ESI MSn Research Articles

Chemical characterization of Centaurium erythrea L. and its effects on carbohydrate and lipid metabolism in experimental diabetes

Ethnopharmacological relevanceCentaurium erythrea L. fam. Gentianaceae (CE) has been traditionally used for centuries in folk medicine of Balkans as a bitter medicinal herb for digestive complications and for treating febrile conditions and diabetes. The aim of this study was to gain insight into the chemical composition and underlying biochemical mechanism of action of the antihyperglycemic and antilipidemic activities of the dry extract of Centaurium erythrea L., wildly growing and traditionally used medicinal plant in the Republic of Macedonia. Materials and methodsAn ultrasonic methanol maceration of the aerial parts of the dried plant was performed and the extract was freeze-dried. HPLC–DAD–ESI–MSn was carried out on 150mm×4.6mm, 5μm RP-18 Eclipse XDB column, at 40°C. Mobile phase: water with 1% formic acid (A) and methanol (B) with linear gradient starting with 10% B was used to reach 15% at 5min, 40% B at 25min, 55% of B at 50min and 100% at 60min, with flow rate of 0.4mLmin−1. Normal and streptozotocin (STZ) hyperglycemic Wistar rats were used for assessment of the antihyperglycemic and antilipidemic activity by measurement of the key carbohydrate-related enzymes and substrates, as well as lipid state of the organism. ResultsHPLC–DAD–ESI–MSn analyses revealed presence of four different secoiridoids, seven flavonoid glycosides and seven xanthones in the freeze-dried extract of CE representing 53%, 25% and 22% of all compounds, respectively. The short-term (12 days) treatment of the STZ-diabetic rats with CE-extracts resulted in a 74% reduction of the produced hyperglycemia, which is only 6% less than the reduction caused by glibeclamide (GLB, positive control). The CE-extract had a significant impact on the hepatic carbohydrate metabolism enhancing the direct synthesis of glycogen, normalizing phosphorylase a activity and reducing the activity of glucose-6-phosphatase, which further causes reduction in production of blood glucose level. The long-term (45 days) treatment showed that the HbA1c in CE-treated group of animals was even lower than in the GLB-treated groups. The antilipidemic assessment of the CE-extract revealed decrease of total cholesterol, triglycerides, HDL and LDL level in the blood of the normal and STZ-hyperglycemic rats. ConclusionThe obtained results indicate that treatment with CE extract in STZ-diabetic rats regulates the elevated level of blood glucose and carbohydrate-related disturbances slightly better than the effect of glibenclamide. There was also regulation of the serum lipid status in diabetic rats. Identified groups of bitter compounds in the extract (flavonoides, iridoids and xanthones) probably have influence on the expressed antihyperglycaemic effect.

Characterization of oxidized phospholipids in oxidatively modified low density lipoproteins by nanoflow liquid chromatography–tandem mass spectrometry

Oxidized low density lipoproteins (Ox-LDLs) have an important role in the development of age-related vascular disease, such as atherosclerosis. Ox-LDLs are defined as oxidatively modified LDLs in the blood by enzymatic or non-enzymatic oxidation of phospholipids (PLs). For the characterization of Ox-LDLs at molecular level, oxidation patterns of oxidized PL (Ox-PL) products were systematically examined with standard PL molecules (16:0/22:6-PC, 18:0/22:6-PA, and 18:0/22:6-PG), by the formation of bilayer vesicles of each standard, followed by oxidation of PL vesicles using a Cu2+ solution. This oxidative modification was applied to LDL standard materials. Nanoflow liquid chromatography–electrospray ionization–tandem mass spectrometry (nLC–ESI–MS/MS) analysis of the extracted PL mixtures resulted in the identification of 276 PLs both in the modified and non-modified LDL, including 139 Ox-PL species. Examination of the identified PL species from the standard LDL before and after oxidation supported the postulate that the specific location of an acyl chain of LPL can be exchanged between the sn-1 and -2 positions. This exchange occurs when the neighboring acyl chain is cleaved during oxidation and the polar head group of PL molecules can be dissociated to form PA molecules that result in the formation of various Ox-PA products. This study demonstrates that nLC–ESI–MSn can be utilized for the separation and structural characterization of complicated Ox-PL mixtures, including long chain products with hydroxylation or hydroperoxylation at an unsaturated acyl chain, and short chain products from the cleavage of unsaturated fatty acyl chains to form lysophospholipids (LPLs) or the truncation of an acyl chain into a shorter chain terminated with aldehyde or carboxylic acid.

