Development of a CE-ESI-microTOF-MS method for a rapid identification of phenolic compounds in buckwheat

A sensitive CE-ESI-MS analytical method for the identification of buckwheat antioxidants has been developed. CE and ESI-TOF parameters (e.g. buffer composition and pH, sheath liquid composition, sheath liquid and gas flow rates, electrospray voltage) were optimized to obtain an optimal analytical separation and identification. The results confirmed the presence of phenolic acids, procyanidins and galloylated propelargonidins. The identification of swertiamacroside and 2-hydroxy-3-O-β-D-glucopyranosil-benzoic acid, found for the first time in our previous work, has been confirmed. Furthermore, 5,7,4'-trimethoxyflavan and dihydroxy-trimethoxyisoflavan have also been tentatively identified for the first time in buckwheat.     

Capillary electrophoresis-electrospray ionization-mass spectrometry method to determine the phenolic fraction of extra-virgin olive oil

We describe the first analytical method involving SPE and CZE coupled to ESI-IT MS (CZE-ESI-MS) used to identify and characterize phenolic compounds in olive oil samples. The SPE, CZE and ESI-MS parameters were optimized in order to maximize the number of phenolic compounds detected and the sensitivity of their determination. To this end we have devised a detailed method to find the best conditions for CE separation and the detection by MS of the phenolic compounds present in olive oil using a methanol-water extract of Picual extra-virgin olive oil (VOO). Electrophoretic separation was carried out using an aqueous CE buffer system consisting of 60 mM NH(4)OAc at pH 9.5 with 5% of 2-propanol, a sheath liquid containing 2-propanol/water 60:40 v/v and 0.1% v/v triethylamine. This method offers to the analyst the chance to study important phenolic compounds such as phenolic alcohols (tyrosol (TY), hydroxytyrosol (HYTY) and 2-(4-hydroxyphenyl)ethyl acetate), lignans ((+)-pinoresinol and (+)-1-acetoxypinoresinol), complex phenols (ligstroside aglycon (Lig Agl), oleuropein aglycon, their respective decarboxylated derivatives and several isomeric forms of these (dialdehydic form of oleuropein aglycon, dialdehydic form of ligstroside aglycon, dialdehydic form of decarboxymethyl elenolic acid linked to HYTY, dialdehydic form of decarboxymethyl elenolic acid linked to TY) and 10-hydroxy-oleuropein aglycon) and one other phenolic compound (elenolic acid) in extra-VOO by using a simple SPE before CE-ESI-MS analysis.     

Advantages of using a modified orthogonal sampling configuration originally designed for LC-ESI-MS to couple CE and MS for the determination of antioxidant phenolic compounds found in virgin olive oil

A ThermoFinnigan sheath liquid flow capillary electrophoresis-mass spectrometry system designed for coupling via a co-axial interface was coupled through an adapted via an alternative, commercially available interface for orthogonal sampling. The affordable, reversible structural alterations made in the commercial LC-MS interface resulted in improved analytical performance.The results of a conventional capillary electrophoresis (CE) method using a commercial co-axial source to determine antioxidant phenolic acids present in virgin olive oil, were compared with those obtained by using a modified orthogonal sampling position. In both cases, separations were done using a 10 mM ammonium acetate/ammonium hydroxide buffer solution at pH 10.0 and a constant applied voltage of 25 kV. The operating variables for the mass spectrometry interface were re-optimized for the modified orthogonal orientation. This allowed the sheath liquid, sheath gas flow rates and capillary voltage to be lowered with respect to the co-axial coupling configuration. In addition, the orthogonal sampling position provided a higher selectivity by effect of ion sampling excluding larger droplets—with an increased momentum along the axis—which were drained through the sink at the bottom of the ion source. Also, the new configuration facilitated sample ionization, improved electrospray stability and led to stronger signals as a result.The new system was validated in terms of precision (repeatability), linearity, and limits of detection and quantification. A comparison of the validation data with the results previously obtained by using a commercial co-axial configuration revealed the adapted orthogonal sampling position to provide better repeatability in both migration times and relative peak areas (<1% and 7% respectively with n = 15 replicates), a good linear range (with levels in the microgram-per-litre region) and lower limits of detection—especially for the compounds detected with the lowest sensitivity when co-axial ESI was used, as HFA, GEN, FER and VAN finding LOD among 24-3.0 μg L⁻¹ respectively.     

