XAD-2 Column Research Articles

Cytotoxicity and anti-inflammatory effects of root bark extracts of Acanthopanax henryi

AimTo investigate the cytotoxicity, anti-inflammatory activity, and action mechanism of root bark extracts of Acanthopanax henryi. MethodThe hot methanol extract of the root bark of A. henryi was subjected to XAD-4 column chromatography eluting with a gradient of methanol in water. The cytotoxicity and anti-inflammatory effects of the MeOH fractions were evaluated on the inhibition on lipopolysaccharide (LPS)-induced nitric oxide, prostaglandin E2, interleukin-1β, and interleukin-6 production in RAW 264.7 macrophages. ResultsThe 80% MeOH fraction was a better inhibitor of LPS-induced NO, PGE2, IL-1β, and IL-6 production, and expression of inducible nitric oxide synthase (iNOS) at the protein levels in a concentration-dependent manner. ConclusionThe 80% MeOH fraction of A. henryi root bark has significant anti-inflammatory activity. This provides a pharmacological basis for clinical application for the treatment of inflammation.

Free and Bound Volatile Compounds in Juice and Peel of Eureka Lemon

Free and glycosidically bound volatile compounds in different parts of Eureka lemon were studied. Free volatile compounds were extracted by solid phase microextraction (SPME). Bound volatile compounds were isolated from juice by adsorption onto an Amberlite XAD-2 column, and then hydrolyzed by almond β-glucosidase. Both the free volatile compounds and the released aglycones were analyzed by using gas chromatography-mass spectromerty (GC-MS). Gas chromatography-olfactometry (GC-O) was also used to determine the aroma-active compounds in juice and peel. Totally 29 and 34 free volatiles were found in juice and peel, respectively. Terpenes and aldehydes were the most abundant compounds in free fractions. Four and six bound volatile compounds were found in juice and peel with a total concentration of 127.8 and 1161.52 µg/L. Benzenic and terpenic compounds were the main bound volatiles found in Eureka lemon fruit. Mannose and glucose were found as sugar moiety in juice, and glucose was found the only sugar linked to bound volatiles in peel.

Large-scale isolation of highly pure malaxinic acid from immature pear (Pyrus pyrifolia Nakai) fruit

Malaxinic acid [4-(O-β-d-glucopyranosyl)-3-(3′-methyl-2′-butenyl)benzoic acid; MA] is one of the main compounds contained in pear fruit. The antifungal, antibacterial, and anticancer effects of MA have been reported. However, additional evaluations on the biological activity of MA have not been fully performed due to the lack of an MA standard. Therefore, in the present study, we established a large-scale isolation method to obtain highly pure MA from immature pear (Pyrus pyrifolia Nakai) fruit. The EtOAc-soluble acidic fraction (134.75 g) of pear fruit (18 kg fresh wt.) 60% EtOH extract (1,081.9 g) was purified by Amberlite XAD-2 (H2O/EtOH, stepwise), Diaion HP-20 (H2O/MeOH, stepwise), and Sephadex LH-20 (80% MeOH) column chromatographies. Then, the fractions containing MA were finally purified by high performance liquid chromatography using an Amide RP-C16 column on semi-preparative scale. The isolated MA (793 mg) was >99% pure, and total recovery was estimated to be 61.7%.

Novel GHB-derived natural products from European mistletoe (Viscum album)

