Authentic Compounds Research Articles

An integrated strategy to quantitatively differentiate chemome between Cistanche deserticola and C. tubulosa using high performance liquid chromatography–hybrid triple quadrupole-linear ion trap mass spectrometry

It is important to conduct large-scale detection, identification, and quantitation of metabolites in a given sample. Herein, a practical strategy was proposed to quantitatively compare the chemome between Cistanche deserticola (CD) and C. tubulosa (CT), which have been widely believed as the ideal edible and medicinal plants for conquering the deserts. The entire workflow was implemented on high performance liquid chromatography–hybrid triple quadrupole-linear ion trap mass spectrometer and consisted of three primary steps: (1) component detection and identification, various mass spectrometric approaches were applied to globally screen the chemical constituents, and structural elucidation was achieved by comparing with authentic compounds, analyzing MS2 spectra, and referring to the literature along with accessible databases; (2) comprehensive relative quantitation, scheduled multiple reaction monitoring algorithm was introduced for relative quantitation of all detected ingredients; and (3) chemome comparison, the quantitative dataset was subjected for multivariate statistical analysis to carry out comparative study. A total of 513 metabolites were detected and relatively quantitated, and 379 ones were annotated. Betaine, Krebs cycle intermediates, phenylethanoid glycosides, and iridoids were picked out as the chemical markers being responsible for the discrimination of the chemical profiles between CD and CT. Above all, the quantitative chemome of CD and CT were exhaustively characterized and compared, which could advance their values concerning drug development, economics, and desertification control. The proposed strategy is expected as a reliable choice for widely targeted metabolomics of plants.

English

Five compounds were reported for the first time in the tribe Malouetieae from the leaves and stems of Pachypodium lamerei viz., fatty acid methyl ester (stearic acid methyl ester 1), a mixture of two steroidal aglycones (β-sitosterol 2 and stigmasterol 3), a pentacyclic triterpene (ursolic acid 4) and a steroidal glucoside (β-sitosterol-3-O-β-D-glucopyranoside 5). The structure elucidation was based on comparison of their physical, chemical, chromatographic properties, spectral data with literature as well as direct comparison with authentic compounds. The petroleum ether fraction and the total ethanolic extract of (leaves and stems) exhibited minimum inhibitory concentrations (MICs) against Pseudomonas aeruginosa (1.06 and 2.52 μg/ml), respectively. These results are comparable to that obtained by amikacin (MIC, 2.38 μg/ml). Moreover, the petroleum ether fraction displayed the lowest MIC (16.45 μg/ml) against Candida albicans. This result is greatly promising since the standard drug (ketoconazole) exhibited MIC (185.87 μg/ml). This is the first time to estimate both antibacterial and antifungal activities of any of Pachypodium species. Key words: Pachypodium lamerei, Apocynaceae, phytochemical, antimicrobial, antibacterial, antifungal.

Real Time Online Correction of Mass Shifts and Intensity Fluctuations in Extractive Electrospray Ionization Mass Spectrometry.

Real time online calibration of mass shift and intensity fluctuation to improve the accuracy of measurements for identification and quantitation in trace mass spectrometric analysis was demonstrated using extractive electrospray ionization mass spectrometry (EESI-MS). The signals of authentic compounds (e.g., lysine (Lys), proline (Pro), and histidine (His)) spiked into the extractive solution for the EESI process were used as the references to calibrate the signal of analytes (e.g., methionine (Met)) in the untreated sample solution. The intensity of the analyte signal was recorded simultaneously with the reference signals. The analyte signals at a given time point were calibrated on the basis of these correlation factors and real time signal response of the reference. The calibrated signal of Met at 10 μg L(-1) was improved with a better signal-to-noise ratio (S/N from 2.3 to 4.3), better linearity (R(2) from 0.9758 to 0.9980), and reduced relative standard deviation (RSD from 9.8% to 6.0%). The shift of mass-to-charge ratio of Met signal between the detected and theoretical values was decreased from 247 ± 133 to -7 ± 167 ppm for 50 min of detection using a linear ion trap mass analyzer and was reduced from -0.27 ± 0.60 to -0.12 ± 0.23 ppm for 50 min of detection using an Orbitrap mass analyzer (P = 95%). This method has been validated using a certified standard amino acids solution (GBW(E)100062) and applied for quantitative detection of amino acids in chicken feed, urine, nutritional drink, and facial mask samples, showing that the method is useful to improve the accuracy of mass spectrometric analysis.

