High-performance Liquid Chromatography Coupled With Mass Spectrometry Research Articles

Effect of Phenyl-Acyl Compounds on the Growth, Morphology, and Toxin Production of Microcystis aeruginosa Kützing

The proliferation of cyanobacteria and, consequently, the production of cyanotoxins is a serious public health concern; for their control, several alternatives have been proposed, including physical, chemical, and biological methods. In the search for new alternatives and a greater understanding of the biochemical process involved in the blooms’ formation, we report here the effect of eight phenyl-acyl compounds in the growth of Microcystis aeruginosa Kützing (assesed as cell density/count and Chl a fluorescence concentration) morphology, and production of the toxin microcystin-LR (MC-LR). Caffeic acid and eugenol decreased the growth of M. aeruginosa Kützing and the levels of Chl a. However, 3,5-dimethoxybenzoic acid and syringic acid caused the opposite effect in the growth; 2′and 4′only affected the Chl a. A reduction in the concentration of the MC-LR toxin was detected after treatment with syringic acid, caffeic acid, and eugenol. According to HPLC/MS (High Performance Liquid Chromatography coupled to Mass Spectrometry), a redox process possibly occurs between caffeic acid and MC-LR. The optical microscopy and Scanning Electron Microscopy analyses revealed morphological changes that had been exposed to caffeic acid and vanillin, specifically in the cell division and presence of mucilage. Finally, assays in Daphnia pulex De Geer neonates indicated that caffeic acid had a non-toxic effect at concentrations as high as 100 mg/L at 48 h.

Analysis of monosaccharide composition in polysaccharides from Lycium barbarum L. by precolumn labeling under mild conditions and high-performance liquid chromatography coupled with mass spectrometry

A method for the separation, identification, and determination of fructose and various aldehyde monosaccharides was established by precolumn labeling with 1-phenyl-3-methyl-5-pyrazolone (PMP) and high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS). The separation was performed on a Kromasil-C18 column (100 mm×4.6 mm, 3.5 μm) with gradient elution. The detection was performed by electrospray ionization mass spectrometry in selected ion monitoring (SIM) mode. In this study, the derivatization mechanism of PMP-labeled fructose was proposed under mild NH3·H2O conditions. The suggested method showed good linearity in the corresponding mass concentration ranges, with the correlation coefficients (r2) > 0.9947. The limits of detection (LODs) and limits of quantification (LOQs) were in the ranges 0.003 to 0.05 mg/L and 0.01 to 0.15 mg/L, respectively. The recoveries in spiked Lycium barbarum L. samples were 65.1% to 116.2%, with relative standard deviations (RSDs) of less than 10.2%. By virtue of its simplicity, high sensitivity, and good repeatability, the method could be successfully applied to the analysis of the monosaccharide composition in polysaccharides of Lycium barbarum L. from four planting areas. Results showed that the isolated polysaccharides comprise mannose, fructose, rhamnose, galactose, glucose, xylose, arabinose, and ribose. The concentration distribution of various monosaccharides differed notably depending on the planted environmention. The proposed method is expected to be of great significance in standardizing the quality control of polysaccharides.

Determination of Phenolic Endocrine Disruptors in Cosmetics by High-Performance Liquid Chromatography Mass Spectrometry

ABSTRACTAn analytical method for the simultaneous determination of bisphenol A, 4-t-octylphenol, 4-n-octylphenol, and 4-n-nonylphenol in cosmetic samples has been developed. These compounds have toxic effects on human health as they have shown to produce endocrine disrupting properties. Therefore, their presence in cosmetics should be avoided according to the current European Regulations on cosmetic products. The method is based on high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) detection. Standard addition calibration was used to avoid matrix effects. The limits of detection values ranged between 7 and 15 ng mL−1 (threefold of the residual standard deviation of regression lines). The proposed method was validated, and good recovery (90–106%) and repeatability values (2.7–8.2%) were obtained. Finally, the method was successfully applied to ten commercially available cosmetic samples. The good analytical features of the proposed method make it useful to carry out the quality control of cosmetic products and raw materials to assure the safety of users.

