Capillary Electrophoresis-ion Trap Mass Spectrometry Research Articles

Fast determination of harmala alkaloids in edible algae by capillary electrophoresis mass spectrometry.

The use of algae as a foodstuff is rapidly expanding worldwide from the East Asian countries, where they are also used for medical care. Harmala alkaloids (HAlk) are a family of bioactive compounds found in the extracts of some plants, including wakame (Undaria pinnatifida), an edible marine invasive algae. HAlks are based on a characteristic β-carboline structure with at least one amino ionizable group. In this work, we report the successful separation of a mixture of six HAlks (harmine, harmaline, harmol, harmalol, harmane, and norharmane) by capillary electrophoresis ion-trap mass spectrometry (CE-IT-MS) in less than 8 min. Optimum separation in fused-silica capillaries and detection sensitivity in positive-ion mode were achieved using a background electrolyte (BGE) with 25 mmol L(-1) ammonium acetate (pH 7.8) and 10% (v/v) methanol, and a sheath liquid with 60:40 (v/v) isopropanol-water and 0.05% (v/v) formic acid. The separation method was validated in terms of linearity, limits of detection and quantification, repeatability, and reproducibility. Later, a sample pretreatment was carefully optimized to determine HAlks in commercial wakame samples with excellent recovery and repeatability. For the complex wakame extracts, the MS-MS fragmentation patterns of the different HAlks were useful to ensure a reliable identification. The complete procedure was validated using the standard-addition calibration method, determining matrix effects on the studied compounds. Harmalol, harmine, and harmaline were naturally present in the samples and were quantified at very low concentrations, ranging from 7 to 24 μg kg(-1) dry algae.

Identification of bioactive peptides in hypoallergenic infant milk formulas by capillary electrophoresis–mass spectrometry

In this study, we use capillary electrophoresis–mass spectrometry (CE–MS) for the identification of bioactive peptides in hypoallergenic infant milk formulas (IF), which are complex bovine milk protein hydrolysates. A sample clean-up pretreatment with a citrate buffer containing dithiothreitol and urea followed by solid-phase extraction (SPE) with different reversed-phase commercial cartridges was investigated to achieve optimum detection sensitivity in CE–MS. SPE with C18, StrataX and Oasis HLB cartridges allowed detection of the largest number of low molecular mass components, but combination of C18 and StrataX results was enough to achieve an excellent coverage of the studied IF. The monoisotopic molecular mass values of the low molecular mass components obtained by capillary electrophoresis ion-trap mass spectrometry (CE–IT–MS) allowed the tentative identification of nine bioactive sequences. Only the identification of five of them could be confirmed when accurate mass measurements were performed by capillary electrophoresis time-of-flight mass spectrometry (CE–TOF–MS), namely LKP, IPY, ALPM, PGPIHN and VAGTWY, which were reported to present angiotensin-converting enzyme (ACE) inhibitory and antimicrobial activity (only VAGTWY).

Ion-trap versus time-of-flight mass spectrometry coupled to capillary electrophoresis to analyze biogenic amines in wine

In this work, two different capillary electrophoresis–mass spectrometry (CE–MS) methods, namely, capillary electrophoresis–ion-trap mass spectrometry (CE–IT-MS) and capillary electrophoresis–time-of-flight mass spectrometry (CE–TOF-MS), applied to analyze biogenic amines in wine samples are investigated. A group of five amines was selected as case study (namely, putrescine, cadaverine, histamine, phenylethylamine and tyramine) since they are the most frequently biogenic amines found in wines. The possibilities of both instruments in terms of sensitivity, selectivity and quantitation during the determination of the mentioned biogenic amines in wine samples was studied and their results corroborated by HPLC. After optimization of the analytical conditions, CE–IT-MS and CE–TOF-MS allowed the identification of biogenic amines in wines without any previous treatment except diluting 1:1 with water and filtering. Biogenic amines were determined in three red wines and one white wine showing, as expected, a higher concentration in red wines. Moreover, CE–IT-MS and CE–TOF-MS were compared regarding their capacity to detect other biogenic amines different to the selected ones in wine samples, showing CE–TOF-MS a much better capability (i.e., putrescine, cadaverine, histamine, phenylethylamine, tyramine, triptamine, spermidine, spermine, ethanolamine and isoamylamine were identified by CE–TOF-MS in a single analysis). Moreover, CE–TOF-MS allowed the quantitation of biogenic amines with limits of detection as low as 10 ng/mL, comparable to those obtained using HPLC with fluorescence detection, without any previous derivatization step and with analysis times fivefold faster (40 min by HPLC and 8 min by CE–TOF-MS).