Study on the Degradation of the Highly Reactive Hypervalent Trifluoromethylation Iodine Reagent PhI(OAc)(CF3)

AbstractDegradation of the highly reactive hypervalent trifluoromethylation iodine reagent PhI(OAc)(CF3), which can only be generated in situ with mixing PhI(OAc)2 and TMSCF3 in the presence of CsF, was studied by ESI‐MS and GC‐MS combined with 19F‐NMR. The important transient intermediate PhICF3+ was determined by ESI‐MS, and the major volatile products containing CF3 were identified with the authentic compounds by using GC‐MS, such as trifluoromethylbenzene, 2‐iodobenzotrifluoride, 3‐iodobenzotrifluoride, 4‐iodobenzotrifluoride. Meanwhile, more evidences obtained with 19F‐NMR were given for such degradation reaction. A possible rapid CF3 radical transfer reaction pathway was proposed to clarify such degradation progress based on the experimental results. Therefore, this study may be helpful in elucidating the intrinsic reactivity of PhI(OAc)(CF3) and the possible competing side reactions caused by such self‐degradation pathway.

Abstract A74: Modulation of oxidative stress and exosome activity by phytoagent deoxyelephantopin (DET) and its derivative treatment in suppressing triple negative breast cancer cell functions

Abstract Breast cancer is one of the frequently diagnosed and life-threatening cancer diseases in women worldwide, especially, the triple negative breast cancer (ER-/PR-/HER2- TNBC) is a clinically challenging breast cancer disease due to lack of efficient targeted therapeutics. Recently, the development of phytocompounds derived from traditional medicinal herbs into potential chemotherapeutic or chemopreventive agent for human cancer management has aroused a great interest. In our previous study, we identified deoxyelephantopin (DET), a major germacranolide sesquiterpene lactone from a traditional medicinal herb Elephantopus scaber L., which could significantly suppress TS/A (ER+) mammary cancer cell growth, motility and metastasis in vitro andin vivo; however, a relatively less suppressive effect was observed in human TNBC cell line, MDA-MB-231. We thus designed and created a DET derivative (designated DETD) by semi-organic synthesis, which exhibited a 4-fold less in IC50 value than DET in inhibiting MDA-MB-231 cell proliferation. To address the modes of action of both authentic DET and novel DETD compounds against TNBC, we established and used mass spectrometry (MS)-based quantitative proteomics, direct binding assay of DET-near infrared (NIR) fluorescent compound conjugate, and mammary tumor model to gain the mechanistic insight. Our data showed that DET and DETD can markedly induce the reactive oxygen species (ROS) production in early stage of treatment, that may further promote nonautophagic cell death through cytoplasmic vacuoles production. Furthermore, we observed the release of exosome vesicles from MDA-MB-231 cells was elevated by DET or DETD treatment compared to the vehicle control. Quantitative investigation of exosomal proteome showed that DET and DETD responsive exosomal proteins were involved in the regulation of EIF2 signaling, mTOR signaling, regulation of eIF4 and p70S6K signaling, CDK5 signaling, and inhibition of angiogenesis. Of note, the immunofluorescence cell staining showed that DET-NIR was co-localized with exosomes of MDA-MB-231 cells, on the other hand, several nuclear exosomal proteins of mouse TNBC 4T1 cells could interact directly with DET. Together results of this study suggest that DET and DETD compounds may serve as a good candidate in treatment of TNBC disease, through modulation of oxidative stress and specific exosomal proteins in cancer cells. Citation Format: Jeng-Yuan Shiau, Kyoko Nakagawa-Goto, Kuo-Hsiung Lee, Wai-Leng Lee, Jacquelyn Gervay-Hague, Lie-Fen Shyur. Modulation of oxidative stress and exosome activity by phytoagent deoxyelephantopin (DET) and its derivative treatment in suppressing triple negative breast cancer cell functions. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A74.

A Novel Aldo-Keto Reductase, HdRed, from the Pacific Abalone Haliotis discus hannai, Which Reduces Alginate-derived 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid to 2-Keto-3-deoxy-d-gluconate

Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-D-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of ∼ 40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-D-gluconate with an optimal temperature and pH at around 50 °C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, α-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18-60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family.

