Chromatography-electrospray Ionization-mass Spectrometry Analysis Research Articles

C-Methylation Catalyzed by Fom3, a Cobalamin-Dependent Radical S-adenosyl-l-methionine Enzyme in Fosfomycin Biosynthesis, Proceeds with Inversion of Configuration.

Fom3, a cobalamin-dependent radical S-adenosyl-l-methionine (SAM) methyltransferase, catalyzes C-methylation at the C2 position of cytidylylated 2-hydroxyethylphosphonate (HEP-CMP) to afford cytidylylated 2-hydroxypropylphosphonate (HPP-CMP) in fosfomycin biosynthesis. In this study, the Fom3 reaction product HPP-CMP was reanalyzed by chiral ligand exchange chromatography to confirm its stereochemistry. The Fom3 methylation product was found to be ( S)-HPP-CMP only, indicating that the stereochemistry of the C-methylation catalyzed by Fom3 is ( S)-selective. In addition, Fom3 reaction was performed with ( S)-[2-2H1]HEP-CMP and ( R)-[2-2H1]HEP-CMP to elucidate the stereoselectivity during the abstraction of the hydrogen atom from C2 of HEP-CMP. Liquid chromatography-electrospray ionization mass spectrometry analysis of the 5'-deoxyadenosine produced showed that the 2H atom of ( R)-[2-2H1]HEP-CMP was incorporated into 5'-deoxyadenosine but that from ( S)-[2-2H1]HEP-CMP was not. Retention of the 2H atom of ( S)-[2-2H1]HEP-CMP in HPP-CMP was also observed. These results indicate that the 5'-deoxyadenosyl radical stereoselectively abstracts the pro-R hydrogen atom at the C2 position of HEP-CMP and the substrate radical intermediate reacts with the methyl group on cobalamin that is located on the opposite side of the substrate from SAM. Consequently, it was clarified that the C-methylation catalyzed by Fom3 proceeds with inversion of configuration.

PEGylated Precision Segments Based on Sequence-Defined Thiolactone Oligomers.

A straightforward access route to multifunctional block copolymers, combining a poly(ethylene glycol) (PEG) block and a monodisperse segment with discrete monomer sequence based on thiolactone chemistry, is described. Exploiting an inverse conjugation strategy on a PEG preloaded poly(styrene) synthesis resin enables the convenient introduction of a predefined PEG-block at the α-terminus of thiolactone-based sequence-defined oligomers. Reaction conditions for the stepwise, submonomer synthesis at polar solid supports are optimized, using sequential synthesis on a model resin that enables to isolate and determine the purity of the oligomer segments by liquid chromatography-electrospray ionization mass spectrometry analysis. The reaction conditions are used to synthesize PEGylated 5mer precision polymers with defined monomer sequence in good yields and high purity to offer an interesting platform of macromolecules with potential for biomedical applications.

Intracellular GSH of Streptococcus thermophilus shows anti-oxidative activity against low-density lipoprotein oxidation in vitro and in a hyperlipidaemic hamster model.

Streptococcus thermophilus YIT 2001 (ST-1), a lactic acid bacterial strain, was shown to have inhibitory effects on the oxidation of low-density lipoprotein (LDL) and the development of aortic fatty lesions in an animal model, and lower the serum levels of malondialdehyde-modified LDL, an oxidative modification product of LDL, in a clinical trial. This study aimed to identify the intracellular active component of ST-1 associated with anti-oxidative activity against LDL oxidation. High-performance liquid chromatography-electrospray ionisation mass spectrometry analysis after fractionation of the cellular extract by reversed-phase chromatography demonstrated that the active fraction contained reduced glutathione (GSH). GSH showed anti-oxidative activity in a dose-dependent manner, while this activity disappeared following thiol derivatisation. ST-1 had the strongest anti-oxidative activity against LDL oxidation and the highest level of intracellular GSH among five strains of S. thermophilus. In addition, the anti-oxidative activity of ST-1 after thiol derivatisation decreased by about half, which was similar to that of three other strains containing poor or no intracellular GSH or thiol components. Moreover, anti-oxidative activity against LDL oxidation was observed in hyperlipidaemic hamsters fed with high GSH ST-1 cells but not in those given low GSH cells. These findings suggest that intracellular GSH in ST-1 may provide beneficial effects via anti-oxidative activity against LDL oxidation and excess oxidative stress in the blood.