A capillary electrophoresis-tandem mass spectrometry methodology for the determination of non-protein amino acids in vegetable oils as novel markers for the detection of adulterations in olive oils

A new analytical methodology based on capillary electrophoresis-mass spectrometry (CE-MS(2)) is presented in this work, enabling the identification and determination of six non-protein amino acids (ornithine, β-alanine, GABA, alloisoleucine, citrulline and pyroglutamic acid) in vegetable oils. This methodology is based on a previous derivatization with butanol and subsequent separation using acidic conditions followed by on-line coupling to an ion trap analyzer for MS(2) detection established through an electrospray-coaxial sheath flow interface. The electrophoretic and interface parameters were optimized obtaining the separation of all compounds in less than 15 min and with resolutions higher than 5. The proposed method was validated by assessing its accuracy, precision (RSD<7% for corrected peak areas), LODs and LOQs (between 0.04-0.19 ng/g and 0.06-0.31 ng/g, respectively) and linearity range (R(2)>0.99), and it was used in order to identify the selected non-protein amino acids in soybean oils, sunflower oils, corn oils and extra virgin olive oils. MS(2) experiments performed the fingerprint fragmentation of these compounds allowing to corroborate ornithine and alloisoleucine in seed oils but not in olive oils. The method was applied to identify and quantify olive oil adulterations with soybean oil detecting in a single run the amino acids in mixtures up to 2% (w/w). The results showed a high potential in using these compounds as novel markers for the detection of adulterations of extra virgin olive oils with seed oils. Thus, the developed method could be considered a simple, rapid and reliable method for the quality evaluation of extra virgin olive oil permitting its authentication.     

Pressurized liquid extraction-capillary electrophoresis-mass spectrometry for the analysis of polar antioxidants in rosemary extracts

A method based on capillary electrophoresis-electrospray-mass spectrometry (CE-ESI-MS) was developed to qualitatively characterize natural antioxidants from rosemary (Rosmarinus officinalis L.) in different fractions obtained by pressurized liquid extraction (PLE) using subcritical water. The parameters of CE-ESI-MS were adjusted allowing the separation and characterization of different compounds from rosemary in the PLE fractions. These parameters for CE are kind, pH and concentration of the separation buffer, parameters for ESI-MS are dry gas temperature and flow, nebulizing gas pressure, and make-up flow. The following analytical conditions were found most favorable: aqueous CE buffer (40 mM ammonium acetate/ammonium hydroxide, pH 9); sheath liquid containing 2-propanol-water (60:40, v/v) and 0.1% (v/v) triethylamine at a flow rate of 0.24 mL/h; drying gas flow rate equal to 7 L/min at 350 degrees C, nebulizing gas pressure of 13.8 kPa (2 psi), using a compound stability of 50%. Different antioxidant compounds (e.g., rosmarinic acid and carnosic acid) could be detected in the rosemary extracts by CE-ESI-MS without any additional treatment, enabling the determination of variations in the extract composition caused by the different PLE conditions (i.e., 60 and 100 degrees C). The results provide complementary information to HPLC analysis.     

Presence of virgin olive oil phenolic metabolites in human low density lipoprotein fraction: determination by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

The biological benefits of olive oil in preventing the oxidation of low density lipoprotein (LDL) would seem to be linked to its high monounsaturated fatty acid contents, but also to its respective phenolic compounds contents. One prerequisite to assess the in vivo physiological significance of phenolic compounds is to determine their presence in human LDL following the ingestion of virgin olive oil.In this work, olive oil phenolic metabolites were identified using high-performance liquid chromatography in tandem with electrospray mass spectrometry (HPLC-ESI-MS/MS) detection, after solid phase extraction (SPE). Quantitative methods were developed in carrying out linearity, precision, sensitivity and recovery tests. The results from two methods of LDL separation were compared and shorter LDL isolation procedure showed a better recovery for antioxidants compounds in LDL. The metabolites identified in LDL were: hydroxytyrosol monoglucuronide, hydroxytyrosol monosulfate, tyrosol glucuronide, tyrosol sulfate and homovanillic acid sulfate. The fact that olive oil phenolic metabolites are able to bind LDL strengthens claims that these compounds act as in vivo antioxidants.     