Hypotensive Activity of <i>Moringa oleifera</i> Lam (Moringaceae) Root Extracts and its Volatile Constituents

Purpose : To explore the hypotensive activity and chemical composition of Moringa oleifera Lam (Moringaceae) roots. Methods : The fresh roots of M. oleifera was cut into small pieces and successively extracted with petroleum ether (PE) and dichloromethane (DC). PE extract was further divided into MRP and MRP -1. DC extract showed a thick mass during evaporation which was separated as MRDC - IN. The mother liquor left was divided into MRDC and MRDC -1. All residues were analyzed by gas chromatographymass spectroscopy (GC-MS) using ZB-5 column. Identification of each extract and fraction was based on comparison of their retention indices (RI), by co-injecting authentic compounds, as well as by comparing literature data available in NIST Standard Reference Database. Hypotensive activity was determined on urethane-anesthetized normotensive Sprague Dawly rats. Results : Petroleum ether (MRP) and dichloromethane (MRDC) extracts of M. oleifera roots showed 50.06 ± 3.48 and 48.16 ± 1.79 % fall in mean arterial blood pressure (MABP), respectively, at a dose of 30 mg/kg (p < 0.01 and p < 0.05, respectively) compared with control. GC-MS analysis of MRP and MRDC extracts and fractions resulted in the identification of seventy four (74) compounds. Methyl hexadecanoate (7, 20.3 %) , stigmastan - 3, 5, diene (24, 19.32 %), methyl 14-hydroxy-5-tetradecenoate (9, 19.22 %), 1 , 11 diphenyl undecane (47, 18.78 %) and cyclopentanyl hexadecane (39, 14.44 %) were the major constituents among the various hydrocarbons, fatty acids, esters, alcohols, aldehydes, isothiocyanate, aromatics, steroids, terphenyl and sulphur-containing compounds. Conclusion : The findings reveal the hypotensive potential of M. oleifera roots and the presence of specific hydrocarbons, fatty acid esters, thioureides, steroids and isothiocyanates in active fractions. Further study is required to determine the suitability of the plant as an antihypertensive remedy. Keywords : Moringa oleifera , Methyl hexadecanoate, Methyl 14-hydroxy-5-tetradecenoate, Petroleum ether, Stigmastan - 3, 5, diene, Cyclopentanyl hexadecane

Large-scale qualitative and quantitative characterization of components in Shenfu injection by integrating hydrophilic interaction chromatography, reversed phase liquid chromatography, and tandem mass spectrometry

It is of great importance to clarify in depth the chemical composition, including qualitative and quantitative aspects, of traditional Chinese medicine (TCM) injection that contains a great number of hydrophilic and hydrophobic ingredients to guarantee its safe medication in clinic. Column-switching hydrophilic interaction liquid chromatography-reversed phase liquid chromatography coupled with tandem mass spectrometry (HILIC-RPLC-MS/MS) has been revealed to be advantageous at simultaneous measurement of compounds covering a broad polarity range. Previous studies have profiled the hydrophobic components, mainly aconite alkaloids and ginsenosides, in Shenfu Injection (SFI); however, the hydrophilic substances haven’t been taken into account. In the present study, we aim to holistically characterize the hydrophilic constituents and to simultaneously quantitate both hydrophilic and hydrophobic components in SFI. A strategy integrating predefined multiple reaction monitoring, step-wise multiple ion monitoring, and enhanced product ion scans was proposed to universally screen the hydrophilic substances using a hybrid triple quadrupole-linear ion trap mass spectrometer. Structural identification was carried out by comparing with authentic compounds, analyzing MS2 spectra, and referring to accessible databases (e.g., MassBank, METLIN and HMDB). A total of 157 hydrophilic compounds were detected from SFI, and 154 ones were identified as amino acids, nucleosides, organic acid, carbohydrates, etc. A column-switching HILIC-RPLC-MS/MS system was developed and validated for simultaneously quantitative analysis of 40 primary hydrophilic and hydrophobic ingredients in SFI, including eleven amino acids, nine nucleosides, nine aconite alkaloids, and eleven ginsenosides. Taken together, the findings obtained could provide meaningful information for comprehensively understanding the chemical composition and offer a reliable approach for the quality control of SFI.

Novel Bioactivation Pathway of Benzbromarone Mediated by Cytochrome P450.