Context: The European white-berry mistletoe [Viscum album L. (Loranthaceae)] is among the oldest known medicinal plants. At present the most important application of mistletoe extracts is in the treatment of cancer. However, natural products specific to mistletoe have rarely been encountered in the current literature.Objective: To discover novel natural products specific to European mistletoe.Materials and methods: European mistletoe was extracted with methanol, purified to partition against diethyl ether and further purified with XAD-7 column chromatography. Pure compounds were separated by Sephadex column chromatography and preparative HPLC. The structures of the novel compounds were established using a combination of several 2D NMR spectroscopic techniques and mass spectrometry.Results: A new type of natural product derived from the methyl ester of γ-hydroxybutyric acid (GHB) coupled to hydroxybenzoic acids, namely 3-(3′-carbomethoxypropyl) gallic acid and 3-(3′-carbomethoxypropyl)-7→3″-protocatechoyl galloate were characterized from European white-berry mistletoe. Condensation of the 3-hydroxyl of gallic acid with the 4-hydroxyl of GHB significantly reduced the radical scavenging properties of the former compound.Discussion and conclusion: The characterized compounds define a novel group of natural products that may be of particular interest because it appears that the two new compounds are not closely related to any known natural product.

Coumaroyl quinic acid derivatives and flavonoids from immature pear (Pyrus pyrifolia nakai) fruit

Fourteen compounds were isolated from 60% ethanol extracts of immature pear (Pyrus pyrifolia Nakai cv. Chuhwangbae) fruit using Amberlite XAD-2 column HPLC with guided DPPH radical scavenging assay. Based on MS and NMR analysis, the isolated compounds were identified as 5-O-trans-caffeoyl quinic acid methyl ester (1), malaxinic acid (2), 5-O-trans-p-coumaroyl quinic acid methyl ester (3), 5-O-cis-p-coumaroyl quinic acid methyl ester (4), 5-O-trans-p-coumaroyl quinic acid (5), trans-p-coumaric acid (6), methyl cis-p-coumarate (7), methyl trans-p-coumarate (8), 3,5-O-dicaffeoyl quinic acid (9), (-)-epicatechin (10), (S)-(+)-2-cis-abscisic acid (11), isorhamnetin 3-O-β-d-galacto-pyranoside (12), isorhamnetin-3-O-β-d-glucopyranoside (13), and isorhamnetin 3-O-α-l-rhamnopyranosyl (1→6)-O-β-d-glucopyranoside (14). Six compounds (1, 2, 6, 9, 10, and 13) were identified previously, but other compounds (3–5, 7, 8, 11, 12, and 14) were isolated for the first time from pear.

Metal etching industry wastewater pretreatment by coagulation and ion exchange resins

The removal of dissolved organic compounds (DOC) in the metal etching industry wastewater was tested by ion-exchange resins (IERs), coagulation and their combination. The appropriate IER was selected by batch and column tests out of Amberlite IR-120, IRA-900 and XAD-4. XAD-4 was the most efficient IER for the DOC removal in addition to removing the added surfactant, showing that the DOC was mostly hydrophobic in nature. The XAD-4 column treated 250 bed volumes. The minimum effluent total organic carbon (TOC) was 30 mg/L during first 50 BV and gradually increased to 90 mg/L at 250 BV. It showed that a column with 0.1 m3 bed volume would be required for the influent flow rate of 24 m3/ day, with daily regeneration. The coagulation with iron salt removed hydrophilic DOC and the minimum and optimum coagulant doses for DOC removal were 2.1 and 15.5 mg Fe(III)/mg TOC, respectively. A combination of coagulation and XAD-4 adsorption decreased minimum effluent TOC to 25 mg/L and 7 mg/L after 2.1 and 15.5 mg Fe(III)/mg TOC pretreatments, respectively. The bed life of XAD-4 was not increased because adsorption capacity is a specific property. Combination process can be used as pretreatment before RO process.

Separation of bulk Y from 89Y(n,p) produced 89Sr by extraction chromatography using TBP coated XAD-4 resin

89Sr was produced using the 89Y(n, p) 89Sr reaction by irradiating yttria target in the fast breeder test reactor (FBTR). An analytical scale procedure was standardized for the removal of the bulk target yttrium by solvent extraction using the tri-n-butyl phosphate (TBP). The final purification of 89Sr source was carried out by ion exchange chromatography. However, extraction chromatography is preferred to solvent extraction due to its low waste generation and ease of operation. This paper reports the separation of Sr from bulk Y and other radioactive impurities produced during irradiation by extraction chromatography using TBP coated XAD-4 resin. Initially the separation procedure was standardized using 85Sr and 88Y tracers. The 89Sr in the dissolver solution of the irradiated yttria target was separated under the same standardized conditions. The study established the separation of Sr from Y in the dissolver solution of the irradiated target yttria by the TBP coated XAD-4 column. However the evaluation of the column after every use for about three separation studies exhibited the reduction in its breakthrough capacity for yttrium.