Structural features, antioxidant and tyrosinase inhibitory activities of proanthocyanidins in leaves of two tea cultivars

ABSTRACTIn this study the structural features, antioxidant, and tyrosinase inhibitory activities of proanthocyanidins in leaves of two tea cultivars, namely Huangjingui and Qilan, were investigated. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and thiolysis-high performance liquid chromatography-electrospray ionization mass spectrometry analyses confirmed that these proanthocyanidins were built up a mixture of procyanidins, propelargonidins, and prodelphinidins, with the predominance of procyanidins. The proanthocyanidins in leaves of Huangjingui and Qilan cultivars exhibited potent antioxidant activities with IC50 values of 87.36 ± 0.83 and 97.33 ± 0.61 µg/mL in 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay, 43.57 ± 0.25 and 55.01 ± 0.22 µg/mL in 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging assay, and with ferric reducing antioxidant power values of 4.83 ± 0.03 and 3.97 ± 0.02 mmol ascorbic acid equivalent/g in ferric reducing antioxidant power assay. It was also found that these proanthocyanidins had strong inhibition on tyrosinase activity with IC50 values of 54.8 ± 1.27 and 20.0 ± 0.89 µg/mL for Huangjingui and Qilan cultivars, respectively. The results indicated that the proanthocyanidins in leaves of two tea cultivars could be considered as natural antioxidants and tyrosinase inhibitors applied in pharmaceutical and cosmetic industries in the future.

Induction of apoptosis in HepG2 by Vitex agnus-castus L. leaves extracts and identification of their active chemical constituents by LC-ESI-MS

ObjectiveTo evaluate the cytotoxic activity and cytopathological changes of Vitex agnus-castus L. (V. agnus-castus) leaves extracts and characterize their bioactive chemical constituents. MethodsThe dried leaves powder of V. agnus-castus was extracted using 85% methanol (MeOH). The methanolic extract was defatted using petroleum ether and fractionated using ethyl acetate (EtOAc) and butanol (BuOH). The anticancer potential of different extracts was evaluated by neutral red assay, cytopathological changes of apoptosis and caspase-3 expression in hepatoma cell line (HepG2). The chemical constituents of most active extracts were identified using liquid chromatography-electrospray ionisation mass spectrometry analysis. ResultsThe butanolic fraction was the most active in inhibiting the proliferation of HepG2 cells [IC50 = (13.42 ± 0.17) mg/mL] compared with MeOH extract [IC50 = (17.61 ± 0.15) mg/mL) and EtOAc fraction [IC50 = (22.51 ± 0.26) mg/mL]. The cytopathological examinations demonstrated the morphology of apoptosis and caspase-3 expression was more evident in HepG2 cells treated with BuOH than cells treated with MeOH and EtOAc. The liquid chromatography-electrospray ionisation mass spectrometry analysis exhibited that the defatted MeOH extract and BuOH fraction had different bioactive secondary metabolites, such as phenolic acids, flavonoids, and iridoids. ConclusionsThe butanolic fraction has higher contents of secondary metabolites than the defatted methanolic extract. The cytotoxic activities, apoptotic changes, and caspase-3 activation may be due to the presence of these bioactive secondary metabolites (iridoids, flavonoid, and phenolic acids) in these extracts. These results would suggest V. agnus-castus to be used as an adjuvant in cancer therapy.