Antagonism between Byssochlamys spectabilis (anamorph Paecilomyces variotii) and plant pathogens: Involvement of the bioactive compounds produced by the endophyte

AbstractFungal endophytes can be part of the defensive system of plants against multiple pathogens by competing for resources, hyperparasitism or producing bioactive compounds with antimicrobial properties. There is an ever‐increasing interest for obtaining new and environmentally friendly products to use in the fight against pathogens. With this purpose, Byssochlamys spectabilis (anamorph Paecilomyces variotii), which is a fungal species that commonly occurs, was evaluated as an antagonistic organism towards three phytopathogens (Biscogniauxia mediterranea, Fusarium moniliforme and Phytophthora cinnamomi). First, an in vitro experiment was designed to test the effect that the endophyte filtrate had on the three pathogens. The endophyte filtrate decreased the radius growth rate of F. moniliforme by nearly 10%. Consequently, the antagonism between B. spectabilis and F. moniliforme was evaluated in Lolium rigidum plants under greenhouse conditions by means of co‐inoculations. The endophyte produced a decrease of 50–75% in the disease severity caused by the pathogen in the earliest infection stages. Crude extracts of B. spectabilis were obtained to determine the secondary metabolites responsible for such an effect. The bioactivity‐guided chromatography and HPLC‐MS (high performance liquid chromatography coupled with mass spectrometry) of the active fraction suggested that the antibiotic activity was caused by viriditoxin. In conclusion, the fungal endophyte B. spectabilis and/or its bioactive compounds showed antagonism towards several phytopathogens and deserves further study to investigate its actual potential for use as a biocontrol agent.

Veronica officinalis Product Authentication Using DNA Metabarcoding and HPLC-MS Reveals Widespread Adulteration with Veronica chamaedrys.

Studying herbal products derived from local and traditional knowledge and their value chains is one of the main challenges in ethnopharmacology. The majority of these products have a long history of use, but non-harmonized trade and differences in regulatory policies between countries impact their value chains and lead to concerns over product efficacy, safety and quality. Veronica officinalis L. (common speedwell), a member of Plantaginaceae family, has a long history of use in European traditional medicine, mainly in central eastern Europe and the Balkans. However, no specified control tests are available either to establish the quality of derived herbal products or for the discrimination of its most common substitute, V. chamaedrys L. (germander speedwell). In this study, we use DNA metabarcoding and high performance liquid chromatography coupled with mass spectrometry (HPLC-MS) to authenticate sixteen V. officinalis herbal products and compare the potential of the two approaches to detect substitution, adulteration and the use of unreported constituents. HPLC-MS showed high resolution in detecting phytochemical target compounds, but did not enable detection of specific plant species in the products. DNA metabarcoding detected V. officinalis in only 15% of the products, whereas it detected V. chamaedrys in 62% of the products. The results confirm that DNA metabarcoding can be used to test for the presence of Veronica species, and detect substitution and/or admixture of other Veronica species, as well as simultaneously detect all other species present. Our results confirm that none of the herbal products contained exactly the species listed on the label, and all included substitutes, contaminants or fillers. This study highlights the need for authentication of raw herbals along the value chain of these products. An integrative methodology can assess both the quality of herbal products in terms of target compound concentrations and species composition, as well as admixture and substitution with other chemical compounds and plants.

Comparative authentication of Hypericum perforatum herbal products using DNA metabarcoding, TLC and HPLC-MS

Many herbal products have a long history of use, but there are increasing concerns over product efficacy, safety and quality in the wake of recent cases exposing discrepancies between labeling and constituents. When it comes to St. John’s wort (Hypericum perforatum L.) herbal products, there is limited oversight, frequent off-label use and insufficient monitoring of adverse drug reactions. In this study, we use amplicon metabarcoding (AMB) to authenticate 78 H. perforatum herbal products and evaluate its ability to detect substitution compared to standard methods using thin-layer chromatography (TLC) and high performance liquid chromatography coupled with mass spectrometry (HPLC-MS). Hypericum perforatum was detected in 68% of the products using AMB. Furthermore, AMB detected incongruence between constituent species and those listed on the label in all products. Neither TLC nor HPLC-MS could be used to unambiguously identify H. perforatum. They are accurate methods for authenticating presence of the target compounds, but have limited efficiency in detecting infrageneric substitution and do not yield any information on other plant ingredients in the products. Random post-marketing AMB of herbal products by regulatory agencies could raise awareness among consumers of substitution and would provide an incentive to manufacturers to increase quality control from raw ingredients to commercialized products.