Analytical determination of polyphenols in olive oils

The increasing popularity of olive oil is mainly attributed to its high content of oleic acid, which may affect the plasma lipid/lipoprotein profiles, and its richness in phenolic compounds, which act as natural antioxidants and may contribute to the prevention of human disease. An overview of analytical methods for the measurement of polyphenols in olive oil is presented. In principle, the analytical procedure for the determination of individual phenolic compounds in virgin olive oil involves three basic steps: extraction from the oil sample, analytical separation, and quantification. A great number of procedures for the isolation of the polar phenolic fraction of virgin olive oil, utilizing two basic extraction techniques, LLE or SPE, have been included. The reviewed techniques are those based on spectrophotometric methods, as well as analytical separation (gas chromatography (GC), high-performance liquid chromatography (HPLC), and capillary electrophoresis (CE)). Many reports in the literature determine the total amount of phenolic compounds in olive oils by spectrophometric analysis and characterize their phenolic patterns by capillary gas chromatography (CGC) and, mainly, by reverse phase high-performance liquid chromatography (RP-HPLC); however, CE has recently been applied to the analysis of phenolic compound of olive oil and has opened up great expectations, especially because of the higher resolution, reduced sample volume, and analysis duration. CE might represent a good compromise between analysis time and satisfactory characterization for some classes of phenolic compounds of virgin olive oils.     

Characterization of transferrin glycoforms in human serum by CE-UV and CE-ESI-MS

Human transferrin (Tf) is a model glycoprotein for congenital disorders of glycosylation (CDG) diagnosis. In the last few years, new CE-UV methods for intact Tf glycoforms analysis have been developed using nonvolatile BGEs and organic modifiers. However, the use of these BGEs does not allow the coupling of these procedures with electrospray MS (ESI-MS). In this study, a new CE-UV separation method of Tf glycoforms is developed, using a double-layer stable coating and a volatile BGE based on ammonium acetate. The separation method is optimized using standard Tf and their potential is demonstrated applying the method to the analysis of sera Tf from healthy individuals and CDG patients. The CE-UV separation method has been coupled to ESI-MS detection. Main parameters such as sheath liquid composition are optimized in order to obtain a good sensitivity. The CE-ESI-MS method has also been used in serum samples obtaining the separation of the different proteins present in serum and partial separation of Tf glycoforms. Different mass spectra and deconvoluted molecular masses were obtained for each sialoform, allowing unequivocal glycoform identification.     

Combined microwave-assisted isolation and solid-phase purification procedures prior to the chromatographic determination of phenolic compounds in plant materials

A fast, sensitive and selective procedure employing a combination of microwave-assisted extraction (MAE) and solid phase extraction (SPE) was applied prior to liquid chromatographic identification and quantification of phenolic compounds in plant materials. MAE has been tested and optimized for the isolation of phenolic acids (gallic, protocatechuic, p-hydroxybenzoic, chlorogenic, vanilic, caffeic, syringic, p-coumaric, ferulic, sinapic, benzoic, m-coumaric, o-coumaric, rosmarinic, cinnamic acids) and 3,4-dihydroxybenzaldehyde, syringaldehyde, p-hydroxybenzaldehyde, and vanillin in various plants. The effects of experimental conditions on MAE efficiency, such as solvent composition, temperature, extraction time, have been studied. The extraction efficiencies were compared with those obtained by computer-controlled, two-step Soxhlet-like extractions. Plant extracts were purified and phenolic compounds were pre-concentrated using SPE on polymeric RP-105 SPE sorbent prior to HPLC analysis. Chromatographic separation was carried out on a Hypersil BDS C₁₈ column using a mobile phase consisted of 0.3% (v/v) acetic acid in water (solvent A) and methanol (solvent B) at flow rate 0.6 ml min⁻¹ and column temperature 30 °C with gradient elution.     

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

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

Direct automatic determination of biogenic amines in wine by flow injection-capillary electrophoresis-mass spectrometry

A capillary electrophoresis-electrospray mass spectrometry (CE-ESI-MS) method for the separation and determination of nine biogenic amines is proposed. Operational variables, such as the voltage, temperature, sheath liquid composition, flow-rate, and MS parameters, were optimized. Samples are injected in the hydrodynamic mode into a 75 cm x 50 microm ID coated capillary and separated by using 25 mM citric acid at pH 2.0. Heptylamine is used as internal standard. The experimental setup includes a flow manifold coupled to the CE system for automatic insertion of samples into the CE vials. The proposed method allows amines to be determined with limits of detection from 0.018 to 0.09 microg x mL(-1) and relative standard deviation (RSD) values from 2.4% to 5.0% (except 6.8% for histamine). The method was successfully used to determine biogenic amines in red and white wines.     

