Trimethylsilyl Ether Derivatives Research Articles

Sterol Fractionation of Refined Olive Oil for Sale in Mediterranean Region with GC-FD, Composition Analysis of Triterpenic Dialkols and Validation

I
 n this study, compositional analysis of the sterol fraction and triterpenic alcohols of olive oil was made assess the degree
 of purity of the oil and the absence of admixture with other plant oils. This determination also permited characterization
 of the type of olive oil. In the present work, 30 samples of olive oil supplied from the markets of Adana, Osmaniye and
 Mersin in 2015 and 2016 were analyzed, and the validation studies were carried out in the analysis method used. The sterol
 fractions were separated from the unsaponifiable fraction by silica gel plate chromatography, and later they were analyzed
 as the trimethylsilyl ether derivatives by capillary column gas chromatography. The validation results obtained were evaluated in accordance with TGK-Olive Oil and Pirina Oil Communiqué (Communiqué No: 2010-35)

GC–MS analysis of D-pinitol in carob: Syrup and fruit (flesh and seed)

D-pinitol (3-O-methyl-D-chiro-inositol) is a well-known bioactive compound with anti-diabetic and anti-oxidant biological functions. A gas chromatography–mass spectrometry (GC–MS) method was developed for its quantitation in carob syrup, flesh and seed samples originated from Cyprus. The analysis was performed after derivatization of carbohydrates and polyols into trimethylsilyl ether derivatives. D-pinitol was determined in 13 carob syrup samples, in concentrations ranging 65.71 ± 4.60 – 77.72 ± 5.44 mg/g (mean: 68.58 ± 4.80 mg/g, n = 13). In two commercial samples, it was determined in relative medium-low concentrations (21.96 ± 1.54 and 44.71 ± 3.13 mg/g), revealing possible adulteration; however, this needs further investigation. Similarly, it was determined in high concentrations in carob flesh samples, in concentrations ranging 53.20 ± 3.72 – 54.58 ± 3.82 mg/g (mean: 53.81 ± 3.76 mg/g, n = 3). On the other hand, seed samples proved very poor in D-pinitol (<LOD). Therefore, bioprospecting of carob fruit and syrup is highlighted. Compared to other plants or legumes, carob appears to be the richest source of D-pinitol, highlighting carobs role as a functional organic food. The historical and cultural association of Cyprus with carobs is linked with traditional foods and habits.

Sterols and Stanols in Foods and Dietary Supplements Containing Added Phytosterols: A Collaborative Study

AbstractAn international, multilaboratory collaborative study was carried out to evaluate the performance of Official Method Ce 12‐16 of the American Oil Chemists’ Society (AOCS) for the determination of plant sterols and stanols, collectively referred to as phytosterols, in foods and dietary supplements containing added phytosterols and in the phytosterol food additive concentrates used to prepare such products. AOCS Official Method Ce 12‐16 involves the extraction of free sterols/stanols and saponified steryl/stanol esters followed by the gas chromatographic separation and flame ionization detection of phytosterol trimethylsilyl ether derivatives. A total of 14 laboratories from six countries successfully completed the analysis of collaborative samples of foods (e.g., baked goods, beverages, margarines; n = 9), dietary supplements (n = 5), and phytosterol concentrates (n = 4). Study results for the contents of total phytosterols (weight/weight) were 0.19–8.4% for foods, 8.7–49% for dietary supplements, and 57–97% for concentrates. AOCS Official Method Ce 12‐16 showed acceptable performance for total and individual phytosterols, indicating that this method was suitable for the determination of added phytosterols in a wide variety of market products and concentrates. AOCS Official Method Ce 12‐16 is appropriate for the determination of the five major phytosterols (i.e., campesterol, stigmasterol, β‐sitosterol, campestanol, and sitostanol) that are the subject of the United States Food and Drug Administration's health claim for phytosterols and the reduced risk of coronary heart disease.

Determination of Fecal Sterols Following a Diet with and without Plant Sterols.