HPLC-MS Analysis of Lichen-Derived Metabolites in the Life Stages of Crambidia cephalica (Grote & Robinson).

Tiger moths (Lepidoptera: Erebidae: Arctiinae: Arctiini) are notable for their specialized associations with hosts that produce toxic secondary compounds, and are thus an ideal study system for understanding insect-plant interactions and the evolution of antipredatory defense. Likewise, their sister lineage (Arctiinae: Lithosiini) has been documented feeding on algae and lichens, and is known to sequester lichen-derived secondary compounds from the larval to adult stages. Prevalence of lichenivory in this early radiation (ca. 3000 species) may provide clues to the phylogenetic basis for storied chemical sequestration within all tiger moths. Despite the evolutionary significance of this trait, we lack a basic understanding of the extent of lichenivory among lithosiines, and the distribution of sequestered chemicals among life stages. The dynamics of chemical sequestration throughout the lifecycle for the lichen moth Crambidia cephalica were investigated by testing the hypothesis that lichen-derived metabolites are unequally distributed among life stages, and that laboratory-reared C. cephalica have less metabolite diversity than wild-caught individuals. Crambidia cephalica was reared on Physcia, and analyzed using high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS). Several putative lichen-derived metabolites were detected across three life stages, i.e., larval, pupal, and adult, and differences among life stages and lichen host were observed. These results provide evidence that multiple lichen-derived metabolites are sequestered by C. cephalica; some metabolites are retained through adulthood, and others are lost or modified in earlier life stages. The presence of differing lichen-derived metabolites across life stages may indicate functional properties of the metabolites for C. cephalica with regards to chemical protection from antagonists, and other physiological processes.

LC-MS–MS and GC-MS analyses of biologically active extracts and fractions from Tunisian Juniperus phoenice leaves

Context: Despite some studies related to Juniperus phoenicea L. (Cupressaceae), phytochemical and biological investigations of this plant remain unexplored. Objective: This work is the first report dealing with the identification and characterization of volatile components and flavonoids in hexane and methanol extracts from J. phoenicea leaves Materials and methods: Antioxidant activity of hexane, and methanol extracts from J. phoenicea leaves were determined by DPPH-radical scavenging assay. α-Amylase inhibitory activity was evaluated by enzyme inhibition using in vitro assay (each extract was dissolved in DMSO to give concentrations of 50, 100 and 200 mg/mL). The chemical composition of fractions (Fr1-Fr3) from methanol extract was determined by high-performance liquid chromatography coupled with mass spectroscopy (HPLC-MS) analysis. Results and discussion: The hexane extract was analyzed by GC-MS technique which allowed the identification of 32 compounds. The main constituents were α-humulene (16.9%), pentadecane (10.2%) and α-cubebene (9.7%). Fraction Fr 2 exhibited a strong DPPH radical-scavenging activity (IC50 = 20.1 μg/mL) compared to that of BHT as well as the highest α-amylase inhibitory activity (IC50 = 28.4 μg/mL). Three flavonoids were identified in these fractions using HPLC-MS analysis: Quercetin 3-O-glucoside, isoscutellarein 7-O-pentoside and quercetin 3-O-pentoside. In addition, the more active fraction (Fr 2) was purified with semi-preparative HPLC affording one pure compound (amentoflavone) using 1H NMR analysis. This compound exhibited powerful DPPH radical-scavenging (IC50 = 14.1 μg/mL) and α-amylase inhibition (IC50 = 20.4 μg/mL) effects. Conclusion: This study provides scientific support to some medicinal uses of J. phoenicea found in North Africa.

