Elena Domínguez-Vega

CE‐TOF MS analysis of complex protein hydrolyzates from genetically modified soybeans – A tool for foodomics

A CE‐TOF MS proteomic approach was applied for the analysis of hydrolyzates from complex soybean protein mixtures. After CE‐TOF MS method development, the new approach provided the simultaneous analysis of more than 150 peptides from the soybean protein fraction soluble in ACN‐water (80/20 v/v). The method is fast (about 30 min of analysis per sample) and is characterized by a relatively low running cost. The approach was used to study the substantial equivalence between a genetically modified variety of soybean compared with its traditional counterpart. No significant differences were found between the two studied soybeans based on the protein fraction studied. The capacity of the CE‐TOF MS method to analyze complex mixtures of peptides in short times opens interesting possibilities in the growing Foodomics area.

Enantiomeric separation of FMOC‐amino acids by nano‐LC and CEC using a new chiral stationary phase, cellulose tris(3‐chloro‐4‐methylphenylcarbamate)

A novel polysaccharide‐based chiral stationary phase (CSP), cellulose tris(3‐chloro‐4‐methylphenylcarbamate), also known as Sepapak‐2 or Lux Cellulose‐2, has been evaluated for the enantiomeric separation of FMOC derivatives of amino acids. After mobile‐phase optimization in nano liquid chromatography (nano‐LC) the column enabled the enantiomeric separation of 19 out of 23 amino acids tested, indicating the high chiral recognition power of this new CSP. Subsequently, a comparison of the driving force employed (pressure or voltage) was carried out comparing nano‐LC and CEC under the same conditions. Better peak efficiencies and resolution were observed by using CEC experiments, which enabled the chiral discrimination of 20 out of 23 amino acids tested. Finally, in order to show the potential of this new CSP, the determination of the content and the enantiomeric purity of the non‐protein amino acid citrulline in food supplements was performed. For that purpose, the method was optimized, evaluated and applied to different commercial samples.

Evaluation of new cellulose-based chiral stationary phases Sepapak-2 and Sepapak-4 for the enantiomeric separation of pesticides by nano liquid chromatography and capillary electrochromatography.

Two novel polysaccharide-based chiral stationary phases (CSPs), known as Sepapak-2 (cellulose tris(3-chloro-4-methylphenylcarbamate)) and Sepapak-4 (cellulose tris(4-chloro-3-methylphenylcarbamate)), have been evaluated in this work for the chiral separation of a group of 16 pesticides including herbicides, insecticides and fungicides. The optimization of the mobile phase employed in nano-liquid chromatography (nano-LC) enabled the chiral separation of seven pesticides on Sepapak-2 and of nine pesticides on Sepapak-4. Due to the fact that Sepapak-4 gave better results, this column was selected to compare nano-LC and capillary electrochromatography (CEC) under the same conditions that consisted in the use of a 90/9/1 (v/v/v) ACN/H₂O/ammonium formate (pH 2.5) background electrolyte (BGE). As expected, both the efficiency and the chiral resolution obtained in CEC experiments were higher than in nano-LC for all the analyzed compounds. The analytical characteristics of the CEC developed methodology were evaluated in terms of linearity, LODs, LOQs, precision, selectivity, and accuracy allowing its application to the quantitation of metalaxyl and its enantiomeric impurity in a commercial fungicide product marketed as enantiomerically pure (metalaxyl-M) and in soil and tap water samples after solid phase extraction (SPE). The determined amount of metalaxyl-M was found to be a 26% above the labeled content and it contained an enantiomeric impurity of a 3.7% of S-metalaxyl was determined.

Recent advances in CE analysis of antibiotics and its use as chiral selectors.

Antibiotics are a class of therapeutic molecules widely employed in both human and veterinary medicine. This article reviews the most recent advances in the analysis of antibiotics by CE in pharmaceutical, environmental, food, and biomedical fields. Emphasis is placed on the strategies to increase sensitivity as diverse off‐line, in‐line, and on‐line preconcentration approaches and the use of different detection systems. The use of CE in the microchip format for the analysis of antibiotics is also reviewed in this article. Moreover, since the use of antibiotics as chiral selectors in CE has grown in the last years, a new section devoted to this aspect has been included. This review constitutes an update of previous published reviews and covers the literature published from June 2011 until June 2013.

Development of a CE-ESI-ITMS method for the enantiomeric determination of the non-protein amino acid ornithine

Unequivocal enantiomeric determination by CE‐MS2 of the non‐protein amino acid ornithine (Orn), previously derivatized with FITC, was carried out in this work. A CE‐tandem MS system was used to combine the potential of CE in chiral separations with the sensitivity and selectivity of tandem MS detection. The electrospray‐coaxial sheath flow interface conditions were optimized and an IT analyzer working in the MS2 mode was employed to provide the best sensitivity and selectivity. Satisfactory results were obtained in terms of linearity (r2>0.99), precision (RSD from 1.3 to 2.3% for migration times and from 11.0 to 18.6% for corrected peak areas), and LOD (2×10−9 M). Interestingly, the CE‐MS2 method developed allowed a sensitivity enhancement of 100 folds with respect to UV detection. The results demonstrated the feasibility for carrying out quantitative CE‐ESI‐MS2 determinations of Orn enantiomers in beers. Thus, L‐Orn was found in the 16 beer samples analyzed at concentrations ranging from 1×10−6 to 2×10−5 M, whereas the percentages of D‐Orn with respect to the total amount of Orn reached values between 1.5 and 10.0%.