The aim of this study was to develop a method for neutral fecal sterols determination in subjects receiving a normal diet with or without a plant sterols-enriched beverage using gas chromatography-mass spectrometry (GC/MS). Sample preparation conditions (homogenization of lyophilized feces with water) were evaluated. Sterol determination required direct hot saponification, unsaponifiable extraction with hexane, and the formation of trimethylsilyl (TMS) ether derivatives. The method allows the quantification of cholesterol, plant sterols and their metabolites (coprostanol, coprostanone, cholestanol, cholestanone, methylcoprostanol, methylcoprostanone, ethylcoprostenol, stigmastenol, ethylcoprostanol and ethylcoprostanone). Good linearity was obtained (r>0.96) and interference was only observed for coprostanone, where the standard addition method proved necessary for quantification. The limits of detection (LOD) ranged from 0.10 to 3.88µg/g dry feces and the limits of quantitation (LOQ) from 0.34 to 12.94µg/g dry feces. Intra- and inter-assay precision (RSD %) were 0.9-9.2 and 2.1-11.3, respectively. Accuracy, expressed as percentage recovery (80-119%) was obtained for all determined sterols.

Optimization of a multiresidue and multiclass analysis method for anabolic agents and β2-agonists in bovine urine by GC-MS/MS

A simple multiresidue and multiclass procedure is described for the simultaneous determination of anabolic agents and β2-agonists from 4 classes of prohibited substances of illegal use in food-producing animals, according to Council Directive 96/23/EC. The method involves analysis of hydrolysed urinary anabolic compounds using liquid–liquid extraction, with subsequent conversion to trimethylsilylether derivatives that are analysed by GC-MS/MS.The oven was set to an initial temperature of 140°C, was increased at a rate of 10°C/min to 300°C, and was held 3 min using an Ultra-1 capillary column. Optimization tests demonstrated good peak resolution and shape for all compounds. A pre-validation study was conducted to obtain an analytical test curve, which showed adequate results for all analytes. Cochran's test (C calculated <0.6838, 95% confidence interval) proved the homoscedasticity of all the compounds. The results for the apparent recovery of each analyte, calculated for the five fortification levels of the analytical curve, varied between 94.7% and 109.3%. The limit of quantitation was 0.5 times the minimum required performance limit (MRPL), and the limit of detection was 0.33 times the MRPL.

GC-ECNICI-MS/MS of eicosanoids as pentafluorobenzyl-trimethylsilyl (TMS) derivatives: Evidence of CAD-induced intramolecular TMS ether-to-ester rearrangement using carboxy-18O-labelled eicosanoids and possible implications in quantitative analysis.

GC-MS and GC-MS/MS of pentafluorobenzyl (PFB) ester trimethylsilyl (TMS) ether (PFB-TMS) derivatives of hydroxylated long-chain fatty acids including arachidonic acid metabolites, the eicosanoids, in the electron-capture negative-ion chemical ionization (ECNICI) mode are the most sensitive and accurate approaches to quantify carboxyl groups-containing compounds in complex biological fluids such as plasma and urine. Under ECNICI conditions, PFB-TMS derivatives of eicosanoids ionize to form very few ions, with the carboxylates [M-PFB]- being typically the most intense. Less intense ions may be additionally formed by consecutive neutral loss (NL) of trimethylsilanol (TMSOH, 90Da) groups ([M-PFB-(TMSOH)n]-). By using [1,1-18O2]- and [1,ω-18O2]-eicosanoids, we studied ion processes following collisionally activated dissociation (CAD) of the precursor ions [M-PFB]-. We found that CAD resulted in formation of product ions due to NL of a TMS18OH (92Da) group in monocarboxylic and of a PFB18OH (200Da) group in dicarboxylic eicosanoids. TMS18OH NL implies an intra-molecular transfer of the TMS group from hydroxyl groups to their carboxylate anions [M-PFB]-. From a mechanistic point of view, this rearrangement may explain formation of unique product ions in GC-MS/MS of eicosanoids under ECNICI conditions. From the quantitative point of view, quantification by GC-MS/MS of product ions due to [M-PFB-(TMSOH)n]- and [M-PFB-TMS18OH-(TMSOH)n-1]-would reveal incorrect data, if [1,1-18O2]-eicosanoids are used as internal standards and if no correction for the 18O-loss is performed. In 18O-labelled dicarboxylic eicosanoids, such as the major urinary metabolite (MUM) of E prostaglandins, i.e., [1,ω-18O2]-PGE-MUM), no TMS ester/TMS ether rearrangement was observed. Yet, 18O-loss occurred upon CAD of [M-PFB]- due to NL of PFB18OH (200Da). In both cases the extent of 18O-loss needs to be determined and considered for accurate quantification of monocarboxylic acids such as 8-isoprostaglandin F2α (8-iso-PGF2α) and dicarboxylic eicosanoids such as PGE-MUM.