Benzbromarone (BBR) is a hepatotoxic drug, but the detailed mechanism of its toxicity remains unknown. We identified 2,6-dibromohydroquinone (DBH) and mono-debrominated catechol (2-ethyl-3-(3-bromo-4,5-dihydroxybenzoyl)benzofuran; CAT) as novel metabolites of BBR in rat and human liver microsomal systems by comparison with chemically synthesized authentic compounds, and we also elucidated that DBH is formed by cytochrome P450 2C9 and that CAT is formed mainly by CYP1A1, 2D6, 2E1, and 3A4. Furthermore, CAT, DBH, and the oxidized form of DBH are highly cytotoxic in HepG2 compared with BBR. Taken together, our data demonstrate that DBH, a novel reactive metabolite, may be relevant to BBR-induced hepatotoxicity.

Homolog-focused profiling of ginsenosides based on the integration of step-wise formate anion-to-deprotonated ion transition screening and scheduled multiple reaction monitoring

Homolog-focused profiling is a favored option to bridge targeted metabolomics toward non-targeted metabolomics. In current study, an attempt was made for the large-scale ginsenoside-specific analysis in ginseng (G) and American ginseng (AG). When formic acid (0.1%, v/v) was introduced as the mobile phase additive, formate anion-to-deprotonated ion transitions ([M+HCOO]−>[M−H]−) with an optimal collision energy (−32eV) could result in satisfactory responses for ginsenosides. Therefore, a step-wise multiple reaction monitoring (MRM)-based method employing [M+HCOO]−>[M−H]− ion pairs was constructed to screen ginsenosides among 501–1250u (for Q1) with a step-size of 2u, and MRM also served as a survey experiment to trigger enhanced product ion scans for MS2 spectrum acquisition on a hybrid triple quadrupole-linear ion trap mass spectrometer; then, the identification of those observed ginsenosides was achieved on the basis of the well-defined mass cracking patterns for ginsenosides; afterwards, scheduled MRM was introduced for large-scale relatively quantitative analysis of all detected ginsenosides. Finally, comparative metabolomics were performed to differentiate G, AG, and their processed products. Method validation was carried out using thirteen authentic compounds. A total of 221 ginsenosides, among which 185 ones were annotated, were observed and relatively quantitated. All crude materials were obviously classified into groups I–III. Above all, the MRM-based homolog-focused profiling of ginsenosides could be used as a reliable tool to gain an in-depth view for ginsenoside-enriched herbal products.

Chemical fingerprinting of Gardenia jasminoides Ellis by HPLC–DAD–ESI-MS combined with chemometrics methods

A fingerprint analysis method has been developed for characterization and discrimination of Gardenia jasminoides Ellis from different areas. The chemometrics methods including similarity evaluation, principal components analysis (PCA) and hierarchical clustering analysis (HCA) were introduced to identify more useful chemical markers for improving the quality control standard of dried ripe fruits of G. jasminoides Ellis. Then the selected chemical markers were analyzed by high performance liquid chromatography–diode array detection–electrospray ionization mass spectrometry (HPLC–DAD–ESI-MS) qualitatively and quantitatively. 23 characteristic peaks were assigned while 19 peaks of them were identified by comparing retention times, UV and MS spectra with authentic compounds or literature data. Moreover, 14 of them were determined quantitatively which could effectively evaluate the quality of G. jasminoides Ellis. This study was expected to provide comprehensive information for the quality evaluation of G. jasminoides Ellis, which would be a valuable reference for further study and development of this herb and related medicinal products.

Serotonin 5-O-β-Glucoside and Its N-Methylated Forms in Citrus Genus Plants

Citrus genus is characterized by a specific presence of indole metabolites deriving from the N-methylation of tryptamine and its hydroxylated form, 5-hydroxytryptamine (serotonin), which are likely involved in plant defense mechanisms. In this study, we identified for the first time the occurrence in Citrus plants of serotonin 5-O-β-glucoside and all its N-methylated derivatives, that is, N-methylserotonin 5-O-β-glucoside, N,N-dimethylserotonin (bufotenine) 5-O-β-glucoside, and N,N,N-trimethylserotonin (bufotenidine) 5-O-β-glucoside. The identification of the glucosylated compounds was based on mass spectrometric studies, hydrolysis by glucosidase, and in some cases, comparison to authentic compounds. Beside leaves, the distribution of the glucosylated forms and their aglycones in some Citrus species was evaluated in flavedo, albedo, juice, and seeds. The simultaneous presence of serotonin and its N-methylated derivatives, together with the corresponding glucosylated forms, is consistent with the occurrence of a metabolic pathway, specific for Citrus, aimed at potentiating the defensive response to biotic stress through the optimization of the production and use of the most toxic of such metabolites.