Enantiomeric separation of ornithine in complex mixtures of amino acids by EKC with off-line derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate

A new analytical methodology was developed by EKC enabling the fast enantiomeric separation of Ornithine in complex mixtures of amino acids. A previous derivatization step with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was achieved to enable the sensitive UV detection of amino acids as well as to make possible their interaction with the CDs employed as chiral selectors. A dual CD system containing an anionic and a neutral CD in phosphate buffer at acid pH showed a high resolving power allowing the enantiomeric separation of 18 protein amino acids and Orn. The method was applied to the analysis of fermented foods to investigate the extent of the presence of Orn enantiomers.

Potential of vancomycin for the enantiomeric resolution of FMOC‐amino acids by capillary electrophoresis‐ion‐trap‐mass spectrometry

The potential of the antibiotic vancomycin (VC) as chiral selector for the enantiomeric separation of amino acids by CE‐ESI‐MS/MS2 was investigated for the first time in this work. Derivatization of amino acids with FMOC‐Cl was carried out to enable their interaction with VC as well as the formation of precursor ions with larger m/z which were employed in MS2 experiments. The partial filling of a coated capillary was employed to avoid the loss in MS sensitivity originated by the introduction of VC in the ionization source. Under optimized conditions, the simultaneous enantiomeric separation and unequivocal identification of 17 amino acids (two of them being nonprotein amino acids) took place in about 20 min with LODs in the micromolar range.

Low-picomolar analysis of peptides by on-line coupling of fritless solid-phase extraction to sheathless capillary electrophoresis-mass spectrometry.

A novel fritless solid-phase extraction (SPE) microcartridge was designed for combination with sheathless capillary electrophoresis-mass spectrometry (sheathless CE-MS) employing a prototype porous-tip capillary for nanoelectrospray ionization (nanoESI). The inlet of the separation capillary (30μm inner diameter (id), 150μm outer diameter (od)) was inserted in a 4mm long SPE microcartridge (150μm id, 365μm od) packed with a C18 sorbent of 55-105μm particle size. Performance of the SPE-CE-MS system was evaluated using diluted solutions of the three opioid peptides dynorphin A (1-7) (DynA), endomorphin 1 (End1) and met-enkephalin (Met). Sample volumes of 1.5μL were loaded on the SPE microcartridge and the retained peptides were eluted with 22nL of an acidic methanol/water (60:40, v/v) solution. Using a pressure of 50mbar during separation to speed up the analysis, good peptide resolution was obtained with acceptable plate numbers (between 53,000 and 92,000). Intraday relative standard deviations (% RSD) for peptide migration times and peak areas were below 4% and 9%, respectively. The SPE-CE-MS method showed good linearity in the 0.05-5ngmL(-1) range and limits of detection (LODs) were 10pgmL(-1). However, loading a larger volume of sample (8μL), LODs could be decreased down to 2pgmL(-1) (2.2-3.5pM). This represents an improvement of up to 5000-fold with respect to the LODs achieved by sheathless CE-MS without on-line preconcentration demonstrating the potential of on-line SPE for further enhancing sensitivity.

Fast derivatization of the non-protein amino acid ornithine with FITC using an ultrasound probe prior to enantiomeric determination in food supplements by EKC.

An EKC method for the determination of ornithine (Orn) enantiomers has been developed after a fast pre‐capillary derivatization with FITC. The derivatization step was needed to provide a chemical moiety to the Orn molecule, enabling a sensitive UV detection and the interaction with the CDs used as chiral selectors. To accelerate the derivatization reaction, an ultrasound probe was used. For the development of the chiral method, the influence of different experimental conditions (type and concentration of the chiral selector, temperature, and separation voltage) was investigated. Due to the anionic nature of the analyte (FITC‐Orn), five neutral CDs were employed as chiral selectors. The native γ‐CD showed the highest chiral separation power, observing that a low concentration of this CD (1 mM), using a working temperature of 25°C and a separation voltage of 20 kV, enabled to obtain the highest enantioresolution for Orn and its separation from other amino acids usually present in food supplements. After optimizing the method for the preconditioning of the capillary, the analytical characteristics of the chiral method were established. Linearity, LOD and LOQ, precision, and accuracy were evaluated previously to the determination of Orn enantiomers contained in ten commercial food supplements. No interferences from other amino acids present in these samples were observed.

First approach based on direct ultrasonic assisted enzymatic digestion and capillary-high performance liquid chromatography for the peptide mapping of soybean proteins.

This work proposes, for the first time, the use of a high intensity ultrasonic probe to accelerate the tryptic digestion of soybean proteins. Different digestion parameters were optimized: protein extracting solution, reduction, and alkylation conditions (time, concentration, and temperature), trypsin:protein ratio, and ultrasonic conditions (sonication amplitude and time). Separation of peptide profiles was carried out by capillary-HPLC. The effect of the variation of chromatographic conditions (elution gradient, column temperature, and injection volume) on peptide separation was also studied using two capillary-HPLC columns with different column diameters and particle sizes. Moreover, samples were focused at the top of the column in order to obtain an increasing sensitivity without loss of efficiency. This method was successfully applied to the profiling of soybean peptides from transgenic and non-transgenic soybeans and from different pigmented beans commercialized as soybeans.