Development of a Method for the Analysis of Sterols in Sterol-Enriched Deli-Style Turkey with GC-FID

Plant sterols have been recognised by the European Food Safety Authority for their cholesterol-lowering properties and are currently added to several food formulations. The objective of this study was to develop a method based on formation of sterol trimethylsilyl ether derivatives and separation and quantification by GC for the determination of three phytosterols (β-sitosterol, stigmasterol and campesterol) in sterol-enriched deli-style turkey. The assay was linear (concentration range 4.3–172.1 μg/ml, R 2 ≥ 0.9868) and accuracy and precision were within the acceptance criteria of the U.S. Food and Drug Administration (USFDA) guidelines for method validation, set at <20 % RSD at the lower limit of quantification (LLOQ) and <15 % RSD for all other standards. Accuracy measured by relative response factor (RRF) also met the USFDA validation criteria. No matrix effects were observed. The response factors (RFs) of the three sterols differed significantly to that of the internal standard (ISTD) used, leading to RRF dissimilar to 1 (campesterol = 1.0167, stigmasterol = 1.4458, β-sitosterol = 0.9029). This method is suitable for quantification in meat matrix, and it has been successfully applied to the determination of sterols in sterol-enriched deli-style turkey (21 mg sterols/0.5 g sample).

Gas chromatography mass spectrometry based profiling of alkyl coumarates and ferulates in two species of cattail (Typha domingensis P., and Typha latifolia L.)

Several long-chain n-alkyl coumarates and ferulates were identified in cattails (Typha domingensis and Typha latifolia) from the Florida Everglades. Characterization of these compounds was achieved based on the interpretation of mass spectra obtained by GCMS as their trimethylsilyl ether derivatives, comparison with published mass spectra and available standards. Both n-alkyl p-coumarates and n-alkyl ferulates were identified in roots and leaves of both Typha species, featuring unique distribution patterns and differences between leaf and root biomass. For both Typha species, roots have higher concentrations and a much greater diversity of n-alkyl p-coumarates and ferulates but with different side chain carbon numbers ranging from C14 to C28. Typha domingensis leaves only contained n-alkyl ferulates with traces of n-alkyl p-coumarates, while both types of compounds were present in Typha latifolia leaf material. These chemicals were not found in the other dominant wetland vegetation, which suggests their potential for application as phytochemical tracers of fresh cattail-derived organic matter in the Everglades ecosystem.

Identification, quantification and distribution of substituted phenols in the dissolved and suspended phases of water samples by gas chromatography tandem mass spectrometry: Derivatization, mass fragmentation and acquisition studies