Structural characterization and identification of five triterpenoid saponins isolated from Momordica cochinchinensis extracts by liquid chromatography/tandem mass spectrometry

In this work, we reported the use of high-performance liquid chromatography (HPLC) with electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn) to study the characterization of five triterpenoid saponins from Momordica cochinchinensis extracts in positive ion mode and negative ion mode, revealed the fragmentation behavior of these five M. cochinchinensis saponins in different ion modes. Results showed that in negative ion mode, fragmentation on the M. cochinchinensis triterpenoid saponins at positions C28 and O bond were the primary pathways. In positive ion mode, the predominant diagnostic ions were a series of ions resulting from the rupture of the saccharic chain on C28. Comparison of different fragmentation patterns showed that the results from positive and negative ion ESI MSn were complementary. Both modes can yield structurally different information for the characterization of triterpenoid saponins. This study provides a powerful method for the online structural identification of five triterpenoid saponins from M. cochinchinensis, and some of which have not been reported.

Widespread distribution and molecular diversity of diatom frustule bound aliphatic long chain polyamines (LCPAs) in marine sediments

The siliceous skeletal remains of diatoms are a primary component of sediments, covering vast areas of the sea floor. We used high performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI–MSn) under high resolution time-of-flight MS (TOF-MS) conditions, as well as nuclear magnetic resonance spectroscopy (NMR) and Fourier transform infrared spectroscopy (FTIR), to investigate the distribution and molecular diversity of long chain polyamines (LCPAs) entrapped in sedimentary diatom biosilica in sediments from the Indian sector of the Southern Ocean, the Bering Sea and the Northeast Pacific. This revealed the existence of complex polyamine populations with both known and unknown structural features, including the presence of ketone and carboxylic acid groups in the LCPA backbone, functional groups that likely modulate the silica precipitating properties of LCPAs in the environment and which may constitute points of initial silica deposition (Wallace, A.F., De Yoreso, J.J., Dove, P.M., 2009. Kinetics of silica nucleation on carboxy- and amine-terminated surfaces: insights for biomineralization. Journal of the American Chemical Society 131, 5244–5250). Although various LCPA species overlapped between samples (putrescine-based LCPAs with various degrees of methylation and N-methyl propylamine repeat units were ubiquitous in all samples), clear regional differences were shown in composition and structural characteristics, likely reflecting the various diatom species contributing to the total LCPA pool for each sedimentary environment. We further show, using scanning electron microscopy and energy dispersive X-ray analysis (SEM–EDX) that LCPAs purified from diatom skeletal remains rapidly re-direct the formation of silica nanospheres in vitro, and become embedded in the precipitated silicified matrix, forming an LCPA–silica composite material. The results show that aliphatic polyamines produced by diatoms for morphogenesis of their cell walls are widespread in sediments where the fossil frustules are preserved. These newly assigned components constitute useful biological markers of diatom input to sediments.

Analysis of natural diatom communities reveals novel insights into the diversity of long chain polyamine (LCPA) structures involved in silica precipitation

The intricately nanopatterned siliceous frustules of diatoms are formed under the control of template organic molecules, some of which are tightly incorporated into the frustule during formation. Long chain polyamines (LCPAs) attract dissolved silicic acid species, enhance silica precipitation and play a role in frustule nanopattern formation. LCPAs thus play a central biogeochemical role, linking the cycling of organic matter with the cycling of silicic acid in the photic zone of the upper ocean. Their distribution and molecular diversity in the environment are as yet unknown. We used high performance liquid chromatography electrospray ionization–mass spectrometry (HPLC–ESI–MSn) to investigate the distribution and molecular diversity of aliphatic LCPAs extracted from the frustules of diatoms in mixed marine plankton communities collected within the euphotic zone of the northeastern Pacific coast, Bering Sea, and Puget Sound estuary. An astonishing variety of over 100 natural LCPAS with complex distribution patterns was found in each sample. Structural variation included degree of methylation, overall chain length, presence of secondary amine functionalities, incorporation of quaternary ammonium and occurrence of sulfonated derivatives. Putrescine-based LCPAs with various degrees of methylation and N-methyl propylamine repeat units were ubiquitous. Although some LCPA species overlapped between samples, there were regional differences in composition and structural characteristics. The structural variety of LCPAs employed by diatoms to produce their nanopatterned silicified exoskeleton could inform biomimetic studies of silica precipitation.

Identification and analysis of isothiocyanates and new acylated anthocyanins in the juice of Raphanus sativus cv. Sango sprouts

The freeze-dried sprouts’ juice of Raphanus sativus (L.) cv. Sango was prepared and analysed for the first time. HPLC analysis of total isothiocyanates, after protein displacement, resulted in 77.8±3.0μmol/g of dry juice while GC–MS analysis of hexane and acetone extracts showed E- and Z-raphasatin (8.9 and 0.11μmol/g, respectively) and sulforaphene (11.7μmol/g), summing up to 20.7±1.7μmol/g of free isothiocyanates. Sprouts’ juice contained an unprecedented wealth of anthocyanins and a new fractionation methodology allowed us to isolate 34mg/g of acylated anthocyanins (28.3±1.9μmol/g), belonging selectively to the cyanidin family. Analysis was performed by HPLC–PDA–ESI–MSn and extended to deacylated anthocyanins and aglycones, obtained, respectively, by alkaline and acid hydrolysis. This study identified 70 anthocyanins, 19 of which have never been described before and 32 of which are reported here in R. sativus for the first time. Sango radish sprouts are exceptional dietary sources of heath-promoting micronutrients.