Influence of pretreatment of piperazine ferulate on pharmacokinetic parameters of methotrexate in methotrexate-induced renal injury model rats by HPLC-MS

The present study was designed to investigate the influence of the pretreatment of piperazine ferulate on pharmacokinetic parameters of methotrexate in methotrexate-induced renal injury rats. A simple and efficient high performance liquid chromatography coupled with mass spectrometry (HPLC-MS) method was developed to determine methotrexate in rat plasma. Methotrexate and syringic acid (internal standard) were extracted from rat plasma samples by protein precipitation with acetonitrile. The analysis was performed on a CAPCELL PAK C18 column (150 mm × 4.6 mm, 5 µm) with acetonitrile and 5 mmol/l ammonium acetate aqueous (10:90, v/v). The linear range was 5.0 × 10−2 to 100.0 µg/ml for methotrexate. Other parameters were all within the acceptance criteria. The validated method was successfully applied the pharmacokinetic study of methotrexate between two methotrexate treated groups (with and without the pretreatment of piperazine ferulate). Compared with the methotrexate treated alone group, the pharmacokinetic parameters in the methotrexate with the pretreatment of piperazine ferulate group showed significantly lower MRT(0-t), MRT(0-∞) and T1/2. Results suggested that methotrexate can be rapidly eliminated, cleared or metabolized in rat blood, which might be related to the pretreatment of piperazine ferulate. The method provided deeper insights into rational clinical use of methotrexate with the pretreatment of piperazine ferulate on cancer patients with renal dysfunction.

Effect of Fuke Qianjin tablets on pharmacokinetics of Azithromycin in rats with chronic pelvic inflammatory disease

To investigate the effect of Fuke Qianjin tablets on the pharmacokinetics of Azithromycin(AZM) in rats with chronic pelvic inflammation, and evaluate the rationality of the drug combination. The animal models of chronic pelvic inflammatory disease were established by intrauterine injection of mixed bacterial suspension plus mechanic injury in female Sprague-Dawley(SD) rats. The model rats were randomly divided into control group and drug combination group. The rats in control group were given with Azithromycin 10 mg•kg⁻¹•d⁻¹ by intragastric administration, while the rats in drug combination group were given with Fuke Qianjin tablets 1.66 g•kg⁻¹•d⁻¹ by intragastric administration 2 hours after the equivalent dose of azithromycin, once a day, consecutively for 7 days. Plasma samples were collected at different time points and the concentration of AZM in plasma was determined by high performance liquid chromatography coupled with mass spectrometry(HPLC-MS). ADAPT 5.1 software was used to calculate the pharmacokinetic parameters of each group by Inverse Gaussian Function model. SPSS software was used for the statistical analysis of data in each group. The results showed that Fuke Qianjin tablets had no significant effects on the main pharmacokinetic parameters of AZM, including CLt, CLd, MIT and F. The study showed that Fuke Qianjin tablets can be combined with azithromycin for treatment of chronic pelvic inflammation disease.

SOURCES, FORMATION, REACTIVITY AND DETERMINATION OF QUINONES IN THE ATMOSPHERE

Quinones are a group of semi-volatile organic substances, ubiquitous in nature, belonging to the class of oxygenated polycyclic aromatic hydrocarbons (Oxygenated PAH). These compounds are formed in biogenic processes, through the oxidative metabolism of endogenous compounds, such as catecholamines, estrogen hormones and xenobiotics, as well as directly emitted to the atmosphere, through the incomplete combustion of organic matter, especially the fossil fuels. Additionally, the quinones are formed in the atmosphere by photooxidation of Polycyclic Aromatic Hydrocarbons (PAH). Their reduction products (semiquinones and hydroquinones) are of toxicological interest, due to their ability in generate reactive oxygen species, which may cause damage to membranes, proteins and DNA and, among others damages, may lead to carcinogenesis and induce apoptosis. The most frequently used analytical methods for quantification of quinones in the atmosphere, either in its original form or as derivatized compounds, are high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) and gas chromatography coupled to mass spectrometry (GC-MS). This review aims to highlight the chemical properties of 1,2-naphthoquinone, 1,4-naphthoquinone, 1,4-benzoquinone, 9,10-phenanthraquinone and 9,10-anthraquinone, the more volatile, reactive and abundant quinones in the atmosphere, as well as the methods usually employed for their determination.