In this paper, due to our new working strategy approach, the identification and quantification of 26 substituted phenols (SPs), including chlorophenols (CPs), methyl and nitro phenols (MPs, NPs, respectively), the technical mixture of 4-nonylphenol, triclosan and bisphenol-A were described. Selected species were determined both in the dissolved and suspended phases of samples as their trimethylsilyl-ether derivatives, developing a step by step, cautiously tuned, gas chromatographic tandem mass spectrometric (GC–MS/MS) method applying multiple reaction monitoring (MRM) acquisition. This procedure proved to be suitable for the quantitation of candidate species in the range of 0.29ng/L (4-chloro-3,5-dimethylphenol) to 14ng/L (2-methylphenol) levels. As a novelty to the field, the characteristic presence of SPs in tap waters as well as their distribution between the dissolved and suspended phases (henceforth: dissolved substituted phenols (DSPs) and suspended substituted phenols (SSPs) of Danube River and wastewater samples were confirmed.It means that SSPs - obtained by filtering water samples on glass fiber papers, prior to their solid phase extraction (SPE), these insoluble, however integral and unfortunately in the overwhelming cases of analyses discarded part of water pollutants - were identified and quantified, simultaneously. For this purpose our new, time, labor, cost effective and quantitative, ultrasound assisted extraction process for the analysis of SSPs was introduced and validated.Reproducibility, reliability and practical applicability of our working strategy were proved by the proportionality of the DSP and SSP contents, determined simultaneously from different sample volumes, characterized with the relative standard deviation percentages (RSD%) of analyses. The analytical performance characterization of our protocol was completed also by the classical recovery process. We added to 1L Danube River waters 150 and 400ng/L, while to 100mL effluent wastewater samples 500 and 1000ng/L SP standards; in all cases performing three parallel spiking tests of each average recoveries proved to be 86% (Danube River) and 84% (effluent wastewater), respectively.Partition of SPs between the dissolved and suspended phases of Danube River and influent and effluent wastewaters, meaning, the total amount of SPs (DSPs+SSPs) that the ecosystem is faced of, was defined for the first time. Distribution of found SPs revealed that their considerable part, in thorough correlation with their solubilities, is present in suspended phases: in average, 82% in wastewater and 57% in Danube River samples.

Elucidation of electron ionization mass spectrometric fragmentation pathways of trimethylsilyl ether derivatives of vicinal diols deriving from haplamine by collision‐induced dissociation gas chromatography/tandem mass spectrometry and 18O labelling

Formation of vicinal diols was observed after in vitro and in vivo studies of the natural product haplamine (9-methoxy-2,2-dimethyl-2,6-dihydropyrano[3,2-c]quinolin-5-one). These compounds, identified as trans- and cis-3,4-dihydroxy-9-methoxy-2,2-dimethyl-2,3,4,6-tetrahydropyrano[3,2-c]quinolin-5-ones and trans- and cis-3,4,9-trihydroxy-2,2-dimethyl-2,3,4,6-tetrahydropyrano[3,2-c]quinolin-5-ones, have a potential interest in oncology. It is therefore essential to elucidate their electron ionization mass spectrometric (EIMS) fragmentation pathways. EIMS fragmentation pathways of trimethylsilyl (TMS) derivatives of 3,4-dihydroxy- and 3,4,9-trihydroxyhaplamines were investigated. These pathways have been substantiated by: (i) comparison with EI mass spectra of structural homologues (silylated diols obtained from various chromenes and 1,2-dihydronaphthalene), (ii) low-energy collision-induced dissociation (CID) gas chromatography/tandem mass spectrometry (GC/MS/MS) and (iii) (18)O labelling. CID-MS/MS analyses and (18)O labelling demonstrated that EI mass spectral fragmentation of these TMS derivatives involves a transannular cleavage of the pyran ring with formation of a characteristic intense cyclic ion. The study of the mass spectra of TMS derivatives of different chromenes and 1,2-dihydroxynaphthalene allowed to confirm the proposed fragmentation pathways and to show that they act only when the pyran ring is present. Elimination of the neutral element [(CH3)2=C(H)OSi(CH3)3] and formation of cyclic ions play a key role during EI mass spectral fragmentation of the TMS derivatives of 3,4-dihydroxy- and 3,4,9-trihydroxyhaplamines. These fragmentation pathways could be generalized to TMS derivatives of cyclic compounds possessing vicinal diols close to a pyran ring.