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

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

Toward a screening method for the analysis of small intact proteins by CE-ESI-TOF MS

Capillary electrophoresis-mass spectrometry (CE-MS) more and more gains in importance as an analytical technique for the identification and characterization of intact proteins in the biopharmaceutical area. Thus, a CE-ESI-MS method was optimized and validated systematically with respect to the improved screening and characterization of intact proteins. The optimization was accomplished by variation of different CE-MS parameters, such as capillary coating, background electrolyte, sheath liquid, and nebulizer gas pressure, while monitoring both the resolution and signal intensities. Achievable separation is discussed quantitatively in the context of the coating and the resulting EOF, the protein mobilities, and the suction effect of the sprayer. The observed precisions of the optimized method regarding the migration times (mean RSD = 1.4%) and peak areas (mean RSD = 12.3%) and an extensive principal component analysis revealed that the presented method is reliable and useful for the quantitation of intact proteins and protein isoforms. The applicability of this method to various proteins showing different characteristics (pI value, molecular mass, hydrophobicity, etc.) is discussed. The presented method will contribute to the improved characterization of a large variety of intact proteins in the biomedical and pharmaceutical area.     

Capillary zone electrophoresis and capillary electrophoresis-mass spectrometry for analyzing qualitative and quantitative variations in therapeutic albumin

The present study describes a reproducible and quantitative capillary zone electrophoresis (CZE) method, which leads to the separation of nine forms (native, oxidized and glycated) of human serum albumin (HSA). In an attempt to identify the different species separated by this CZE method, the capillary electrophoresis was coupled to mass spectrometry using a sheath liquid interface, an optimized capillary coating and a suitable CE running buffer. CE-MS analyses confirmed the heterogeneity of albumin preparation and revealed new truncated and modified forms such as Advanced Glycation End products (AGEs). Assignment of the CZE peaks was carried out using specific antibodies, carboxypeptidase A or sample reduction before or during the CE separation. Thus, five HSA forms were unambiguously identified. Using this CZE method several albumin batches produced by slightly different fractionation ways could be discriminated. Furthermore, analyses of HSA preparations marketed by five pharmaceutical industries revealed that two therapeutic albumins, including that marketed by LFB, contained the highest proportion of native form and lower levels of oxidized forms.     

CE-ESI-MS separation of divalent organic and inorganic anions using a tricationic complexing reagent

A tricationic ion-pairing reagent, 1,3,5-1-butyl-3-methyl-1H-imidazol-3-ium-2,4,6-trimethylbenzene, was used to form complexes with doubly charged anions for their subsequent analysis by capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) in the positive-ion mode. This methodology offers the advantages of greater versatility and sensitivity relative to direct detection of the anions in negative-ion mode, and it can be realized by a number of possible complexation strategies, including pre-column, on-column, and post-column modes. Three model anions, sulfate [SO?2?], thiosulfate [TSFA, S?O?2?], and benzenedisulfonate [BZDS, C?H?(SO?)?2?], were amenable to complexation with the tricationic reagent, yielding singly charged cations with greater mass-to-charge (m/z) ratios than the native analytes. By utilizing optimized parameters obtained through previous work with dicationic reagents and singly charged anions, including the CE separation buffer composition and pH, the concentration of the dicationic reagent, the mode of complexation, the nebulizing gas pressure, and the sheath liquid composition, it was possible to develop a robust CE-ESI-MS method appropriate for the analysis of divalent anions in a mixture.     

Analysis of benzalkonium chloride by capillary electrophoresis-tandem mass spectrometry

Conditions for the separation and determination of benzalkonium chloride (BAC) homologues by CE with UV-detection and CE coupled to MS (IT) using electrospray as ionization source were established. The separation was performed using fused-silica capillaries of 50 microm id and 100 mM acetic acid-ammonium acetate buffer solution at pH 4.5 with 80% of ACN as carrier electrolyte. CE-MS coupling parameters were optimized and methanol-10 mM acetic acid (90:10 v/v) was selected as sheath liquid. Detection limits, based on an S/N of 3:1, were calculated, and values between 0.8 and 1.3 mg/L with CE-ESI/MS and around 0.5 mg/L with CE-ESI-MS/MS, using hydrodynamic injection (15 s, 3.5 kPa), were obtained. Good run-to-run and day-to-day precisions on concentration were achieved with RSDs lower than 8%. Quantitative analysis was carried out by the internal standard method and the calibration curves showed good linearities (r(2) > 0.98). The CE-ESI-MS/MS method was successfully applied to the analysis of BAC in different ophthalmic solutions, allowing the direct determination, identification and confirmation of the BAC homologues presented in these samples.     