An integrated electrophoretic mobility control device with split design for signal improvement in liquid chromatography–electrospray ionization mass spectrometry analysis of aminoglycosides using a heptafluorobutyric acid containing mobile phase

Electrophoretic mobility control (EMC) was used to alleviate the adverse effect of the ion-pairing agent heptafluorobutyric acid (HFBA) in the liquid chromatography–electrospray ionization mass spectrometry (LC-ESI-MS) analysis of aminoglycosides. Aminoglycosides separated by LC were directed to a connecting column before their detection via ESI. Applying an electric field across the connecting column caused the positively charged aminoglycosides to migrate toward the mass spectrometer whereas the HFBA anions remained in the junction reservoir, thus alleviating the ion suppression caused by HFBA. To accommodate the flow rate of a narrow-bore column, minimize the effect of electrophoretic mobility on separation, and facilitate the operation, an integrated EMC device with a split design was fabricated. With the proposed EMC device, the signals of aminoglycosides were enhanced by a factor of 5–85 without affecting the separation efficiency or elution order. For the analysis of aminoglycosides in bovine milk, the proposed approach demonstrates a sensitivity that is at least 10 times below the maximum residue limits set by most countries.

Liquid Chromatography-Electrospray Ionization Mass Spectrometry Analysis of Limonoids and Flavonoids in Seeds of Grapefruits, Other Citrus Species, and Dietary Supplements.

A selective UHPLC-DAD-QToF-MS method was developed to screen grapefruit seeds, and the seeds of other Citrus species for limonoid aglycones, acids, glucosides, and flavonoids. These classes of compounds were identified in positive and negative ion modes over a mass-to-charge range from 100-1500. Accurate mass values, elution times, and fragmentation patterns obtained by QToF-mass spectrometry were used to identify or tentatively characterize the compounds detected in the sample of this study. Limonin was the major limonoid in most of the seeds of Citrus species, followed by nomilin. This analytical method was successfully applied for the analysis of commercial extracts and dietary supplements claiming to contain grapefruit seed extract, or extracts made from the seed and other fruit parts such as the peel or pulp. Many commercial products contained large numbers of flavonoids, indicating the use of peel, pulp, or seed coat. This method also permitted detection of synthetic preservatives such as benzethonium chloride, methylparaben, and triclosan in commercial grapefruit seed extract products. Out of the 17 commercial products analyzed, two contained the synthetic antimicrobial agent benzethonium chloride.

Acute Hepatitis Caused by Green Tea Infusion: A Case Report

The green tea is obtained by an unfermented process of leaves of Camellia sinensis and the main chemical components are polyphenols, particularly epigallocatechin-3-gallate and epicatechin-3-gallate that could be associated to adverse hepatic reactions. We present a case of acute hepatitis caused by the use of green tea.A 62-year-old woman was hospitalized because of the persistent high levels of liver function tests. After the hospitalization a lot of instrumental exams and blood checks were performed. The search of metallic elements in the used green tea infusions was performed using an inductively coupled-plasma mass spectrometry; high performance liquid chromatography-electro spray ionization-mass spectrometry analysis was done to characterize the metabolite profiles of the infusions of green tea. The blood check showed in particular alanine aminotransferase (780 U/L) and total bilirubin (1.15 mg/dL) levels abnormal. The abdominal echography and other blood parameters were normal, but liver biopsy described a “drug toxic damage”. Every day over the previous 9 months the patient drank two or three cups of several brands of green tea infusions and she stopped this behavior when abdominal pain was persistent. Her medical history didn’t report the use of other drugs or toxic products. After four months of stopping the use of green tea infusions, the liver function tests were normalized. The presence of metallic elements in tea infusion cannot justify the observed liver toxicity in our patient. Instead, the highest levels of epigallo catechin methyl gallate derived from epigallocatechin-3-gallate observed in one of the samples consumed by the patient, arise a possible correlation between some of the catechins in green tea and the hepatotoxic effect. It is conceivable that the mechanism of damage can be idiosyncratic-metabolic or allergic.

Enhancement of anti-inflammatory activity of Aloe vera adventitious root extracts through the alteration of primary and secondary metabolites via salicylic acid elicitation.