LC–MS method for detecting prostratin in plant extracts and identification of a high-yielding population of Euphorbia fischeriana

Prostratin, a tigliane phorbol ester with promise for the treatment of HIV, was identified and quantified in Euphorbia fischeriana root extracts obtained from several different sites in China. The greatest yield was recovered from root samples collected from Yakeshi, Inner Mongolia (63.54μg/g dry weight, or 0.00635% by mass). Prostratin was not detected in extracts of Euphorbia sp., E. esula, E. lucorum or Stellera chamaejasme. The presence of prostratin was verified by 1H and 13C NMR. Major fragmentation products were identified from ESI MSn spectra after HPLC separation, providing a reproducible fingerprint for unambiguously recognizing the compound in plant extracts.

Quantitative analysis of triterpenoids in different parts of Ilex hainanensis, Ilex stewardii and Ilex pubescens using HPLC–ELSD and HPLC–MSn and antibacterial activity

An HPLC–ELSD method was first developed for the quantitative analysis of principle triterpenoids in Ilex hainanensis, Ilex stewardii and Ilex pubescens. The established method, with excellent precision, repeatability and recovery, was successfully applied to determine four triterpenoids in 24 samples from different species and medicinal parts of Ilex, and the changing trend was discussed. HPLC–ESI–MSn was used for the identification of constituents in samples. The proposed method was simple, effective and suitable for investigations of these plants. Furthermore, the ethanol extracts from leaves of Ilex species, as well as their main components, were assessed for their antibacterial activities. The results indicated that the extracts of I. hainanensis, I. stewardii and I. pubescens could inhibit the growth of the tested oral pathogens, Streptococcus mutans and Actinomyces viscosu. Particularly, ilexgenin A was the most effective with MIC values of 7.8 and ⩽3.9μg/ml, respectively.

Differentiation of isomeric Lewis blood groups by positive ion electrospray tandem mass spectrometry

Lewis histo-blood group antigens are one of the major classes of biologically active oligosaccharides. In this work, underivatized Lewis blood groups were studied by electrospray tandem mass spectrometry (ESI–MSn) in the positive mode with three different mass analyzers: Q-TOF (quadrupole time-of-flight), QqQ (triple quadrupole), and LIT (linear ion trap). It was observed that, under collision-induced fragmentations, type 1 Lewis antigens (Lea and Leb) could be distinguished from type 2 (Lex and Ley) on the basis of specific fragmentations of the GlcNAc unit. Whereas O-4-linked sugars of the GlcNAc are lost as residues, the O-3-linked sugars undergo fragmentation both as sugar units and as sugar residues (unit −18Da). Type 2 Lewis antigens also showed a characteristic cross-ring cleavage 0,2A2 of the GlcNAc. As a result, the product ions at m/z 388 and 305, characteristic of Lex, and m/z 372, characteristic of Lea, are proposed to distinguish the trisaccharide isomers Lex/Lea. Also, the product ions at m/z 534 and 305, characteristic of Ley, and m/z 372, characteristic of Leb, are proposed to distinguish the tetrasaccharide isomers Leb/Ley. These diagnostic fragment ions were further applied in the identification of Lewis type 2 antigens (Lex and Ley) in the lipopolysaccharide of the human gastric pathogen, Helicobacter pylori.

Simultaneous Determination of Six Steroidal Alkaloids of Veratrum dahuricum by HPLC–ELSD and HPLC–MSn

A high performance liquid chromatography coupled with evaporative light scattering detection (HPLC–ELSD) and electrospray ionization multistage mass spectrometry (HPLC–ESI–MSn), respectively, has been performed for the simultaneous determination of six steroidal alkaloids, including pseudojervine, veratrosine, jervine, veratramine, 3-veratroylzygadenine, 3-angeloylzygadenine, in Veratrum dahuricum collected in different seasons. The plants were soaked in methanol and extracted ultrasonically. The six steroidal alkaloids were obtained by silica gel column chromatography, eluting with gradient petroleum and acetyl acetate. The intra-day and inter-day precisions of the method were evaluated and were less than 1.4%. The content of steroidal alkaloids in the plant varied significantly from spring to autumn, confirming the necessity to control the quality of V. dahuricum during its preparation and application.

P.2.d.015 The effects of desvenlafaxine succinate and paroxetine on the pharmacokinetics of desipiramine in healthy adults

This paper provides analytical chemical information on a range of psycho-active drugs. This analytical chemical information on liquid chromatography–electrospray ionisation-mass spectrometry (HPLC–ESI-MS), ion trap mass spectrometry (ESI–MSn), gas chromatography–flame ionisation detection (GLC–FID) and polarographic behaviour is then incorporated into a database which is of use in drug characterisation. Application is found in the determination of selected drug compounds in hair samples.