1.6 to 2.6 microg/L of NSAIDs with only 5 mL of sample. The precision of the method for the determination of real spiked urine samples ranged from 5.4 to 7.4% and the recoveries from 98.6 to 102.2%.     

Antioxidant compounds of propolis determined by capillary electrophoresis-mass spectrometry

Propolis is a resinous hive product rich in antioxidant compounds. Capillary electrophoresis coupled to mass spectrometric detection can provide selective information about the analytes present in complex extracts of propolis and has turned out to be an attractive alternative to HPLC methods. Therefore, a CE-ESI-MS method has been developed for the analysis of antioxidant compounds obtained from propolis. For this purpose, different electrophoretic parameters such as the nature, pH, and concentration of the separation buffer, as well as electrospray parameters (dry gas temperature and flow, nebulising gas pressure, and make-up flow) have been carefully optimised. Different phenolic compounds (e.g. pinobanksin 3-acetate, naringenin, pinocembrin, chrysin, daidzein, quercetin 3',7-dimethyl ether, apigenin, and kaempferid) could be detected. To confirm the identity of the phenolic compounds in propolis extracts, accurate mass data of the molecular ions were obtained by TOF MS. Limits of detection ranging from 6 mg/100 g of raw propolis for chrysin to 58 mg/ 100 g of raw propolis for luteolin, were obtained.     

Determination of low-molecular-mass quaternary ammonium compounds by capillary electrophoresis and hyphenation with mass spectrometry

The determination of quaternary ammonium ions by capillary electrophoresis (CE) is reviewed. The analytes include tetraalkylammonium and alkylbenzyldimethylammonium compounds frequently used as antiseptic and antibacterial agents as well as in various household products, several plant growth regulators and herbicides, by-products in bile acid sequestrants, and a range of anticholinergic drugs. Besides direct and indirect UV detection, hyphenation with electrospray mass spectrometry is particularly suited for quaternary ammonium ions and may lower the detection limits by two orders of magnitude. In comparison with established liquid chromatographic techniques, CE may exhibits superior separation efficiency. Applications in routine analysis have demonstrated that CE is reliable and robust enough to represent a real alternative to chromatography.     

Simulation and optimization of peptide separation by capillary electrophoresis-mass spectrometry

The potential of capillary electrophoresis (CE) for the separation of peptides has been extensively demonstrated in the last decade. Their correct characterization and sequenciation is a difficult task that can be accomplished using CE-mass spectrometry (CE-MS). An important limitation of CE-MS is the buffer choice since it should provide an adequate CE separation without ruining the MS signal. In this work, a new strategy is used to help to solve this limitation based on the combination of two different methodologies. Namely, an ab initio semiempirical model that relates electrophoretic behavior of peptides to their sequence is first used to obtain in a fast and easy way adequate CE buffers compatible with MS analysis. Next, CE-MS is used to separate and characterize peptides via the determination of their relative molecular masses. The usefulness of this procedure is demonstrated analyzing in a single CE-MS run a group of 10 standard peptides of very different nature (i.e., relative molecular masses ranging from 132 to 1037 and isoelectric points ranging from 5.69 to 10.62). It is concluded that the use of this strategy can help to overcome the buffer limitation in CE-MS.     

Sample treatments prior to capillary electrophoresis-mass spectrometry

Sample preparation is a crucial part of chemical analysis and in most cases can become the bottleneck of the whole analytical process. Its adequacy is a key factor in determining the success of the analysis and, therefore, careful selection and optimization of the parameters controlling sample treatment should be carried out. This work revises the different strategies that have been developed for sample preparation prior to capillary electrophoresis-mass spectrometry (CE-MS). Namely the present work presents an exhaustive and critical revision of the different samples treatments used together with on-line CE-MS including works published from January 2000 to July 2006.     

Analysis of glycans derived from glycoconjugates by capillary electrophoresis-mass spectrometry

The high structural variation of glycan derived from glycoconjugates, which substantially increases with the molecular size of a protein, contributes to the complexity of glycosylation patterns commonly associated with glycoconjugates. In the case of glycoproteins, such variation originates from the multiple glycosylation sites of proteins and the number of glycan structures associated with each site (microheterogeneity). The ability to comprehensively characterize highly complex mixture of glycans has been analytically stimulating and challenging. Although the most powerful MS and MS/MS techniques are capable of providing a wealth of structural information, they are still not able to readily identify isomeric glycan structures without high-order MS/MS (MS(n) ). The analysis of isomeric glycan structures has been attained using several separation methods, including high-pH anion-exchange chromatography, hydrophilic interaction chromatography and GC. However, CE and microfluidics CE (MCE) offer high separation efficiency and resolutions, allowing the separation of closely related glycan structures. Therefore, interfacing CE and MCE to MS is a powerful analytical approach, allowing potentially comprehensive and sensitive analysis of complex glycan samples. This review describes and discusses the utility of different CE and MCE approaches in the structural characterization of glycoproteins and the feasibility of interfacing these approaches to MS.     