Aloe vera (Asphodeloideae) is a medicinal plant in which useful secondary metabolites are plentiful. Among the representative secondary metabolites of Aloe vera are the anthraquinones including aloe emodin and chrysophanol, which are tricyclic aromatic quinones synthesized via a plant-specific type III polyketide biosynthesis pathway. However, it is not yet clear which cellular responses can induce the pathway, leading to production of tricyclic aromatic quinones. In this study, we examined the effect of endogenous elicitors on the type III polyketide biosynthesis pathway and identified the metabolic changes induced in elicitor-treated Aloe vera adventitious roots. Salicylic acid, methyl jasmonate, and ethephon were used to treat Aloe vera adventitious roots cultured on MS liquid media with 0.3 mg/L IBA for 35 days. Aloe emodin and chrysophanol were remarkably increased by the SA treatment, more than 10–11 and 5–13 fold as compared with untreated control, respectively. Ultra-performance liquid chromatography-electrospray ionization mass spectrometry analysis identified a total of 37 SA-induced compounds, including aloe emodin and chrysophanol, and 3 of the compounds were tentatively identified as tricyclic aromatic quinones. Transcript accumulation analysis of polyketide synthase genes and gas chromatography mass spectrometry showed that these secondary metabolic changes resulted from increased expression of octaketide synthase genes and decreases in malonyl-CoA, which is the precursor for the tricyclic aromatic quinone biosynthesis pathway. In addition, anti-inflammatory activity was enhanced in extracts of SA-treated adventitious roots. Our results suggest that SA has an important role in activation of the plant specific-type III polyketide biosynthetic pathway, and therefore that the efficacy of Aloe vera as medicinal agent can be improved through SA treatment.

Synthesis and evaluation of two novel 2-nitroimidazole derivatives as potential PET radioligands for tumor imaging

Nitroimidazole (azomycin) derivatives labeled with radioisotopes have been developed as cancer imaging and radiotherapeutic agents based on the oncological hypoxic mechanism. By attaching nitroimidazole core with different functional groups, we synthesized new nitroimidazole derivatives and evaluated their potentiality as tumor imaging agents. Starting with commercially available 2-nitroimidazole, 2-fluoro-N-(2-(2-nitro-1H-imidazol-1-yl)ethyl)acetamide (NEFA, [(19)F]7) and 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 2-fluoroacetate (NEFT, [(19)F]8), as well as radiolabeling precursors, the bromo-substituted analogs were quickly synthesized through a three-step synthetic pathway. The precursors were radiolabeled with [(18)F]F(-)/18-crown-6/KHCO(3) in dimethyl sulfoxide at 90°C for 10 min followed by purification with an Oasis HLB cartridge. Biodistribution studies were carried out in EMT-6 tumor-bearing mice. The uptake (%ID/g) in tumors and normal tissues were measured at 30 min postinjection. Liquid chromatography-electrospray ionization mass spectrometry (LC/MS) was used to distinguish metabolites from parent drugs in urine and plasma of rat injected with "cold" NEFA ([(19)F]7) and NEFT ([(19)F]8). Two radiotracers, [(18)F]NEFA ([(18)F]7) and [(18)F]NEFT ([(18)F]8), were prepared with average yields of 6%-7% and 9%-10% (not decay corrected). Radiochemical purity for both tracers was >95% as determined by HPLC. Biodistribution studies in EMT-6 tumor-bearing mice indicated that the tumor to blood and tumor to liver ratios of both [(18)F]7 (0.96, 0.61) and [(18)F]8 (0.98, 1.10) at 30 min were higher than those observed for [(18)F]FMISO (1) (0.91, 0.59), a well-investigated azomycin-type hypoxia radiotracer. Liquid chromatography-electrospray ionization mass spectrometry analysis demonstrated that fluoroacetate was the main in vivo metabolite for both NEFA ([(19)F]7) and NEFT ([(19)F]8). In this research, two new fluorine-18 labeled 2-nitroimidazole derivatives, [(18)F]7 and [(18)F]8, both of which containing in vivo hydrolyzable group, were successfully prepared. Further biological evaluations are warranted to investigate their potential as PET radioligands for imaging tumor.

