The Quantitative Measurement of Peptidoglycan Components Obtained from Acidic Hydrolysis in Gram-Positive and Gram-Negative Bacteria via Hydrophilic Interaction Liquid Chromatography Coupled with Mass Spectrometry.

The high throughput in genome sequencing and metabolic model (MM) reconstruction has democratised bioinformatics approaches such as flux balance analysis. Fluxes' prediction accuracy greatly relates to the deepness of the MM curation for a specific organism starting from the cell composition. One component is the cell wall, which is a functional barrier (cell shape, exchanges) with the environment. The bacterial cell wall (BCW), including its thickness, structure, and composition, has been extensively studied in Escherichia coli but poorly described for other organisms. The peptidoglycan (PG) layer composing the BCW is usually thinner in Gram- bacteria than in Gram+ bacteria. In both bacteria groups, PG is a polymeric mesh-like structure of amino acids and sugars, including N-acetylglucosamine, N-acetylmuramic acid, and amino acids. In this study, we propose a high-throughput method to characterise and quantify PG in Gram-positive and Gram-negative bacteria using acidic hydrolysis and hydrophilic interaction liquid chromatography coupled with mass spectrometry (HILIC-MS). The method showed a relatively short time frame (11 min analytical run), low inter- and intraday variability (3.2% and 4%, respectively), and high sensitivity and selectivity (limits of quantification in the sub mg/L range). The method was successfully applied on two Gram-negative bacteria (Escherichia coli K12 MG1655, Bacteroides thetaiotaomicron DSM 2079) and one Gram-positive bacterium (Streptococcus salivarius ssp. thermophilus DSM20259). The PG concentration ranged from 1.6% w/w to 14% w/w of the dry cell weight. The results were in good correlation with previously published results. With further development, the PG concentration provided by this newly developed method could reinforce the curation of MM.

Quantitative Analysis of Oat (Avena sativa L.) and Pea (Pisum sativum L.) Saponins in Plant-Based Food Products by Hydrophilic Interaction Liquid Chromatography Coupled with Mass Spectrometry.

This work presents the sample extraction methods for solid and liquid sample matrices for simultaneous quantification of oat (Avena sativa L.) and pea (Pisum sativum L.) saponins: avenacoside A, avenacoside B, 26-desglucoavenacoside A, and saponin B and 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) saponin, respectively. The targeted saponins were identified and quantified using a hydrophilic interaction liquid chromatography with mass spectrometric detection (HILIC-MS) method. The simple and high-throughput extraction procedure was developed for solid oat- and pea-based food samples. In addition, a very simple extraction procedure for liquid samples, without the need to use lyophilisation, was also implemented. Oat seed flour (U-13C-labelled) and soyasaponin Ba were used as internal standards for avenacoside A and saponin B, respectively. Other saponins were relatively quantified based on avenacoside A and saponin B standard responses. The developed method was tested and successfully validated using oat and pea flours, protein concentrates and isolates, as well as their mixtures, and plant-based drinks. With this method, the saponins from oat- and pea-based products were separated and quantified simultaneously within 6 min. The use of respective internal standards derived from U-13C-labelled oat and soyasaponin Ba ensured high accuracy and precision of the proposed method.

Development of a capillary electrophoresis method with direct UV detection for the analysis of thiodiglycol and its oxidation products.

A novel method based on CE with precolumn derivatization and direct UV detection for the determination of thiodiglycol (TDG), TDG sulfoxide, and TDG sulfone in water samples was developed. The lack of a UV chromophore of target analytes was overcome by derivatization with phthalic anhydride. The reactant concentrations, as well as the derivatization dependence on heating temperature and time, were carefully investigated. The baseline separation of three derivatives was achieved in less than 8 min by applying a simple BGE composed of a 30 mM borate buffer at pH 8.5. Several parameters affecting the separation efficiency (buffer pH and concentration, capillary temperature, and applied voltage) were evaluated. Calibration curves of all compounds showed good linear correlations (R(2) > 0.9994). The LODs of the TDG and its oxidation products were in the range of 98-154 ng/mL. The precision tests resulted in RSDs for migration times and peak areas of less than 1.2 and 3.6%, respectively. The developed method was successfully applied for the analysis of TDG and oxidation products in seawater, utilizing the carbon aerogel-based adsorbents for sample purification and concentration. Additionally, the method has the potential to be transformed into a portable CE format.

Fingerprinting postblast explosive residues by portable capillary electrophoresis with contactless conductivity detection.

A portable capillary electrophoretic system with contactless conductivity detection was used for fingerprint analysis of postblast explosive residues from commercial organic and improvised inorganic explosives on various surfaces (sand, concrete, metal witness plates). Simple extraction methods were developed for each of the surfaces for subsequent simultaneous capillary electrophoretic analysis of anions and cations. Dual-opposite end injection principle was used for fast (<4 min) separation of 10 common anions and cations from postblast residues using an optimized separation electrolyte composed of 20 mM MES, 20 mM l-histidine, 30 µM CTAB and 2 mM 18-crown-6. The concentrations of all ions obtained from the electropherograms were subjected to principal component analysis to classify the tested explosives on all tested surfaces, resulting in distinct cluster formations that could be used to verify (each) type of the explosive.

Capillary electrophoresis--a new tool for ionic analysis of exhaled breath condensate.

Exhaled breath condensate has been analyzed for its ionic content by capillary electrophoresis with capacitively coupled contactless conductometric detection. A simple device for collection of small volumes (100-200 µL) of exhaled breath condensate in less than 2 min was developed. A method for simultaneous determination of inorganic cations, inorganic anions and organic anions from the samples using dual-opposite end injection principle with a short fused silica capillary (35 cm, 50 µm I.D.) was developed. A background electrolyte composed of 20mM 2-(N-morpholino)ethanesulfonic acid, 20 mM l-histidine, 30 µM cetyltrimethylammonium bromide and 2mM 18-crown-6 was used. The analysis time was less than 3 min with limits of detection reaching low µM levels for most of the anions and cations. It has been shown that changes of nitrite could be observed in acute inflammation of upper airways and in a person with diagnosed mild chronic obstructive pulmonary disease, while changes of other ions could also be observed. Lactate concentrations could also be monitored and about 4-fold increase of lactate concentration in exhaled breath condensate was determined following an exhaustive cycling exercise. The developed non-invasive sampling of exhaled breath condensate, followed by rapid capillary electrophoretic analysis, could be very useful in lung inflammatory disease screening as well as in monitoring fast metabolic processes such as lactate build-up and removal.