Characterization and quantitative analysis of phenylpropanoid amides in eggplant (Solanum melongena L.) by high performance liquid chromatography coupled with diode array detection and hybrid ion trap time-of-flight mass spectrometry.

Eggplant (Solanum melongena L.) is a famous edible and medicinal plant. Despite being widely cultivated and used, data on certain parts other than the fruit are limited. The present study focused on the qualitative and quantitative analysis of the chemical constituents, particularly phenylpropanoid amides (PAs), in eggplant. The mass fragmentation patterns of PAs were proposed using seven authentic compounds with the assistance of a hybrid ion trap time-of-flight mass spectrometer. Thirty-seven compounds (27 PAs and 10 others) were detected and plausibly assigned in the different parts of eggplant. Afterward, a reliable method based on liquid chromatography coupled with diode array detection was developed, validated, and applied for the simultaneous determination of seven PAs and three caffeoylquinic acids in 17 batches of eggplant roots with satisfactory accuracy, precision, and reproducibility, which could not only provide global chemical insight of eggplant but also offer a reliable tool for quality control.

Characterization of oxygenated metabolites of ginsenoside Rb1 in plasma and urine of rat.

Oxygenated metabolites have been suggested as the major circulating metabolites of ginsenosides. In the current study, 10 oxygenated metabolites of ginsenoside Rb1 in plasma and urine of rat following iv dose were characterized by comparison with chemically synthesized authentic compounds as quinquenoside L16 (M1 and M2), notoginsenoside A (M3), ginsenoside V (M4 and M7), epoxyginsenoside Rb1 (M5 and M9), notoginsenoside K (M6), and notoginsenoside C (M8 and M10), 9 of which were detected as in vivo metabolites for the first time. After oral administration of ginsenoside Rb1, M3, M4, and M7 were observed as major circulating metabolites and presented in the bloodstream of rat for 24 h. Characterization of the exact chemical structures of these circulating metabolites could contribute greatly to our understanding of chemical exposure of ginsenosides after consumption of ginseng products and provide valuable information for explaining multiple bioactivities of ginseng products.

Composition and Antibacterial Activity of the Essential Oil of Phlomidoschema parviflorum from Iran

The monotypic genus Phlomidoschema Vved. belongs to the family Lamiaceae, subfamily Lamioideae and tribe Stachydeae [1]. Phlomidoschema parviflorum (Benth.) Vved. grows in restricted parts of Iran (Khorasan Province) as well as Afghanistan, west of Pakistan, Pamir Mountains, and Punjab of India. Morphologically, the species is characterized by perennial, multi-stemmed and subshrub habit and white subfloccose and canescent hairs on the leaves and stem [2]. The species is considered vulnerable and rare according to IUCN categories [3]. The infusion of the aerial flowering part of the plant is used locally for the treatment of gastrointestinal disorders, colic and stomachache. From the phylogenetic point of view, the genus is closely allied with the genus Stachys. The most relative and closest species of the genus Stachys to the genus Phlomidoschema was found to be S. lavandulifolia Vahl. [1]. As far as our literature survey could ascertain, there is no report on the oil composition and biological activity of P. parviflorum. So, in continuation of our research on the composition and biological activity of Iranian aromatic and essential oil-bearing plants of the mint family [4–10], we have investigated the composition and antibacterial activity of the essential oil of P. parviflorum. The aerial full flowering parts of Phlomidoschema parviflorum were collected from Khorasan-e Razavi Province, Ghayen towards Gonabad, Zirkuh region with an altitude of 1300–1350 m. A voucher specimen was identified and deposited (MPH-1220) in the Medicinal Plants and Drugs Research Institute Herbarium (MPH), Shahid Beheshti University of Tehran, Iran. Air-dried aerial parts (200 g) were subjected to hydrodistillation for 3 h using a Clevenger-type apparatus. The oil was dried over anhydrous sodium sulfate and stored in sealed vials at 4C until analysis and tested. GC analysis was carried out on a Thermoquest-Finnigan Trace GC instrument equipped with a capillary DB-5 fused silica column (60 m 0.25 mm i.d., film thickness 0.25 m). The oven temperature was raised from 60C to 250C at a rate of 5C/min, then held at 250C for 10 min. Nitrogen was used as the carrier gas at a flow rate of 1.1 mL/min; the split ratio was adjusted to 1:50. The injector and detector (FID) temperatures were kept at 250C and 280C, respectively. GC-MS analysis was carried out on a Thermoquest-Finnigan Trace GC-MS instrument equipped with a DB-5 fused silica capillary column. Helium was used as the carrier gas at a flow rate of 1.1 mL/min with a split ratio of 1:50. The quadrupole mass spectrometer was scanned over 45–465 amu with an ionizing voltage of 70 eV. Gas chromatographic conditions were the same as given above for GC. The constituents of the oil were identified by calculation of their retention indices under temperature-programmed conditions for n-alkanes (C 6 –C 24 ), and the essential oil on a DB-5 column. Identification of individual compounds was made by comparison of their mass spectra with those of the internal reference mass spectra library or with authentic compounds and confirmed by comparison of their retention indices with those reported in the literature [11]. In vitro antibacterial activity of the essential oil was assessed against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 as models of Gram positive and Gram negative bacteria, respectively. The broth microdilution susceptibility method using 96 well trays was used to determine the minimum concentration of essential oil required for inhibition of visible growth (MIC) or killing (MBC) of the test strains. For this purpose, the standard protocol of CLSI (Clinical Laboratory and Standards Institute) was used with some modifications [12]. The inoculants of the bacterial strains were prepared from freshly cultured strains using sterile normal saline, which were adjusted to 0.5 McFarland standard turbidity and then further diluted (1:1000 for bacteria) by sterile Mueller-Hinton broth (MHB) just before adding to the wells containing a desired range of diluted essential oil.