FORECASTING AND EXPERIMENTAL DETERMINATION OF DESTRUCTION IMPURITIES AND IN THE PHARMACEUTICAL COMPOSITION OF THE HYPOTENSIVE ACTION

Determination results of impurities verapamil hydrochloride and lisinopril dihydrate using computer modeling and HPLC coupled with mass spectrometry (HPLC-MS) reported in this article. Prediction and calcu-lation of the thermodynamic characteristics of the substances carried by the method of RM1 and programs HyperChem. Mass spectrometric analysis was performed on a quadrupole mass analyzer in the recording mode of the positive ions within the range m / z 100-1000. The study found that described in the regulations extrane-ous verapamil and lisinopril are the products of degradation of materials. Prediction of degradation products of lisinopril dihydrate fully confirmed the data of the ion current chromatograms and mass spectra of the substanc-es. However, in the case of verapamil simulation results coincided with the degradation products by HPLC-MS analysis for two of the four predicted substances

Phenolic Content, Antioxidant and Astroprotective Response to Oxidative Stress of Ethanolic Extracts of Mentha longifolia from Sinai

The aerial parts ofMentha longifolia L. are used as herbal remedies for curing different diseases through traditional Bedouin medicine. The antioxidant activity of the ethanolic extracts of M longifolia was investigated measuring peroxyl radical-scavenging activity by ORAC assay, with Trolox (a water-soluble analogue of α-tocopherol) employed as reference compound. In addition, the total content of phenolic compounds estimated by the Folin-Ciocalteau method and the identification of the polyphenols using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) have been performed. Furthermore, the effect of these extracts on cell viability and intracellular ROS production was assayed using the U373-MG human astrocytoma cell line in a H2O2-induced oxidative stress model. Results showed that the major type of polyphenols found were benzoic acids, cinnamic acids, flavones and flavanones. The total phenolic content was 37.7 mg gallic acid/g sample and the ORAC value was 1.355 .mol TE/mg sample. The data obtained in cellular assays demonstrated that these ethanolic extracts protected H2O2-induced astrocyte damage by increasing cell viability and inhibiting production of intracellular ROS. These results suggest that the investigated extracts obtained from the aerial parts of M longifolia have antioxidant potential related to their phenol content which have important beneficial health effects, especially in those disease associated with ROS.

Novel tracer method to measure isotopic labeled gas-phase nitrous acid (HO15NO) in biogeochemical studies.

Gaseous nitrous acid (HONO), the protonated form of nitrite, contributes up to ∼60% to the primary formation of hydroxyl radical (OH), which is a key oxidant in the degradation of most air pollutants. Field measurements and modeling studies indicate a large unknown source of HONO during daytime. Here, we developed a new tracer method based on gas-phase stripping-derivatization coupled to liquid chromatography-mass spectrometry (LC-MS) to measure the 15N relative exceedance, ψ(15N), of HONO in the gas-phase. Gaseous HONO is quantitatively collected and transferred to an azo dye, purified by solid phase extraction (SPE), and analyzed using high performance liquid chromatography coupled to mass spectrometry (HPLC-MS). In the optimal working range of ψ(15N)=0.2-0.5, the relative standard deviation of ψ(15N) is <4%. The optimum pH and solvents for extraction by SPE and potential interferences are discussed. The method was applied to measure HO15NO emissions from soil in a dynamic chamber with and without spiking 15) labeled urea. The identification of HO15NO from soil with 15N urea addition confirmed biogenic emissions of HONO from soil. The method enables a new approach of studying the formation pathways of HONO and its role for atmospheric chemistry (e.g., ozone formation) and environmental tracer studies on the formation and conversion of gaseous HONO or aqueous NO2- as part of the biogeochemical nitrogen cycle, e.g., in the investigation of fertilization effects on soil HONO emissions and microbiological conversion of NO2- in the hydrosphere.