Determination of sterol and fatty alcohols in unsaponifiable matter of Roystonea regia fruits oil

Roystonea regia (Kunth) F. Cook fruit is a good source of oil with possible application in the nutritional and pharmaceutical industries. The chemical constituent of the unsaponifiable matter of this oil, however, has not been studied yet. The aim of this work was to assess the content of sterols and alcohols in this fraction using GC-MS. Samples were submitted to basic hydrolysis with KOH/EtOH solution and then the unsaponifiable fractions extracted with n-hexane, and then alcohols and sterols were analyzed as trimethylsilylether derivatives using cholestane as the internal standard. The average content of the unsaponifiable matter of R. regia was 1.3%. The major fraction (66%), was composed of sterols, like s-sitosterol, stigmasterol, campesterol, 24-methylencycloartanol, Δ5-avenasterol, cycloartanol and cycloartenol, while the fatty alcohol fraction (33.9%) contained C18OH-C34OH alcohols.

Systematic derivatization, mass fragmentation and acquisition studies in the analysis of chlorophenols, as their silyl derivatives by gas chromatography–mass spectrometry

An exhaustive GC–MS sample preparation, derivatization, mass fragmentation and acquisition study was performed, for the simultaneous analysis of chlorophenols (CPs). Selected species were 2-CP, 3-CP, 4-CP, 3,5-dichlorophenol (diCP), 2,5-diCP, 2,6-diCP, 2,4-diCP, 2,3-diCP, 3,4-diCP 2,4,6-trichlorophenol (triCP), 2,4,5-triCP, 2,3,4-triCP, 2,3,4,6-tetrachlorophenol (tetraCP) and pentachlorophenol (pentaCP), in total 14 compounds. As novelties to the field, basic researches, like systematic derivatization, mass fragmentation and acquisition methods have been optimized for the trimethylsilyl (TMS) ether derivatives of CPs. The reactivity of chlorophenols with silylating agents has not been systematically analyzed. Here, we studied the reactivity of 14 chlorophenols with five silylating reagents. The three acquisition techniques, the full scan (FS), the multiple ion monitoring (MIM), and the currently optimized multiple reaction monitoring (MRM) methods, have been compared. We developed a new analytical approach, simultaneously monitoring the fragmentation pattern of the 35Cl and the 37Cl containing fragment ions both as precursor and as product ions. This principle resulted in remarkable specificity and sensitivity of detection and quantification; particularly in the cases of the tetraCP and pentaCP derivatives containing the 35Cl and the 37Cl fragment ions at an approximate ratio of <1:1. Detailed documentation of the loss of HCl via fragmentation processes, without decomposition of the benzene ring, was attributed to the “ring-walk” mechanism described first for monochlorophenol. Critical evaluation of the derivatization and acquisition protocols was collated and validated with the same characteristics. Data of six point calibration along with the corresponding relative standard deviation percentage (RSD%) values, in the line of FS, MIM and MRM methods (r2: 0.9987, 0.9992, 0.9989; RSD%: 8.7, 5.6, 8.1), proved to be independent on the acquisition processes. The practical utility of the optimized MRM acquisition techniques was confirmed by the quantitation of the CP contents of Danube River, tap water and distilled water samples. Results confirmed at the first time the primary importance of the MRM acquisition method, even in comparison to the MIM one: we revealed that distilled water contains higher chlorophenol content than tap water, which might have a great significance for the water industry.

Cyclic glycolipids from glandular trichome exudates of Cerastium glomeratum

Fourteen cyclic glycolipids, named glomerasides A-N, have been isolated from the glandular trichome exudate of Cerastium glomeratum (Caryophyllaceae). Their structures were determined by spectroscopic analysis of the glycolipids, as well as by application of the Ohrui-Akasaka method to the fatty acid methyl esters derived from the glycolipids and GCMS studies of trimethylsilyl ether derivatives of the methyl esters. The various glomerasides have a glycosidic linkage between the anomeric hydroxy group of the glucose and the C-11, C-10 or C-9 positions of the docosanoyl moiety. They also contained an ester linkage between the C-6 hydroxy group of the glucose ring and the carboxyl group of the oxygenated fatty acid to form their macrocyclic structures. The glucose moiety was optionally acetylated and/or malonylated at the C-2 or C-3 hydroxy groups. Among these compounds, the 1,6'-cyclic ester of 11(R)-(2-O-acetyl-β-d-glucopyranosyloxy)docosanoic acid (glomeraside D) was the most abundant (25%).