Determination of three phenylphenols in grapefruit juice by HPLC after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole

o-Phenylphenol is generally utilized as a disinfectant for citrus fruits. The purpose of this study is to develop a high-performance liquid chromatography coupled with ultraviolet detection (380 nm) method for simultaneous determination of o-phenylphenol, and its analogues (m-phenylphenol and p-phenylphenol) in grapefruit juice after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). 2-Hydroxyfluorene was used as an internal standard (IS). Standard curves were obtained after derivatization with NBD-F in borate buffer (pH 8.0) at room temperature for 5 min. The three NBD-F derivatives were almost completely separated on a Cholester column (5 μm, 3.0 mm i.d. × 150 mm). Calibration plots were linear in the range of absolute amount of 1.04 ∼ 2.08 to 41.6 ng/50 μL injection volume, with r2 values ≥0.9981, for the three compounds. The lower limits of detection were 0.3 to 0.7 ng/50 μL injection volume (signal-to-noise ratio of 3 : 1). The coefficients of variation were less than 11.1%. After extraction of grapefruit juice (2.0 mL) with n-pentane, the level of o-phenylphenol in the juice was estimated to be 20.2 ± 2.0 ng/mL (n = 6, mean ± SD), while m-phenylphenol and p-phenylphenol were below the lower limits of quantification. The recovery values of the three phenylphenols from samples spiked with a standard mixture of authentic compounds and IS were satisfactory (99.1 to 118.7%).

Studies on residue-free decontaminants for chemical warfare agents.

Residue-free decontaminants based on hydrogen peroxide, which decomposes to water and oxygen in the environment, are examined as decontaminants for chemical warfare agents (CWA). For the apparent special case of CWA on concrete, H2O2 alone, without any additives, effectively decontaminates S-2-(diisopropylamino)ethyl O-ethyl methylphosphonothioate (VX), pinacolyl methylphosphorofluoridate (GD), and bis(2-choroethyl) sulfide (HD) in a process thought to involve H2O2 activation by surface-bound carbonates/bicarbonates (known H2O2 activators for CWA decontamination). A plethora of products are formed during the H2O2 decontamination of HD on concrete, and these are characterized by comparison to synthesized authentic compounds. As a potential residue-free decontaminant for surfaces other than concrete (or those lacking adsorbed carbonate/bicarbonate) H2O2 activation for CWA decontamination is feasible using residue-free NH3 and CO2 as demonstrated by reaction studies for VX, GD, and HD in homogeneous solution. Although H2O2/NH3/CO2 ("HPAC") decontaminants are active for CWA decontamination in solution, they require testing on actual surfaces of interest to assess their true efficacy for surface decontamination.

Liquid chromatography high-resolution mass spectrometry for fatty acid profiling.

