Simultaneous enrichment and analysis of tobacco alkaloids by microextraction coupled with mass spectrometry using a poly (N-isopropyl-acrylamide-co-divinyl-benzene-co-N, N'-methylene diacrylamide) monolithic column.

Herein, we realized the simultaneous online detection of six tobacco alkaloids (TAs) by in-tube solid-phase microextraction (In-tube SPME) coupled with mass spectrometry by a rapid, sensitive, and matrix effect-free method requiring no chromatographic separation and only minimal sample pre-treatment. A poly (N-isopropylacrylamide-co-divinylbenzene-co-N, N'-methylenediacrylamide) [Poly (NIPAAm-co-DVB-co-MBAA)] monolithic column was designed according to the chemical structures of selected TAs and used as an extraction medium engaging in hydrophobic, π-π, and hydrogen bonding interactions with analytes, allowing them to be effectively extracted. A number of important parameters were systematically optimized to achieve maximal extraction efficiency. The ion intensity of the TAs signals obtained by in-tube SPME-MS were higher than the direct MS mode by about 400 folds with the signal-to-noise ratio improved by 2-7 folds. The detection limits of the six TAs were determined as 1.99-4.06 ng g-1, with good linearity with correlation coefficients exceeding 0.99 obtained under optimal extraction conditions. Besides, TA recoveries in cigarette tobacco spiked at three concentration levels were in the range of 76.4-100.2%, and the corresponding RSDs (n = 5) were obtained as 4.32-7.16%. The extraction performance of the poly (NIPAAm-co-DVB-co-MBAA) monolithic column was well reproducible, with intra- or inter-day precision RSDs determined not to exceed 7.38%. Finally, no marked matrix effects were observed when the developed method was applied to the analysis of both high-abundance and trace-level TAs in practical samples, and the above technique was therefore concluded to be well suited for the detection of TAs in cigarette tobacco or other products.

Targeted Measurements of O- and N-Glycopeptides Show That Proteins in High Density Lipoprotein Particles Are Enriched with Specific Glycosylation Compared to Plasma.

High density lipoprotein (HDL) particles are believed to be protective due to their inverse correlation with the prevalence of cardiovascular diseases. However, recent studies show that in some conditions such as heart disease and diabetes, HDL particles can become dysfunctional. Great attention has been directed toward HDL particle composition because the relative abundances of HDL constituents determine HDL's functional properties. A key factor to consider when studying the structure and composition of plasma particles is the protein glycosylation. Here, we profile the O- and N-linked glycosylation of HDL associated-proteins including the truncated form of Apo CIII and their glycan heterogeneity in a site-specific manner. Apolipoprotein CIII, fetuin A, and alpha 1 antitrypsin are glycoproteins associated with lipoproteins and are implicated in many cardiovascular and other disease conditions. A targeted method (UHPLC-QQQ) was used to measure the glycoprotein concentrations and site-specific glycovariations of the proteins in human plasma and compared with HDL particles isolated from the same plasma samples. The proteins found in the plasma are differentially glycosylated compared to those isolated in HDL. The results of this study suggest that glycosylation may play a role in protein partitioning in the blood, with possible functional implications.

Quantifying non-covalent binding affinity using mass spectrometry: a systematic study on complexes of cyclodextrins with alkali metal cations.

RATIONALE: To date, the quantification of binding affinities for non-covalent complexes between cyclodextrin (CD) and alkali cations including Li(+) , Na(+) , K(+) , Rb(+) , and Cs(+) has not been investigated in detail by electrospray ionization mass spectrometry (ESI-MS) due to the unknown ionization efficiencies of the different species. In this study, the binding constants of CD-Cs(+) complexes were determined by an improved mass spectrometric titration methodology, which was based only on the peak intensities of equilibrium CD. Hence, the discrepancy of ionization efficiencies of CD, alkaline cation and their complex would not affect the measurement. Then the obtained lgKa values were provided as references for competitive ESI-MS. The binding constants for complexes of α-, ß- or γ-CD with Li(+) , Na(+) , K(+) and Rb(+) could be derived directly and quickly. METHODS: The lgKa values between α-, ß- or γ-CD and Cs(+) data were processed by curve fitting. These lgKa values were provided as references for competitive ESI-MS. In addition, linear fit equations for complexes of α-, ß- or γ-CD with Cs(+) were derived. Through the linear fit equations of competitive ESI-MS, the binding constants for complexes of Li(+) , Na(+) , K(+) and Rb(+) with α-, ß- or γ-CD were acquired. RESULTS: Results showed that the binding constant (lgKa ) values for the complexes of Cs(+) with α-, ß- and γ-cyclodextrins were 3.94, 3.88 and 3.80, respectively, revealing that the binding strength decreased with the increase in diameter of cyclodextrins. The competitive ESI-MS results showed a clear trend of decreasing affinity for complexes of cyclodextrins in the order of Li(+) , Na(+) , K(+) , Rb(+) . CONCLUSIONS: The binding constants of non-covalent cyclodextrin-alkali cation complexes have been systematically studied by an improved mass spectrometric titration and competitive ESI-MS. Also, the structural features of the complexes were discussed. Our results are valuable for better understanding of mechanisms driving inclusion chemistry under well-defined conditions.

Negative charge induced dissociation: fragmentation of deprotonated N-benzylidene-2-hydroxylanilines in electrospray ionization mass spectrometry.

Unimolecular reactivities of different N-benzylidene-2-hydroxylaniline anions were investigated in gas phase by electrospray ionization tandem mass spectrometry. All the collision-induced dissociation spectra of N-benzylidene-2-hydroxylaniline anions show similar ions at phenyl anions, neutral loss of benzonitrile and benzoxazole anions, respectively. The possible fragmentation pathway was probed through deuterium labeling and various group substituents experiments. Computational results were applied to shed light on the mechanism of fragmentation patterns. The proton in the CH=N is reactive in the formation of the concerned ions. Its direct transfer to the oxygen results in 2-hydroxyphenyl anion. Proton abstraction between benzoxazole and phenyl anion leads to the formation of benzene and benzoxazole anion.