Reversed-phase High-performance Liquid Chromatography-electrospray Mass Research Articles

Antioxidant and Antidiabetic Activity of Proanthocyanidins from Fagopyrum dibotrys.

Proanthocyanidins are natural glycosidase inhibitors with excellent antioxidant activity. This study aims to search for a new source of proanthocyanidins for the prevention and treatment of type 2 diabetes with higher content and better activity and get their structure elucidated. First, the total proanthocyanidins contents (TOPCs), antioxidant activity, antidiabetic activity of seven common Polygonaceae plants were analyzed and compared. Then proanthocyanidins from the rhizome of Fagopyrum dibotrys were purified, and the detailed structure was comprehensively analyzed by ultraviolet visible spectroscopy (UV-Vis), Fourier transform infrared spectroscopy (FT-IR), 13C nuclear magnetic resonance spectroscopy (13C NMR), reversed-phase high-performance liquid chromatography-electrospray mass spectrometry (RP-HPLC-ESI-MS), and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The rhizome of F. dibotrys showed the highest TOPCs, the strongest antioxidant, and antidiabetic activities; the TOPCs, antioxidant and antidiabetic activities were all very significantly positively correlated. Proanthocyanidins purified from the rhizome of F. dibotrys showed better antidiabetic activity than grape seed proanthocyanidins (GsPs). Seventy-two proanthocyanidins from trimer to undecamer with a mean degree of polymerization (mDP) of about 5.02 ± 0.21 were identified with catechin and epicatechin as the dominant monomers. Conclusion: Proanthocyanidins are the main antioxidant and antidiabetic active substances of F. dibotrys and are expected to be developed into potential antioxidant and hypoglycemic products.

Reversed-phase high-performance liquid chromatography–electrospray mass spectrometry profiling of transgenic and non-transgenic maize for cultivar characterization

A reversed-phase high-performance liquid chromatography–electrospray mass spectrometry (RP-HPLC–ESI-MS (ion trap)) method is developed, for the first time, for profiling transgenic and non-transgenic maize with the aim of cultivar characterization. To optimize chromatographic conditions the following parameters were studied: column, gradient, and ion-pairing reagent. Moreover, the influence in the MS signal of the variation of the capillary voltage and the accumulated ions in the trap was also studied. The developed method was applied to the profiling of different protein fractions (albumin, globulin, prolamin, and glutelin) isolated from Bt transgenic and non-transgenic maize cultivars. Moreover, different maize samples, namely, maize cultivars from different geographical origins (USA, Canada, France, and Spain), transgenic maize samples with certified GMO content, and three transgenic Bt maize cultivars with their isogenic non-transgenic counterparts (Aristis Bt vs. Aristis, PR33P67 vs. PR33P66, and DKC6575 vs. Tietar) were profiled by the developed method. Mass spectra obtained for certain peaks in the maize cultivars studied resulted, in some occasions, useful for cultivar characterization and differentiation. The comparison of UV and MS profiles and mass spectra corresponding to the protein fractions with those of the whole seeds enabled the assignment of some peaks.

Determination of denaturated proteins and biotoxins by on-line size-exclusion chromatography–digestion–liquid chromatography–electrospray mass spectrometry

A multidimensional analytical method for the rapid determination and identification of proteins has been developed. The method is based on the size-exclusion fractionation of protein-containing samples, subsequent on-line trypsin digestion and desalination, and reversed-phase high-performance liquid chromatography–electrospray mass spectrometry detection. The present system reduces digestion times to 20 min and the total analysis time to less than 100 min. Using bovine serum albumin and myoglobin as model proteins, optimization of key parameters such as digestion times and interfacing conditions between the different pretreatment steps was performed. The automated system was tested for the identification of infectious disease agents such as cholera toxin and staphylococcal enterotoxin B. This resulted typically in a positive identification by a total sequence coverage of approximately 40%.

Evaluation of volatile eluents and electrolytes for high-performance liquid chromatography–electrospray ionization mass spectrometry and capillary electrophoresis–electrospray ionization mass spectrometry of proteins: I. Liquid chromatography

Proteins ranging in molecular mass from 14 000 to 80 000 were analyzed by reversed-phase high-performance liquid chromatography–electrospray mass spectrometry (RP-HPLC–ESI-MS) using 60×1.0 mm I.D. microbore-columns packed with 2.3 μm highly crosslinked, octadecylated poly(styrene–divinylbenzene) particles. Proteins were eluted at temperatures of 80–90°C with gradients of acetonitrile in 0.10–0.50% aqueous solutions of trifluoroacetic acid, formic acid or acetic acid. Substitution of trifluoroacetic acid, the most commonly used mobile phase additive for RP-HPLC, by formic acid resulted in a 35–160-fold improvement in analyte detectability at the cost of an only 32–104% increase in peak width at half height of eluting chromatographic peaks. The lower limits of detection for carbonic anhydrase ( M r 29 022.7) in full scan and selected ion monitoring mode were 37 and 2.3 fmol, respectively. Measurement of protein masses by RP-HPLC–ESI-MS was accurate and highly reproducible with maximum mass deviations of 0.025% and relative standard deviations of less than 0.011%. Calibration plots of peak area versus concentration allowed the reliable quantitation of proteins in a concentration range of 0.010–1.0 mg/ml. Finally, the optimized method was applied to the separation, identification and quantification of proteins in real samples such as commercial protein preparations, monoclonal antibody fragments, allergen extracts and whey drinks.