Derivatization and fragmentation pattern analysis of natural and synthetic steroids, as their trimethylsilyl (oxime) ether derivatives by gas chromatography mass spectrometry: Analysis of dissolved steroids in wastewater samples

This paper reports the extension of our multiresidue analysis (MA) procedure with 18 natural and synthetic steroids; permitting the identification and quantification, in total of 81 pollutants from one solution, by a single injection, as their trimethylsilyl (TMS)-oxime ether/ester derivatives, by gas chromatography–mass spectrometry (GC–MS), within 31 min. As a novelty to the field, basic researches, such as fragmentation pattern analysis and derivatization optimization studies were performed for androsterone, transdehydroandrosterone, transandrosterone, mestranol, dihydrotestosterone, ethinylestradiol, testosterone, norethisterone, estriol, 4-androstene-3,17-dione, gestodene, levonorgestrel, etonogestrel, coprostanol, progesterone, cholesterol, medroxy-progesterone-acetate, stigmasterol and β-sitosterol. Results confirmed that (i) the TMS oxime-ether derivatives of the keto steroids provide from 1.40 times (gestodene) up to 4.25 times (norethisterone) higher responses compared to their TMS-ether ones, and (ii) the distribution of syn/anti oximes is characteristic to the ketosteroid species examined. Based on our optimized mass fragmentation, solid phase extraction (SPE) and derivatization studies separations have been performed in the total ion current (TIC) mode, identification and quantification of compounds have been carried out on the basis of their selective fragment ions. Responses, obtained with derivatized standards proved to be linear (hydroxysteroids), or have been calculated from calibration curves (ketosteroids) in the range of 1.88–750 ng/L levels. Limit of quantitation (LOQ) values varied between 1.88 ng/L and 37.5 ng/L concentrations. The most important practical messages of this work are the high androsterone (0.744–4.28 μg/L), transandrosterone (0.138–4.00 μg/L), coprostanol (2.11–302 μg/L), cholesterol (0.308–41 μg/L), stigmasterol (1.21–8.40 μg/L) and β-sitosterol (1.12–11.0 μg/L) contents of influent wastewaters. β-Estradiol (100 ng/L) and estriol (54 ng/L) were found in one influent sample, only. Reproducibilities, characterized with the relative standard deviation percentages (RSD%) of measurements, varied between 1.73 RSD% ( β-estradiol) and 5.4 RSD% (stigmasterol), with an average of 4.82 RSD%.

Determination by GC-MS of Unsaponifiable Compounds: Minor Constituents of D-004, A Lipid Extract from Roystonea regia

D-004, a new lipid extract containing more than 85 % of free fatty acids (C6-C18) obtained from fruit of the Cuban royal palm (Roystonea regia (Kunth) F. Cook) has been shown to be effective in experimental models of prostatic hyperplasia. Taking into account the lipid nature of this extract, the aim of this work was to study the composition of its unsaponifiable fraction using GC-MS. Samples of nine batches of D004 were submitted to the procedure developed by the Institute for Nutraceutical Advancement of USA for determining sterols, which shortly consists on saponification with KOH/Et0H solution and extraction of the unsaponifiable fractions with n-hexane. Quantification of alcohols and sterols, analyzed as trimethylsilylether derivatives, was carried out by the internal standard method. D004’s unsaponifiable fraction content ranged from 0.66 to 7.8 %. This fraction was mainly composed of fatty alcohols: C22-C32 (2.62–58.6 %); sterols: β-sitosterol, campesterol, stigmasterol, Δ5-avenasterol, cycloartanol, cycloartenol, 24-methylencycloartanol (0.65- 17.7 %); triterpenes (0.61–17.6 %) with ursane and oleanane structures; aliphatic hydrocarbons C14-C32 (0.08-2.21%), olefins C16:1-C28:1 and squalene (0.06–1.8%).