Proteomic approach for the analysis of acrylamide–hemoglobin adducts: Perspectives for biological monitoring

The formation of adducts between acrylamide and hemoglobin in vitro was investigated by using mass spectrometric methodologies to identify the amino acid residues sensitive to alkylation. Liquid chromatography–electrospray ionisation mass spectrometry analysis of either intact or trypsin-digested α- and β-globin chains isolated from hemolysate samples incubated in vitro with acrylamide at different molecular ratios allowed us to identify Cys93 of β-globin as the most reactive site in hemoglobin, according to a Michael-type addition reaction between acrylamide and the sulphydryl group of cysteine. The only other reactive sites were Cys104 of α-globin and the N-terminal amino groups of both chains. The method developed, based on electrospray ionisation quadrupole time-of-flight tandem mass spectrometry analysis of intact globin chains was able to specifically detect low levels of adducts. In this way, rapid identification of alkylated portion of Hb was achieved to be potentially used as a biomarker for high-sensitivity biological monitoring.

Matrix effect in liquid chromatography–electrospray ionization mass spectrometry analysis of benzoxazinoid derivatives in plant material

Despite the increasing success of liquid chromatography (LC) coupled to mass spectrometry (MS), matrix effects have limited the ESI applications. Matrix effects result from co-eluting residual matrix components affecting the ionization efficiency of target analytes and can lead to erroneous results. The present work evaluates the LC–electrospray ionization (ESI)-MS behaviour of benzoxazinoid derivatives in wheat plant material as a case where endogenous matrix components were found to interfere with the ionization of target analytes. When LC–MS was applied to the analysis of foliage and root extracts, significant signal suppressions were observed. To compensate for this matrix signal suppression, different methods were applied: the use of internal standards, the application of standard dilution method and the dilution of the extracts before instrumental determination. The results obtained indicated that evaluation of matrix effects should become an integrated part of quantitative LC–ESI-MS method development and validation.

Effects of glucosamine sulfate on intracellular UDP-hexosamine and UDP-glucuronic acid levels in bovine primary chondrocytes

To analyze the effects of exogenously added glucose (Glc), glucosamine (GlcN) and glucosamine sulfate (GS) on the intracellular UDP-hexoses (UDP-Hex), UDP-N-acetylhexosamines (UDP-HexN) and UDP-glucuronic acid (UDP-GlcA) levels in bovine primary chondrocytes. Chondrocytes were incubated with different concentrations of Glc, GlcN and GS either in high- or low-glucose DMEM for up to 120min to analyze the intracellular levels of UDP-Hex, UDP-GlcA and UDP-HexN by a reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry analysis. Glycosaminoglycan (GAG) synthesis rate and aggrecan mRNA expression levels were quantified using (35)S-sulfate incorporation assay and quantitative real-time RT-PCR, respectively. The cells were cultivated for 2 days or 8 days before UDP-sugar analysis. Levels of UDP-HexN and UDP-GlcA were unchanged at 10microM concentration of GS in low-glucose DMEM, while addition of 1mM GlcN or GS in low-glucose DMEM for 10min increased UDP-HexN level. The highest intracellular level of UDP-HexN was reached at 30min after addition of 1mM GS to the cells. The intracellular contents of UDP-HexN and UDP-GlcA related to UDP-Hex were higher after prolonged cultivation of chondrocytes for 8 days compared with 2-day-old cultures. Aggrecan mRNA expression and GAG synthesis remained at control level after the cells were treated with 10, 100microM or 1mM of GS for 24h. Physiologically relevant level of GS could not increase the intracellular UDP-HexN and UDP-GlcA levels in bovine primary chondrocyte, while longer-time culture itself appeared to increase the intracellular UDP-HexN and UDP-GlcA levels.

14,15-Epoxyeicosatrienoic Acid Represents a Transferable Endothelium-Dependent Relaxing Factor in Bovine Coronary Arteries