Abstract 670: A Metabolic Approach to Examining the Hexosamine Biosynthetic Pathway and Its Role in the Development of Diabetes-Associated Atherosclerosis

Introduction: Diabetes is a disease affecting millions of people worldwide, and is a major independent risk factor for cardiovascular disease (CVD). Despite a vast amount of research, the molecular mechanisms that link diabetes to CVD are not well understood. Current evidence suggests that increased flux through the hexosamine biosynthetic pathway (HBP) contributes to the development of hyperglycemia-associated diabetic complications. Our data suggest that increased HBP flux can induce vascular ER stress and accelerate atherogenesis in a mouse model. We hypothesized that this process can be attenuated by inhibiting the first and rate-limiting enzyme in the HBP - glutamine fructose-6-phosphate amidotransferase (GFAT) - using small molecules. Methods and Results: Using high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) we have developed a methodology to monitor and quantify the levels of the end product of the HBP, uridine diphosphate N -acetylglucosamine (UDP-GlcNAc). Treatment of HepG2 cells with glucosamine (0.2-5 mM), or adenovirus-directed overexpression of GFAT caused a 3-7 fold increase in UDP-GlcNAc ( P&lt; 0.001 &amp; P&lt; 0.05, respectively). Inhibition of GFAT with three novel compounds - amrinone, lapachol or alloxan - decreased levels of UDP-GlcNAc by 1.5 ( P&lt; 0.05), 3 ( P&lt; 0.05) and 3.5-fold ( P&lt; 0.001), respectively. Furthermore, we show that by modulating HBP flux, we can regulate ER stress levels in cultured HepG2 cells. The physiological relevance of this mechanism is supported by evidence of HBP augmentation in a hyperglycemic mouse model. Conclusions: These results support a role for the HBP in the development of atherosclerosis. Currently, MALDI imaging mass spectrometry is being performed on tissue sections to compare the levels of UDP-GlcNAc directly in hyperglycemic vs. normoglycemic mice. These studies may lead to the identification and validation of novel targets for the development of new pharmaceuticals to prevent diabetic atherosclerosis.

Evaluation of Antioxidant and Antimicrobial Activities and Phenolic Profile for Hyssopus officinalis, Ocimum basilicum and Teucrium chamaedrys

This study was designed to examine the in vitro antioxidant and antimicrobial activities and to characterize the polyphenolic composition of the ethanolic extracts of Hyssopus officinalis, Ocimum basilicum and Teucrium chamaedrys. Qualitative and quantitative analysis of the major phenolic compounds were conducted using high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS). The total polyphenols, caffeic acid derivatives and flavonoids content was spectrophotometrically determined. The phenolic profile showed the presence of phenolic acid derivatives (caftaric, gentisic, caffeic, p-coumaric, chlorogenic and ferulic acids), flavonoid glycosides (rutin, isoquercitrin and quercitrin) and free flavonoid aglycons (luteolin, quercetin), in different concentrations. DPPH radical scavenging assay, Trolox equivalent antioxidant capacity (TEAC) method, hemoglobin ascorbate peroxidase activity inhibition (HAPX) assay, and electron paramagnetic resonance (EPR) radicals detection were employed, revealing several aspects of the antioxidant activities of these species. The antimicrobial tests were performed using the disk diffusion assay. These extracts contained a large amount of the polyphenolic compounds (77.72, 175.57, and 243.65 mg/g, respectively), and they showed a good antioxidant activity, as witnessed by a number of methods. T. chamaedrys had a high antimicrobial activity. Besides their antioxidant activity, the antimicrobial effect of these extracts confirms the biological activities of these herbal medicinal products.

Closely related phytoplankton species produce similar suites of dissolved organic matter.