Simultaneous Determination of Free and Esterified Fatty Alcohols, Phytosterols and Solanesol in Tobacco Leaves by GC

A convenient gas chromatographic method equipped with flame ionization detection has been achieved for simultaneously determining six free and esterified fatty alcohols, three phytosterols, and solanesol in tobacco leaves. This analytic method consisted of direct alkaline hydrolysis, ultrasonic-assisted extraction and derivatization to trimethylsilyl ether derivatives. These three procedures did not require complicated and laborious preliminary purification steps of samples prior to GC, and the method was simple and convenient. The recovery of all target compounds was in the range of 85–105%, the relative standard deviations (RSDs) were less than 4%, and limits of detection (LOD) varied from 0.1–0.4 μg mL−1. The optimized methodology was successfully applied to determine free and esterified fatty alcohols, phytosterols and solanesol contents of different type tobacco samples.

Biochemical Characterization of the Oxygenation of Unsaturated Fatty Acids by the Dioxygenase and Hydroperoxide Isomerase of Pseudomonas aeruginosa 42A2

We have studied oxygenation of fatty acids by cell extract of Pseudomonas aeruginosa 42A2. Oleic acid ((9Z)-18:1) was transformed to (10S)-hydroperoxy-(8E)-octadecenoic acid ((10S)-HPOME) and to (7S,10S)-dihydroxy-(8E)-octadecenoic acid (7,10-DiHOME). Experiments under oxygen-18 showed that 7,10-DiHOME contained oxygen from air and was formed sequentially from (10S)-HPOME by isomerization. (10R)-HPOME was not isomerized. The (10S)-dioxygenase and hydroperoxide isomerase activities co-eluted on ion exchange chromatography and on gel filtration with an apparent molecular size of approximately 50 kDa. 16:1n-7, 18:2n-6, and 20:1n-11 were also oxygenated to 7,10-dihydroxy fatty acids, and (8Z)-18:1 was oxygenated to 6,9-dihydroxy-(7E)-octadecenoic acid. A series of fatty acids with the double bond positioned closer to ((6Z)-18:1, (5Z,9Z)-18:2) or more distant from the carboxyl group ((11Z)-, (13Z)-, and (15Z)-18:1) were poor substrates. The oxygenation mechanism was studied with [7S-(2)H]18:1n-9, [7R-(2)H]18:2n-6, and [8R-(2)H]18:2n-6 as substrates. The pro-R hydrogen at C-8 was lost in the biosynthesis of (10S)-HPODE, whereas the pro-S hydrogen was lost and the pro-R hydrogen was retained at C-7 during biosynthesis of the 7,10-dihydroxy metabolites. Analysis of the fatty acid composition of P. aeruginosa revealed relatively large amounts of (9E/Z)-16:1 and (11E/Z)-18:1 and only traces of 18:1n-9. We found that (11Z)-18:1 (vaccenic acid) was transformed to (11S,14S)-dihydroxy-(12E)-octadecenoic acid and to a mixture of 11- and 12-HPOME, possibly due to reverse orientation of (11Z)-18:1 at the active site compared with oleic acid. The reaction mechanism of the hydroperoxide isomerase suggests catalytic similarities to cytochrome P450.