Separation and identification of isomeric acidic degradation products of organophosphorus chemical warfare agents by capillary electrophoresis-ion trap mass spectrometry

Capillary electrophoresis (CE) coupled to ion trap mass spectrometry (MS) was evaluated for the separation and identification of chemical warfare agent degradation products (alkylphosphonic acids and alkyl alkylphosphonic acids). Different analytical parameters were optimized in negative ionization mode such as electrolyte composition (15 mM CH 3COONH 4, pH 8.8), sheath liquid composition (MeOH/H 2O/NH 3, 75:25:2, v/v/v), nebulization and ion trapping conditions. A standard mixture of five alkylphosphonic (di)acids and five alkyl alkylphosphonic (mono)acids containing isomeric compounds was used in order to evaluate CE selectivity and MS identification capability. The obtained electropherograms revealed that CE selectivity was very limited in the case of alkyl alkylphosphonic acid positional isomers, whereas isomeric isopropylphosphonic and propylphosphonic acids were baseline-separated. CE–MS–MS experiments provided an unambiguous identification of each isomeric co-migrating alkyl alkylphosphonic acids thanks to the presence of specific fragment ions. On the other hand, CE separation was mandatory for the identification of isomeric alkylphosphonic acids, which led to the same fragment ion and could not be differentiated by MS–MS. The developed method was applied to the analysis of soil extracts spiked with the analytes (before or after extraction treatment) and appeared to be very promising since resolution and sensitivity were similar to those observed in deionized water. Especially, analytes were detected and identified in soil extract spiked at 5 μg mL −1 with each compound before extraction treatment.

Nonaqueous capillary electrophoresis-mass spectrometry of synthetic polymers.

In this work, the separation and characterization of ionizable organic polymers nonsoluble in water is carried out using nonaqueous capillary electrophoresis-ion trap mass spectrometry (NACE-MS). The polymers studied are poly(N(epsilon)-trifluoroacetyl-l-lysine) (poly(TFA-Lys)) obtained by ring-opening polymerization of the corresponding N-carboxyanhydride. Different parameters (i.e., liquid sheath nature and flow rate, electrospray temperature, and separation buffer composition) are optimized in order to obtain both an adequate CE separation and a high MS signal of the samples under study. The optimum NACE-MS separation conditions allow the molecular mass characterization of poly(TFA-Lys) up to a degree of polymerization of 38. NACE-MS provides interesting information on the chemical structure of (i). the polymer end groups and (ii). other final byproducts. The MS spectra obtained by using this CE-MS protocol confirm that the polymerization was initiated by the reaction of n-hexylamine (initiator) on the monomer. CE-MS-MS and CE-MS-MS-MS results demonstrate that two different termination reactions occurred during the polymerization process leading to the transformation of the reactive amine end group into a carboxylic or a formyl groups. Byproducts such as 3-hydantoinacetic acid or diketopiperazine were also detected. To our knowledge, this is the first work in which the great possibilities of NACE-MS and NACE-MS(n) for characterizing synthetic polymers are demonstrated.

Capillary electrophoresis-ion trap mass spectrometry analysis of Ziagen and its phosphorylated metabolites.

A capillary electrophoresis-ion trap mass spectrometry method with a time-segment program was developed to simultaneously analyze Ziagen and its phosphorylated metabolites such as carbovir monophosphate, carbovir diphosphate, and carbovir triphosphate. By using the time-segment program, the positively charged nucleoside analog and negatively charged nucleotides were separated and detected in a single electrophoretic run. The limits of detection were less than 2 micro M for all of the analytes. Calibration curves of the compounds showed excellent linearity over the range of 2-100 micro M. The capability of the method was demonstrated by analyzing Ziagen and its phosphorylated metabolites that were spiked in cellular extracts of human peripheral blood mononuclear cells at 20 micro M levels. Some endogenous nucleotides such as adenosine 5'-triphosphate, adenosine 5'-diphosphate, and adenosine 5'-monophosphate, were also detected in the cellular extracts.

Pressure-assisted capillary electrophoresis–electrospray ion trap mass spectrometry for the analysis of heparin depolymerised disaccharides

A pressure-assisted capillary electrophoresis–ion trap mass spectrometry method was developed for the analysis of eight heparin-derived disaccharides. A 30 m M formic acid buffer at pH 3.20 was selected as running electrolyte, and the separation was performed by the simultaneous application of a CE voltage of −30 kV and an overimposed pressure of 0.5 p.s.i. (3.45 kPa). The application of pressure assistance was needed to provide stable electrospray conditions for successful coupling. The linearity of the CE–MS and CE–MS–MS methods was checked under these conditions. Quality parameters such as run-to-run precision and limits of detection were established in both CE–MS and CE–MS–MS modes. Finally, enzymatically depolymerised bovine and porcine mucosal heparins were analysed in this CE–MS system and the characteristic relative molar percentages of major and minor disaccharides were calculated.