Production of dissolved organic matter (DOM) by marine phytoplankton supplies the majority of organic substrate consumed by heterotrophic bacterioplankton in the sea. This production and subsequent consumption converts a vast quantity of carbon, nitrogen, and phosphorus between organic and inorganic forms, directly impacting global cycles of these biologically important elements. Details regarding the chemical composition of DOM produced by marine phytoplankton are sparse, and while often assumed, it is not currently known if phylogenetically distinct groups of marine phytoplankton release characteristic suites of DOM. To investigate the relationship between specific phytoplankton groups and the DOM they release, hydrophobic phytoplankton-derived dissolved organic matter (DOMP) from eight axenic strains was analyzed using high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS). Identification of DOM features derived from Prochlorococcus, Synechococcus, Thalassiosira, and Phaeodactylum revealed DOMP to be complex and highly strain dependent. Connections between DOMP features and the phylogenetic relatedness of these strains were identified on multiple levels of phylogenetic distance, suggesting that marine phytoplankton produce DOM that in part reflects its phylogenetic origin. Chemical information regarding the size and polarity ranges of features from defined biological sources was also obtained. Our findings reveal DOMP composition to be partially conserved among related phytoplankton species, and implicate marine DOM as a potential factor influencing microbial diversity in the sea by acting as a link between autotrophic and heterotrophic microbial community structures.

Quantitative analysis of ribonucleoside modifications in tRNA by HPLC-coupled mass spectrometry.

Post-transcriptional modification of RNA is an important determinant of RNA quality control, translational efficiency, RNA-protein interactions and stress response. This is illustrated by the observation of toxicant-specific changes in the spectrum of tRNA modifications in a stress-response mechanism involving selective translation of codon-biased mRNA for crucial proteins. To facilitate systems-level studies of RNA modifications, we developed a liquid chromatography-mass spectrometry (LC-MS) technique for the quantitative analysis of modified ribonucleosides in tRNA. The protocol includes tRNA purification by HPLC, enzymatic hydrolysis, reversed-phase HPLC resolution of the ribonucleosides, and identification and quantification of individual ribonucleosides by LC-MS via dynamic multiple reaction monitoring (DMRM). In this approach, the relative proportions of modified ribonucleosides are quantified in several micrograms of tRNA in a 15-min LC-MS run. This protocol can be modified to analyze other types of RNA by modifying the steps for RNA purification as appropriate. By comparison, traditional methods for detecting modified ribonucleosides are labor- and time-intensive, they require larger RNA quantities, they are modification-specific or require radioactive labeling.

Standardization of a protocol to extract and analyze chlorophyll a and carotenoids in Gracilaria tenuistipitata Var. Liui. Zhang and Xia (Rhodophyta)

Chlorophyll a and carotenoids are important pigments in photosynthesis. Several studies have been published describing extraction and analysis protocols of these pigments, mainly in vascular plant species. This study standardizes an extraction and analysis protocol of these substances in Gracilaria tenuistipitata var. liui, a red seaweed. Apical portions grown in vitro were triturated in liquid nitrogen. Extracts were prepared in 1.5 mL solvent and centrifuged. Quantitative and qualitative analyses of pigments were performed by UV/visible light spectrophotometry and high performance liquid chromatography (HPLC) and HPLC coupled to mass spectrometry (HPLC-MS). The parameters assessed were: minimum biomass, best extraction solvent, and number of extraction steps. Methanol was the most efficient solvent, and 50 mg fresh biomass was the amount of sample indicated, submitted to one single extraction step. No significant differences were observed in levels of these pigments by UV-visible light spectrophotometry and HPLC. However, HPLC or HPLC-MS are required to identify the different carotenoids present in this seaweed species.

Degradation and mineralization of 2,4,6-trinitroresorcine in various photochemical systems

Comparison was observed for degradation and mineralization of the explosive 2,4,6-trinitroresorcine (TNR) in different photochemical systems TNR/UV, TNR/UV/TiO2, TNR/UV/H2O2, TNR/UV/O3, TNR/UV/TiO2/H2O2 and TNR/UV/TiO2/O3 using High Performance Liquid Chromatography coupled with Mass Spectrometry (HPLC/MS) and Total Organic Carbon (TOC) analysis. Addition of oxidizing agents such as H2O2 or O3 accelerated the rate of TNR conversion and mineralization. Highest reaction rate was obtained in TNR/UV/TiO2/H2O2 system. The intermediate products were characterized and identified by LS–MS technique. The similarity in intermediate products of TNR suggested the analogous reaction pathways of the TNR degradation by these different systems.