Analysis of sesquiterpene lactones in Eupatorium lindleyanum by HPLC‐PDA‐ESI‐MS/MS

Introduction – The aerial part Eupatorium lindleyanum is commonly used as an antipyretic and detoxicant clinically in traditional Chinese medicine. Our previous research showed that germacrane sesquiterpene lactones were its main active constituents, so the development of rapid and accurate methods for the identification of the sesquiterpene lactones is of great significance. Objective – To develop an HPLC‐PDA‐ESI‐MS/MS method capable for simple and rapid analysis of germacrane sesquiterpene lactones in the aerial part E. lindleyanum. Methodology – High‐performance liquid chromatography‐photodiode array detection‐electrospray ionization‐tandem mass spectrometry was used to analyze germacrane sesquiterpene lactones of Eupatorium lindleyanum. The fragmentation behavior of germacrane sesquiterpene lactones in a Micromass Q/TOF Mass Spectrometer was discussed, and 9 germacrane sesquiterpene lactones were identified by comparison of their characteristic data of HPLC and MS analyses with those obtained from reference compounds. Results – The investigated germacrane sesquiterpene lactones were identified as eupalinolides C (1), 3β‐acetoxy‐8β‐(4′‐hydroxy‐tigloyloxy)‐14‐hydroxy‐costunolide (2), eupalinolides A (3), eupalinolides B (4), eupalinolides E (5), 3β‐acetoxy‐8β‐(4′‐oxo‐tigloyloxy)‐14‐hydroxy‐heliangolide (6), 3β‐acetoxy‐8β‐(4′‐oxo‐ tigloyloxy)‐14‐hydroxy‐costunolide (7), hiyodorilactone B (8), and 3β‐acetoxy‐8β‐(4′‐hydroxy‐tigloyloxy)‐ costunolide (9). Compounds 6, 7 and 9 were reported for the first time. Conclusion – HPLC‐PDA‐ESI‐MS/MS provides a new powerful approach to identify germacrane sesquiterpene lactones in E. lindleyanum rapidly and accurately. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of sesterterpenoids from Aspergillus terreus using ESI‐QTOF and ESI‐IT

Introduction – Biosynthesis of terretonin was studied due to the interesting skeleton of this series of sesterterpenoids. Very recently, López‐Gresa reported two new sesterterpenoids (terretonins E and F) which are inhibitors of the mammalian mitochondrial respiratory chain. Mass spectrometry (MS), especially tandem mass spectrometry, has been one of the most important physicochemical methods for the identification of trace natural products due to it rapidity, sensitivity and low levels of sample consumption. The potential application prospect and unique skeleton prompted us to study structural characterisation using MS. Objective – To obtain sufficient information for rapid structural elucidation of this class of compounds using MS. Methodology – The elemental composition of the product ions was confirmed by low‐energy ESI‐CID‐QTOF‐MS/MS analyses. The fragmentation pathways were postulated on the basis of ESI‐QTOF‐MS/MS/MS and ESI‐IT‐MSⁿ spectra. Common features and major differences between ESI‐QTOF‐MS/MS and IT‐MSⁿ spectra were compared. For ESI‐QTOF‐MS/MS/MS experiments, capillary exit voltage was raised to induce in‐source dissociation. Ammonium acetate or acetic acid were added into solutions to improve the intensity of [M + H]⁺. The collision energy was optimised to achieve sufficient fragmentation. Some fragmentation pathways were unambiguously proposed by the variety of abundance of fragment ions at different collision energies even without MSⁿ spectra. Results – Fragmentation pathways of five representative sesterterpenoids were elucidated using ESI‐QTOF‐MS/MS/MS and ESI‐IT‐MSⁿ in both positive‐ and negative‐ion mode. The key group of characterising fragmentation profiles was ring B, and these fragmentation patterns are helpful to identify different types of sestertepenoids. Conclusion – Complementary information obtained from fragmentation experiments of [M + H]⁺ (or [M + NH₄]⁺) and [M ? H]^? precursor ions is especially valuable for rapid identification of this kind of sesterterpenoid.     

UHPLC/HR‐ESI‐MS/MS Profiling of Phenolics from Tunisian Lycium arabicum Boiss. Antioxidant and Anti‐lipase Activities’ Evaluation

This study was performed in the aim to evaluate nine different extracts from Tunisian Lycium arabicum for their total phenolic and total flavonoid contents, phytochemical analyses as well as their antioxidant and anti‐lipase activities. The in vitro antioxidant property was investigated using three complementary methods (DPPH, ferric reducing antioxidant power (FRAP), and β‐carotene‐linoleic acid bleaching assays) while anti‐lipase activity was evaluated using 4‐methylumbelliferyl oleate method. From all of the tested extracts the most potent found to be the polar MeOH extracts especially those of stems and leaves. In order to investigate the chemical composition of these extracts and possible correlation of their constituents with the observed activities, an UHPLC/HR‐ESI‐MS/MS analysis was performed. Several compounds belonging to different chemical classes were tentatively identified such as rutin and kampferol rutinoside, the major constituents of the leaves, and N‐caffeoyltyramine, lyciumide A, N‐dihydrocaffeoyltyramine as well as fatty acids: trihydroxyoctadecadienoic acid and hydroxyoctadecadienoic acid isomers were detected abundantly in the stems. These results showed that the MeOH extracts of stems and leaves of L. arabicum can be considered as a potential source of biological active compounds.     

