Lipid A foszforilációs- és acilációs izomereinek elválasztása és jellemzése NACE-ESI-MS/MS módszerrel: Separation and characterization of the phosphorylation and acylation isomers of lipid A by NACE-ESI-MS/MS

Lipid A is the anchor of endotoxins on the surface of Gram-negative bacteria. In the human body, it is a prominent stimulator of the immune system, but it may also cause dangerous medical conditions, such as endotoxic shock and sepsis. To reveal the specific structural parts and molecular heterogeneity of lipid A isolates, such as the site of phosphorylation and type of fatty acyl chains, a pressure-assisted non-aqueous capillary electrophoresis – tandem mass spectrometry method was developed. Baseline separation of both phosphorylation and acylation isomers was achieved. Identification was carried out from the tandem mass spectra recorded in the positive ionization mode. B-type ions are formed by diagnostic neutral losses. B2 type ions are confirming the site of phosphorylation, while B1 type ions and other fragments are enabling the characterization of acylation isomers. This novel method should be regarded as an orthogonal technique to formerly developed LC‐MS/MS methods in the screening of bacterial samples or lipid A based therapeutics. Kivonat Az endotoxinok a lipid A részükkel ágyazódnak be a Gram-negatív baktériumok sejtmembránjába. Az emberi szervezetben a lipid A hatékonyan stimulálja az immunrendszert, de akár súlyos egészségi állapotokat is előidézhet, mint az endotoxikus sokk, vagy a szepszis. A szerkezetének részleteinek megismerésére és a lipid A izolátum alkotóinak változatosságát – úgymint a foszforilációs helyek és a kapcsolódó zsírsavláncok típusát – feltárandó kifejlesztettünk egy nyomással segített nemvizes kapilláris elektroforézis–tandem tömegspektrometriás módszert. A foszforilációs- és az acilációs izomereknél is sikeres alapvonali elválasztást értünk el. A szerkezeti információt a pozitív ion módban felvett tandem tömegspektrumok szolgáltatták. Diagnosztikus semleges vesztésekkel B-típusú ionok keletkeznek. A B2-típusú ionok azonosítják a foszforilációs pozíciót, míg a B1 ionok az acilációs izomerek jellemzését teszik lehetővé. Jelen módszer a korábban kifejlesztett LC-MS/MS módszerek kiegészítője lehet a bakteriális eredetű minták vizsgálatában és a lipid A klinikai alkalmazásakor

Determination of Organic Acids in Wines Using Capillary Zone Electrophoresis-Electrospray Ionization /Quadrupole-Time-of-Flight-Mass Spectrometry (CZE-ESI/QTOF-MS)

Organic acids, inlcuding tartaric, malic, lactic, citric, succinic and shikimic, were determined in red wines using capillary zone electrophoresis hyphenated to electrospray ionization/quadrupole-time-of-flight-mass spectrometry (CZE-ESI/QTOF-MS). Separation of the analytes was perfomred using 50 mM ammonium acetate buffer, with pH 6, as a background electrolyte. The capillary was coated with 1 % (m/v) solution of hexadimetrine bromide. The applied voltage for the capillary electrophoretic separation was – 20 kV with anodic detection. The method was validated presenting best recoveries ranged from 98.4 % to 112 % for all organic acids. The calibration curves were linear with correlation coefficients r2 > 0.99, ranging from 0.9902 for shikimic acid to 0.9990 for tartaric acid. Developed method was applied for analysis of Vranec wines from different wine regions. Tartaric acid was the main ogranic acid in wines (range: 2.09 – 4.96 g/L), followd by malic acid (range: 0.29 to 4.03 g/L). The total content of oraganic acids ranged between 3.53 to 8.5 g/L, concluding that climate conditions in the wine areas inlfuenced the acids amount in grapes and wine

CZE-ESI/QTOF-MS analysis of organic acids in red Vranec wines from different locations

Organic acids are important compounds influencing the stability, flavor, aroma and color of grapes and wine and contributing to the pH, chemical and microbiological stability of the wines. In this study, capillary zone electrophoresis hyphenated to electrospray ionization/quadrupole-time-of-flight-mass spectrometry (CZE-ESI/QTOF-MS) was applied for analysis of organic acids (lactic acid, succinic acid, malic acid, tartaric acid, shikimic acid and citric acid) in red wines [1]. All wines were produced from Vranec V. vinifera grapes, grown in different wine areas in Republic of Macedonia, applying same winemaking technology. Results showed that Vranec wines produced under controlled winemaking protocols presented relatively high content of tartaric, citric, lactic, malic and succinic acids compared to commercial Macedonian wines [2], while shikimic acid was present in a significantly low concentration. In fact, high concentration of tartaric acid in Vranec wines is nevertheless, typical for this variety, influencing higher chemical stability and colour, and giving soft freshens of the wines. Thus, tartaric acid ranged from 14.03 to 33.29 mM, followed by malic acid (range: 0.45 – 30.3 mM). Lactic acid was formed during the spontaneous malolactic fermentation, ranging between 1.24 to 6.74 mM. The concentrations of the naturally present citric acid in the samples (ranging from 1.36 to 4.66 mM) were in accordance to the official regulations, i.e., not higher than 1 g/L (5.24 mM). These results here provide a clear view for the organic acids profile in Vranec wines produced from various wine areas and they are useful for winemakers to manage and/or modify the winemaking protocols for this variety in order to obtain stable and high quality wines for the global market. Key words: organic acids, Vranec wines, CZE-ESI/QTOF-MS. Acknowledgement This work was supported by the grants CEEPUS CII-HU-0010-03-0809, NKFIH K-125275 and project titled “Chemical characterization of wine, alcoholic beverages and food by instrumental techniques”, funded by University “Goce Delčev” – Štip. References [1] V. Ivanova-Petropulos, Z. Naceva, V. Sándor, L. Makszin, L. Nagy, B. Berkics, T. Stafilov, and F. Kilár, Electrophoresis, accepted for publication (2018). [2] K. Tašev, M. Stefova, and V. Ivanova-Petropulos, Macedonian Journal of Chemistry and Chemical Engineering, 35, 225-233 (2016)