Development and validation of a nonaqueous capillary electrophoretic method for the enantiomeric purity determination of a synthetic intermediate of new 3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans using a single-isomer anionic cyclodextrin derivative and an ionic liquid

The enantiomeric purity determination of a synthetic intermediate of new 3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans, i.e. 4-amino-2,2-dimethyl-6-ethoxycarbonylamino-3,4-dihydro-2H-1-benzopyran, was successfully carried out using an anionic cyclodextrin (CD) derivative combined with a chiral ionic liquid (IL). In order to obtain high resolution and efficiency values, the addition of a chiral IL, i.e. ethylcholine bis(trifluoromethylsulfonyl)imide (EtChol NTf₂), to the background electrolyte containing heptakis(2,3-di-O-methyl-6-O-sulfo)-β-CD (HDMS-β-CD) was found to be essential. A simultaneous increase in separation selectivity and enantioresolution seems to indicate a synergistic effect of HDMS-β-CD and EtChol NTf₂. The best enantioseparation of the key intermediate was achieved using a methanolic solution of 0.75 M formic acid, 10 mM ammonium formate, 1.5 mM HDMS-β-CD and 5 mM EtChol NTf₂. Levamisole was selected as internal standard. The optimized conditions allowed the determination of 0.1% of each enantiomer in the presence of its stereoisomer using the method of standard additions. The NACE method was then fully validated with respect to selectivity, response function, trueness, precision, accuracy, linearity and limits of detection and quantification.     

Determination of phenolic acids in olive oil by capillary electrophoresis

A CZE method for the separation and quantitation of phenolic acids (cinnamic, syringic, p-coumaric, vanillic, caffeic, 3,4-dihydroxyphenylacetic, protocatechuic), extracted from extra virgin olive oil, was developed. The sample preparation involved the LLE and SPE extraction methods. CE separation was performed in a fused silica capillary of I.D.= 50microm using as a BGE 40 mM borate buffer at pH=9.2. The separation voltage was 18kV with corresponding current of 27-28 microA. Detection was accomplished with UV-detector at lambda=200nm. The proposed method was fully validated. A good repeatability of migration time (RSD% ranged from 0.81 to 1.63) and of corrected peak area (RSD% from 2.89 to 5.77) was obtained. The linearity of detector response in the range from 5 to 50 ppm was checked, obtaining the correlation coefficient R2 values in the range: 0.9919-0.9997. Some phenolic acids in real oil samples were detected and quantified with the proposed method.     

Determination of stimulants and narcotics as well as their in vitro metabolites by online CE-ESI-MS

A simple, rapid and sensitive CE-ESI-MS method for the simultaneous analysis of seven stimulants and narcotics (amphetamine, ephedrine, methadone, pethidine, tetracaine, codeine and heroin) was developed. The CE-ESI-MS experimental conditions were optimized as follows: 20?mmol/L ammonium acetate with pH 9.0 as running buffer, the separation voltage of 22?kV and the sheath liquid of isopropanol/water (1:1 v/v) containing 7.5?mmol/L acetic acid with 3.0?μL/min flow rate. Under the optimized conditions, the stimulants and narcotics were well separated within 4.6?min using a 70-cm length fused-silica capillary (50?μm id). The detection limits (S/N=3) of the CE-ESI-MS analysis were in the range of 0.40-1.0?ng/mL. Method repeatability of intra-day and inter-day was satisfactory. The recoveries obtained from the analysis of spiked urine samples were between 84.1 and 108%. The developed method was successfully applied for the simultaneous analysis of methadone, pethidine and codeine and their in vitro metabolites.     

Direct olive oil authentication: detection of adulteration of olive oil with hazelnut oil by direct coupling of headspace and mass spectrometry, and multivariate regression techniques

Control of adulteration of olive oil, together with authentication and contamination, is one of the main aspects in the quality control of olive oil. Adulteration with hazelnut oil is one of the most difficult to detect due to the similar composition of hazelnut and olive oils; both virgin olive oil and olive oil are subjected to that kind of adulteration. The main objective of this work was to develop an analytical method able to detect adulteration of virgin olive oils and olive oils with hazelnut oil by means of its analysis by a headspace autosampler directly coupled to a mass spectrometer used as detector (ChemSensor). As no chromatographic separation of the individual components of the samples exists, a global signal of the sample is obtained and employed for its characterization by means of chemometric techniques. Four different crude hazelnut oils from Turkey were employed for the development of the method. Multivariate regression techniques (partial least squares and principal components analysis) were applied to generate adequate regression models. Good values were obtained in both techniques for the parameters employed (standard errors of prediction (SEP) and prediction residual error sum of squares (PRESS)) to evaluate its goodness. With the proposed method, minimum adulteration levels of 7 and 15% can be detected in refined and virgin olive oils, respectively. Once validated, the method was applied to the detection of such adulteration in commercial olive oil and virgin olive oil samples.