Histone post‐translational modifications by HPLC‐ESI‐MS after HT29 cell treatment with histone deacetylase inhibitors

The goal of the present work is to establish a correlation between the degree of histone post‐translational modifications and the effects caused by treatment of HT29 colon cancer cells with class I‐selective (MS‐275 and MC1855), class II‐selective (MC1568), and non‐selective (suberoylanilide hydroxamic acid (SAHA) histone deacetylase inhibitors (HDACi). This correlation could afford a mean to better understand the mechanism of action of new, more potent, and selective HDACi directly on the cells. To this end, LC coupled to MS was applied in studies of time and concentration‐dependent treatment with HDACi in HT29 cells. The results were correlated to their potency of histone deacetylase inhibition and to their effects on the cell cycle. The results indicate that the four tested inhibitors show a different pattern of time‐ and concentration‐dependent modification after treatment of HT29 cells. At the selected concentrations, they cause different histone hyperacetylation and different cell cycle effects. In particular, SAHA (non‐selective HDACi) affected hyperacetylation of all histones and caused massive cell death. MC1855 (class I‐selective HDACi, hydroxamate) proved to be more potent and less toxic (cell arrest in G2/M phase) than SAHA. MS‐275 (class I‐selective HDACi, benzamide) exhibited a higher degree of hyperacetylation of H4 and a lower degree of H2A, H2B, and H3 acetylation, causing a cell arrest in G0/G1 phase. On the contrary, MC1568 (class II‐selective HDACi) produced only a modest hyperacetylation of H4, was ineffective on the other histones, and showed no effect on cell cycle in HT29 cells.     

Cyclodimerization of immunosuppressive fragment of HLA‐DR molecule. Design,synthesis and ESI‐MS/MS analysis

The nonapeptide fragment of the HLA‐DR molecule, located in the exposed loop of the alpha‐chain (164–172), having the VPRSGEVYT sequence, suppresses the immune response. Based on the three‐dimensional structure of the HLA‐DR superdimer, we designed a new cyclodimeric analog in which the two parallel peptide chains of VPRSGEVYT sequence are linked through their C‐termini by spacer of (Gly₅)₂‐Lys‐NH₂ and the N‐termini are also linked by poly(ethylene glycol). The (VPRSGEVYTG₅)₂K‐resin analog was synthesized using solid‐phase peptide synthesis protocols. The cyclization was achieved by cross‐linking the N‐terminal positions of the dimeric peptide, attached to a MBHA resin, with alpha, omega‐bis (acetic acid) poly(ethylene glycol), activated by esterification with pentafluorophenol. Our results demonstrate that the cyclodimerization of VPRSGEVYT results in enhanced immunosuppressive activity of the peptide. Mass spectrometry fragmentation analysis of the obtained cyclodimeric peptide is also presented. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Analysis of flumequine enantiomers in rat plasma by UFLC‐ESI‐MS/MS

In this study the analysis and confirmation of flumequine enantiomers in rat plasma by ultra‐fast liquid chromatography coupled with electron spray ionization mass spectrometry (using propranolol as an internal standard [IS]) was developed and validated. Plasma samples were prepared by liquid–liquid extraction using methyl tert‐butyl ether as the extraction solvent. Direct resolution of the R‐ and S‐isomers was performed on a CHIRALCEL OJ‐RH column (4.6 × 150 mm, 5 μm) using acetonitrile / 0.1% formic acid / 1 mM ammonium acetate as the mobile phase. Detection was operated by electron spray ionization in the selected ion monitoring and positive ion mode. The target ions at m/z 262.1 and m/z 260.1 were selected for the quantification of the enantiomers and IS, respectively. The linear range was 0.5–500 ng/mL. The precisions (coefficient of variation, CV%) and recoveries were 1.43–8.68 and 94.24–106.76%, respectively. The lowest quantitation limit for both enantiomers is 0.5 ng/mL, which is sensitive enough to be applied to sample analysis in other related studies.     

GOAT – A simple LC‐MS/MS gradient optimization tool

Modern nano‐HPLC systems are capable of extremely precise control of solvent gradients, allowing high‐resolution separation of peptides. Most proteomics laboratories use a simple linear analytical gradient for nano‐LC‐MS/MS experiments, though recent evidence indicates that optimized non‐linear gradients result in increased peptide and protein identifications from cell lysates. In concurrent work, we examined non‐linear gradients for the analysis of samples fractionated at the peptide level, where the distribution of peptide retention times often varies by fraction. We hypothesized that greater coverage of these samples could be achieved using per‐fraction optimized gradients. We demonstrate that the optimized gradients improve the distribution of peptides throughout the analysis. Using previous generation MS instrumentation, a considerable gain in peptide and protein identifications can be realized. With current MS platforms that have faster electronics and achieve shorter duty cycle, the improvement in identifications is smaller. Our gradient optimization method has been implemented in a simple graphical tool (GOAT) that is MS‐vendor independent, does not require peptide ID input, and is freely available for non‐commercial use at http://proteomics.swmed.edu/goat/