Analysis of nucleic acid constituents by on-line capillary electrophoresis-mass spectrometry

This review is focused on the capillary electrophoresis-mass spectrometric (CE-MS) analysis of nucleic acid constituents in the broadest sense, going from nucleotides and adducted nucleotides over nucleoside analogues to oligonucleotides. These nucleic acid constituents play an important role in a variety of biochemical processes. Hence, their isolation, identification, and quantification will undoubtedly help reveal the process of life and disease mechanisms, such as carcinogenesis, and can also be useful for antitumor and antiviral drug research to provide valuable information about mechanism of action, pharmacokinetics, pharmacodynamics, toxicity, therapeutic drug level monitoring, and quality control related to this substance class. Fundamental investigations into their structure, the search for modifications, the occurrence and biochemical impact of structural variation amongst others, are therefore of great value. In view of the related bioanalytical procedures, the coupling of CE to MS has emerged as a powerful tool for the analysis of the complex mixtures of nucleic acid constituents: CE confers rapid analysis and efficient resolution, while MS provides high selectivity and sensitivity with structural characterization of minute amounts of compound. After an introduction about the biochemical and analytical perspectives on the nucleic acid constituents, the different modes of CE used in this field of research as well as the relevant CE-MS interfaces and the difficulties associated with quantitative CE-MS are briefly discussed. A large section is finally devoted to field-oriented applications.     

Ionization of dichlorophenols for their analysis by capillary electrophoresis-mass spectrometry

The qualitative and quantitative betalain pigment content of two cultivars of prickly pear (Opuntia ficus-indica) fruits grown in southeastern Spain was evaluated. After methanolic extraction of crushed fruits, reversed-phase high-performance liquid chromatography and photodiode array detection were applied simultaneously for the separation, identification and quantification of these pigments. Two main pigments were obtained, which were identified as indicaxanthin (lambda(max) 484 nm) and betanin (lambda(max) 535 nm). Spectrophotometric evaluation of both pigments showed a yield of around 20-30 mg per 100 g of fresh pulp. When the influence of temperature (25 to 90 degrees C) on betacyanin pigment stability was investigated, the results revealed a substantial degree of thermodegradation at temperatures higher than 70 degrees C.     

Separation of quaternary ammonium diastereomeric oligomers by capillary electrophoresis

The separation of novel diastereomeric trimers (3M) and pentamers (5M), derived from quaternary ammonium salts, was studied in conventional, uncoated and coated capillaries using capillary zone electrophoresis (CZE) with a variety of buffers and additives. Resolution of 5M diastereomers was best achieved using gamma-cyclodextrin (gamma-CD) as a chiral selector, while no diastereomeric resolution was realized for the 3M material.     

Chiral capillary electrophoresis-mass spectrometry of amino acids in foods

In this work, the development of a new chiral capillary electrophoresis-mass spectrometry (CE-MS) method to separate D- and L-amino acids is shown. On-line coupling between CE and MS is established through an electrospray-coaxial sheath flow interface. Enantiomer separation is achieved by using a cheap, nonvolatile, chiral selector as beta-cyclodextrin in the background electrolyte (BGE) together with a physically coated capillary that is aimed to prevent contamination of the electrospray. The capillary coating is simple and easy to obtain as it only requires flushing of the capillary with a polymer aqueous solution for 3 min. Optimization of CE parameters (pH of BGE, type and concentration of chiral selector, and capillary inner diameter) and electrospray-MS parameters (nature and flow rate of the sheath liquid, nebulizer pressure) is carried out. Two different derivatization protocols of amino acids using dansyl chloride (DNS) and fluorescein isothiocyanate (FITC) are compared in terms of MS sensitivity and chiral resolution. Under optimum CE-MS conditions it is observed that the MS sensitivity obtained for FITC- and DNS-amino acids is similar (with limit of detection (LOD) in the microM range, corresponding to amounts injected in the fmol range) while chiral resolution is better for FITC-amino acids. The optimized method is demonstrated to provide the simultaneous analysis of 15 selected amino acids (i.e., FITC-D/L-Asp, -Glu, -Ser, -Asn, -Ala, -Pro, -Arg, and FITC-gamma-aminobutyric acid (GABA) in a single chiral CE-MS run, corresponding to the main amino acids that can be found in orange. Moreover, as a result of the high resolution achieved, it is possible to detect down to 2% of D-Asp in the presence of 98% of L-Asp. The good possibilities of chiral CE-MS in food analysis are corroborated through the detection of the main amino acids in a commercial orange juice (i.e., FITC-L-Asp, -Glu, -Ser, -Asn, -Pro, -Arg, and the nonchiral FITC-GABA) as well as the determination of the fraudulent addition of synthetic amino acids (containing D- and L-forms) to a fresh orange juice.