Surface Water Concentrations and Loading Budgets of Pharmaceuticals and Other Domestic-Use Chemicals in an Urban Watershed (Washington, DC, USA)

Pharmaceuticals and domestic-use chemicals (PDCs) are classes of emerging chemical contaminants thought to enter the aquatic environment primarily through wastewater treatment plant (WTP) discharges. The intent of this study was to quantify loadings of PDCs in an urban watershed. The watershed has two major branches but with wastewater discharge occurring in only one of the two major branches. Surface water from the Anacostia River (Washington, DC) was collected in base-flow and storm-flow regimes. Surface water was filtered to separate water and particles, and the PDCs were extracted from water with Oasis HLB solid-phase extraction cartridges and extracted from sediments using microwave-assisted extraction. The PDCs in the extracts were quantified using gas chromatography-mass spectrometry in the form of the trimethylsilyl ether derivatives. The most frequently detected PDC with the highest concentration was bisphenol-A in both branches of the Anacostia River watershed and the least frequently detected PDC was diclofenac. The overall median concentrations for all measured PDCs in surface water ranged from nondetectable to 54.9 ng/l. Alternatively, in the collected WTPs samples, naproxen was observed, with the highest concentration and the median concentrations in WTP effluent ranging from nondetectable to 276 ng/l. Estimates of PDC loadings for February 2006 from WTP effluent showed that <2% of the downstream load in the NE Branch was derived from WTP discharge. PDC sources other than WTP effluent appear to influence surface water concentrations in the urban Anacostia River watershed.

Gas Chromatography/Mass Spectrometry of the Lignans in Resin of Callitris preissii

The total extract of resin from Callitris preissii was analyzed by gas chromatography/mass spectrometry and 34 lignans, underivatized and as trimethylsilyl ether derivatives, were identified by interpretation of the mass spectra. The lignans in the resin include 13 new lignans, i.e., four secoisolariciresinol derivatives, four lariciresinol derivatives, 3′,4′-methylenedioxy-7′-hydroxylariciresinol, 3,4-methylenedioxypinoresinol isomer, and two matairesinol derivatives. Furthermore, 17 lignans of the resin are characterized by methylenedioxy substitution on either or both guaiacylpropyl units or the syringylpropyl unit: i.e., five secoisolariciresinol derivatives, three lariciresinol derivatives, 3′,4′-methylenedioxy-7′-hydroxylariciresinol, three pinoresinol derivatives, and five matairesinol derivatives, have methylenedioxy structures. Eleven lignans, including two secoisolariciresinol derivatives, four lariciresinol derivatives, two pinoresinol derivatives, and three matairesinol derivatives, contain a syringyl moiety.

Convergence of the 5-LOX and COX-2 pathways: heme-catalyzed cleavage of the 5S-HETE-derived di-endoperoxide into aldehyde fragments

Oxygenation of the 5-lipoxygenase product 5S-hydroxyeicosatetraenoic acid by cyclooxygenase-2 yields a bicyclic di-endoperoxide. The di-endoperoxide contains two peroxides spanning from carbons 9 to 11 and 8 to 12, and two hydroxyls at carbons 5 and 15 of arachidonic acid (Schneider C., et al. 2006. Convergent oxygenation of arachidonic acid by 5-lipoxygenase and cyclooxygenase-2. J. Am. Chem. Soc. 128: 720). Here, we report that treatment of the di-endoperoxide with hematin or ferrous chloride results in cleavage of both peroxide O-O bonds and of the bonds between the carbons that carry the peroxide groups, producing the aldehydes 4-hydroxy-2E-nonenal (4-HNE), 8-oxo-5S-hydroxy-6E-octenoic acid, and malondialdehyde (MDA). The hematin- and ferrous iron-catalyzed transformation of the di-endoperoxide proceeded with a similar yield of products as the cleavage of the prostaglandin endoperoxide PGH(2) to 12S-hydroxy-5Z,8E,10E-heptadecatrienoic acid and MDA. Chiral phase HPLC analysis of the 4-HNE cleavage product showed greater than 98% 4S and thus established the S configuration of the 15-carbon of the di-endoperoxide that had not previously been assigned. This transformation of the 5-lipoxygenase/cyclooxygenase-2 derived di-endoperoxide invokes the possibility of a novel pathway to formation of the classic lipid peroxidation products 4-HNE and MDA.