Quantification of fatty acids has been crucial to elucidate lipid biosynthesis pathways in plants. To date, fatty acid identification and quantification has relied mainly on gas chromatography (GC) coupled to flame ionization detection (FID) or mass spectrometry (MS), which requires the derivatization of samples and the use of chemical standards for annotation. Here we present an alternative method based on a simple procedure for the hydrolysis of lipids, so that fatty acids can be quantified by liquid chromatography mass spectrometry (LC-MS) analysis. Proper peak annotation of the fatty acids in the LC-MS-based methods has been achieved by LC-MS measurements of authentic standard compounds and elemental formula annotation supported by (13)C isotope-labeled Arabidopsis. As a proof of concept, we have compared the analysis by LC-MS and GC-FID of two previously characterized Arabidopsis thaliana knock-out mutants for FAD6 and FAD7 desaturase genes. These results are discussed in light of lipidomic profiles obtained from the same samples. In addition, we performed untargeted LC-MS analysis to determine the fatty acid content of two diatom species. Our results indicate that both LC-MS and GC-FID analyses are comparable, but that because of higher sensitivity and selectivity the LC-MS-based method allows for a broader coverage and determination of novel fatty acids.

Isolation and Identification of Potential Antineoplastic Bioactive Phenolic Compounds in Malaysian Honeys

The role of honeys as chemopreventive agents was inferred from their phenolic composition. The present study aims to isolate and identify such phenolic compounds with anticarcinogenic potential from the sugar matrix of Malaysian honeys. Phenolic compounds in Malaysian Acacia, Gelam and Tualang honey samples were isolated using octadecyl (C18) silica solid phase extraction (SPE) technique and identification was performed using high-performance liquid chromatography (HPLC) with diode array detector (DAD). Identification of phenolic compounds was achieved by comparing chromatographic retention times of honey samples with those of authentic standard compounds. HPLC analysis confirmed the presence of anticancer phenolic compounds in all honey samples with considerable variation observed among both different and the same types of honey. Six flavonoids (quercetin, naringenin, kaempferol, rutin, hesperetin, and apigenin), two phenolic acids (p-coumaric acid, and ferulic acid) and two tannins (ellagic acid, and penta-O-galloyl-s-D-glucose [PGG]) were the bioactive anticancer compounds identified. The presence of PGG in Malaysian honey was described for the first time. This study concludes that these three types of Malaysian honey possessed anticancer properties at varying degrees. Their potential usage as natural anticancer therapeutic agents with numerous health benefits could be further explored and considered as an alternative for current anticancer drugs

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency: urinary organic acid profiles and expanded spectrum of mutations.

Mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase (HMCS2) deficiency results in episodes of hypoglycemia and increases in fatty acid metabolites. Metabolite abnormalities described to date in HMCS2 deficiency are nonspecific and overlap with other inborn errors of metabolism, making the biochemical diagnosis of HMCS2 deficiency difficult. Urinary organic acid profiles from periods of metabolic decompensation were studied in detail in HMCS2-deficient patients from four families. An additional six unrelated patients were identified from clinical presentation and/or qualitative identification of abnormal organic acids. The diagnosis was confirmed by sequencing and deletion/duplication analysis of the HMGCS2 gene. Seven related novel organic acids were identified in urine profiles. Five of them (3,5-dihydroxyhexanoic 1,5 lactone; trans-5-hydroxyhex-2-enoate; 4-hydroxy-6-methyl-2-pyrone; 5-hydroxy-3-ketohexanoate; 3,5-dihydroxyhexanoate) were identified by comparison with synthesized or commercial authentic compounds. We provisionally identified trans-3-hydroxyhex-4-enoate and 3-hydroxy-5-ketohexanoate by their mass spectral characteristics. These metabolites were found in samples taken during periods of decompensation and normalized when patients recovered. When cutoffs of adipic >200 and 4-hydroxy-6-methyl-2-pyrone >20 μmol/mmol creatinine were applied, all eight samples taken from five HMCS2-deficient patients during episodes of decompensation were flagged with a positive predictive value of 80% (95% confidence interval 35-100%). Some ketotic patients had increased 4-hydroxy-6-methyl-2-pyrone. Molecular studies identified a total of 12 novel mutations, including a large deletion of HMGCS2 exon 1 in two families, highlighting the need to perform quantitative gene analyses. There are now 26 known HMGCS2 mutations, which are reviewed in the text. 4-Hydroxy-6-methyl-2-pyrone and related metabolites are markers for HMCS2 deficiency. Detection of these metabolites will streamline the biochemical diagnosis of this disorder.