Bradykinin causes arterial relaxation and hyperpolarization, which is mediated by a transferable endothelium-derived hyperpolarizing factor (EDHF). In coronary arteries, epoxyeicosatrienoic acids (EETs) are involved in the EDHF response. However, the role of EETs as transferable mediators of EDHF-dependent relaxation remains poorly defined. Two small bovine coronary arteries were cannulated and perfused in tandem in the presence of the nitric oxide synthase inhibitor, nitro-L-arginine (30 micromol/L), and the cyclooxygenase inhibitor, indomethacin (10 micromol/L). Luminal perfusate from donor arteries with intact endothelium perfused endothelium-denuded detector arteries. Detector arteries were constricted with U46619 and diameters were monitored. Bradykinin (10 nmol/L) added to detector arteries did not induce dilation (5+/-2%), whereas bradykinin addition to donor arteries dilated detector arteries by 26.5+/-7% (P<0.05). These dilations were blocked by donor artery endothelium removal and detector artery treatment with the EET-selective antagonist, 14,15-epoxyeicosa-5(Z)-monoenoic acid (14,15-EEZE; 10 micromol/L, -5+/-6%) but not 14,15-EEZE treatment of donor arteries (20+/-5%). 14,15-EET (0.1 to 10 micromol/L) added to detector arteries induced maximal dilations of 82+/-5% that were inhibited 50% by detector artery treatment with 14,15-EEZE (32+/-12%) but not donor artery treatment with 14,15-EEZE. Liquid chromatography-electrospray ionization mass spectrometry analysis verified the presence of 14,15-EET in the perfusate from an endothelium-intact but not denuded artery. These results show that bradykinin stimulates donor artery 14,15-EET release that dilates detector arteries. 14,15-EEZE blocked the donor artery, endothelium-dependent, bradykinin-induced relaxations, and attenuated relaxations to 14,15-EET. These results suggest that EETs are transferable EDHFs in coronary arteries.

Analysis of medium-chain acyl-coenzyme A esters in mouse tissues by liquid chromatography–electrospray ionization mass spectrometry

Medium-chain acyl-coenzyme A (CoA) esters are key metabolites in lipid metabolism. Liquid chromatography-electrospray ionization mass spectrometry analysis of medium-chain acyl-CoA esters is described. Eight medium-chain acyl-CoA esters were well separated on a C 8-MS reversed-phase column using a linear gradient of ammonium acetate buffer (pH 5.3)–acetonitrile. The positive-ion mass spectra of all the saturated and unsaturated medium-chain acyl-CoA esters gave dominant [M + H] + ions, whereas their negative-ion mass spectra showed abundant [M − H] − and [M − 2H] 2− ions. The positive-ion mode of operation was slightly less sensitive than the negative-ion detection mode. Five medium-chain acyl-CoA esters of C 6:0, C 8:0, C 8:1, C 10:0, and C 10:1 in liver, heart, kidney, and brain from the mouse were identified. The predominant acyl-CoA peaks were C 6:0, C 8:0, and C 10:0. Small amounts of medium-chain acyl-CoAs of C 8:1 and C 10:1 were detected only in heart and kidney. The analytical method is very useful for the analysis of medium-chain acyl-CoA esters in the tissues.

Signal enhancement for gradient reverse-phase high-performance liquid chromatography-electrospray ionization mass spectrometry analysis with trifluoroacetic and other strong acid modifiers by postcolumn addition of propionic acid and isopropanol

Trifluoroacetic acid (TFA) and other volatile strong acids, used as modifiers in reverse-phase high-performance liquid chromatography, cause signal suppression for basic compounds when analyzed by electrospray ionization mass spectrometry (ESI-MS). Evidence is presented that signal suppression is caused by strong ion pairing between the TFA anion and the protonated sample cation of basic sample molecules. The ion-pairing process "masks" the protonated sample cations from the ESI-MS electric fields by rendering them "neutral. " Weakly basic molecules are not suppressed by this process. The TFA signal suppression effect is independent from the well-known spray problem that electrospray has with highly aqueous solutions that contain TFA. This previously reported spray problem is caused by the high conductivity and surface tension of aqueous TFA solutions. A practical method to enhance the signal for most basic analytes in the presence of signal-suppressing volatile strong acids has been developed. The method employs postcolumn addition of a solution of 75% propionic acid and 25% isopropanol in a ratio 1:2 to the column flow. Signal enhancement is typically 10-50 times for peptides and other small basic molecules. Thus, peptide maps that use ESI-MS for detection can be performed at lower levels, with conventional columns, without the need to use capillary chromatography or reduced mass spectral resolution to achieve satisfactory sensitivity. The method may be used with similar results for heptafluorobutyric acid and hydrochloric acid. A mechanism for TFA signal suppression and signal enhancement by the foregoing method, is proposed.