Analysis of codeine, dihydrocodeine and their glucuronides in human urine by electrokinetic capillary immunoassays and capillary electrophoresis–ion trap mass spectrometry

Screening for and confirmation of illicit, abused and banned drugs in human urine is a timely topic in which capillary separation techniques play a key role. Capillary electrophoresis (CE) represents the newest technology employed in this field of analysis. Two rapid competitive binding, electrokinetic capillary-based immunoassays are shown to be capable of recognizing the presence, but not the identity, of urinary opioids, namely codeine (COD), codeine-6-glucuronide, dihydrocodeine (DHC), dihydrocodeine-6-glucuronide, morphine (MOR), morphine-3-glucuronide and ethylmorphine (EMOR). In these approaches, aliquots of urine and immunoreagents of a commercial, broadly cross-reacting fluorescence polarization immunoassay for opiates were combined and analyzed by capillary zone electrophoresis or micellar electrokinetic capillary chromatography with laser induced fluorescence detection. With the fluorescent tracer solution employed, the former method is shown to provide simple electropherograms which are characterized by an opioid concentration dependent magnitude of the free tracer peak. In presence of dodecyl sulfate micelles, however, two tracer peaks with equal opioid concentration sensitivity are monitored. These data suggest the presence of two fluorescent tracers which react competitively with the urinary opioids for the binding sites of the antibody. Assay sensitivities for COD and MOR are comparable (10 ng/ml), whereas those for DHC and EMOR are about four-fold lower. Furthermore, glucuronides are shown to react like the corresponding free opioids. Analysis of urines that were collected after administration of 7 mg COD and 25 mg DHC tested positively in both assay formats. The presence of the free and conjugated codeinoids in these urines and their identification was accomplished by capillary electrophoresis–ion trap mass spectrometry (CE–MS). This confirmatory assay is based upon solid-phase extraction using a mixed-mode polymer cartridge followed by CE hyphenated to the LCQ mass spectrometer with electrospray ionization in the positive ion mode. With this technology, MS 2 is employed for proper identification of COD ( m/z 300.4) and DHC ( m/z 302.4) whereas MS 3 provides unambiguous identification of the glucuronides of COD ( m/z 476.5) and DHC ( m/z 478.5) via their fragmentation to COD and DHC, respectively. MS n ( n≥2) is shown to be capable of properly identifying the urinary codeinoids on the 100–200 ng/ml concentration level.

Confirmation testing of amphetamines and designer drugs in human urine by capillary electrophoresis-ion trap mass spectrometry.

Monitoring of amphetamines and designer drugs in human urine is a timely topic in clinical toxicology, surveillance of drug substitution, forensic science, drug testing at the workplace, and doping control. Confirmation testing of urinary amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) and 3,4-methylenedioxyamphetamine (MDA) by capillary electrophoresis (CE) combined with atmospheric pressure electrospray ionization and ion trap mass spectrometry (MS) is described. Using an aqueous pH 4.6 buffer composed of ammonium acetate/acetic acid, CE-MS and CE-MS2 provided data that permitted the unambiguous confirmation of these drugs in external quality control urines. Furthermore, other drugs of abuse present in alkaline urinary extracts, including methadone and morphine, could also be monitored. The data presented illustrate that the sensitivity achieved with the benchtop MS is comparable to that observed by CE with UV absorption detection. CE-MS2 is further shown to be capable of identifying comigrating compounds, including the comigration of amphetamine with nicotine.

Multianalyte capillary electrophoresis assays for screening and confirmation of urinary drugs of abuse

Screening for and confirmation of drugs of abuse in human urine is a timely topic in which capillary separation techniques play a key role. Capillary electrophoresis (CE) represents the newest technology employed in this field of analysis. Simple multianalyte electrokinetic capillary-based competitive immunoassays, assays based upon drug extraction and achiral–chiral CE with on-column multiwavelength detection, and a capillary electrophoresis-ion trap mass spectrometry assay were developed. These techniques are shown to comprise an analytical scheme beginning with presumptive immunological drug testing, followed by drug differentiation with spectral identification, and ending with complete confirmatory drug testing that is based upon tandem mass spectrometry. With data obtained from the urine of a polydrug abuser who was found to have ingested amphetamine, methadone, and other drugs, the suitability and limitations of the electrokinetic capillary methods for screening and confirmation of urinary methadone, its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine and amphetamine are discussed. CE coupled to tandem mass spectrometry is further shown to represent a comprehensive approach for analysis of other drugs in the patient urine. ©2000 John Wiley & Sons, Inc. J Micro Sep 12: 13–24, 2000

Quantitative Capillary Electrophoresis-Ion Spray Mass Spectrometry on a Benchtop Ion Trap for the Determination of Isoquinoline Alkaloids

The quantitative determination of some isoquinoline alkaloid natural products is described using combined capillary electrophoresis-ion trap mass spectrometry (CE/MS). The benchtop ion trap is a previously described modified commercial Varian Saturn II system equipped with an in-house constructed atmospheric pressure ionzation (API) ion source which was operated in the ion spray CE/MS mode. All relevant operational parameters have been optimized for the CE/MS experiments described in this work. A coaxial sheath flow of solvent buffer was used to facilitate ion spray CE/MS operation. Analysis of synthetic mixtures containing nine related isoquinoline alkaloids provided full-scan mass spectra for these compounds with injected quantities as low as 370 attomole