Optimization of Extraction Parameters by Using Response Surface Methodology, Purification, and Identification of Anthocyanin Pigments in Melastoma malabathricum Fruit

Anthocyanins not just have various benefits in food industry but also have been used as natural colourants in cosmetic, coating products and as potential natural photosensitizers in solar cell. Thus, the main purpose of this study was to obtain information on the maximum yield of anthocyanin that can be recovered from Melastoma malabathricum fruit. Factors such as extraction temperature, extraction time, and solid to liquid ratio were identified to be significantly affecting anthocyanin extraction efficiency. By using three-level three-factor Box-Behnken design, the optimized conditions for anthocyanin extraction by acidified methanol (R 2 = 0.972) were temperature of 60°C, time of 86.82 min, and 0.5 : 35 (g/mL) solid to liquid ratio while the optimum extraction conditions by acidified ethanol (R 2 = 0.954) were temperature of 60°C, time of 120 min, and 0.5 : 23.06 (g/mL) solid to liquid ratio. The crude anthocyanin extract was further purified by using Amberlite XAD-7 and Sephadex LH-20 column chromatography. Identification of anthocyanins revealed the presence of cyanidin dihexoside, cyanidin hexoside, and delphinidin hexoside as the main anthocyanins in M. malabathricum fruit.

Red cabbage anthocyanins: Profile, isolation, identification, and antioxidant activity

Red cabbage is rich in a number of bioactive substances, including anthocyanins. The anthocyanin profile of red cabbage consisting of twenty derivatives of cyanidin glucosides was described by means of HPLC-DAD-MS/MS. The base structure of anthocyanins identified was cyanidin-3-diglucoside-5-glucoside. Their glucoside residues were nonacylated, monoacylated and diacylated. Sinapic, ferulic, caffeic and p-coumaric acids were recognized as main phenolic acids in this structure. The predominant anthocyanin in red cabbage was nonacylated form of cyanidin-3-diglucoside-5-glucoside, followed by cyanidin-3-(sinapoyl)(sinapoyl)-diglucoside-5-glucoside and cyanidin-3-(p-coumaroyl)-diglucoside-5-glucoside. Anthocyanins were isolated and purified by Amberlite XAD-16 column chromatography and by HPLC on C18 semi-preparative column. Among the seven isolated and purified red cabbage anthocyanins, cyanidin-3-diglucoside-5-glucoside diacylated with sinapic acid showed the highest radical-scavenging activities toward both 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) radical cation and superoxide anion radical. The relative contribution of anthocyanins to the antioxidant capacity of red cabbage extract was low. The results obtained indicated that red cabbage has its own characteristic anthocyanin pattern as well as a kind of acylation that affects the antioxidant activity of acylated anthocyanins.

Anti-proliferative activity and protection against oxidative DNA damage by punicalagin isolated from pomegranate husk

Ellagitannins are the most abundant polyphenols in pomegranate (Punica granatum) husk and contribute greatly towards its biological properties. A pre-enriched pomegranate husk powder was extracted with water and then further purified by an Amberlite XAD-16 column. Punicalagin (PC) anomers were eluted using a gradient of methanol and water. Fractions eluted with 20% and 25% methanol yielded 1.08g of light brown powder (purity>97%) from a total of 40g of extract. This fraction was identified as PC by HPLC-UV using reference compounds and confirmed by FTICR-MS analysis. PC (10–40μM) was found to significantly inhibit oxidative DNA products, about 70% inhibition at 40μM (p=0.0017), resulting from Cu2+-catalyzed redox cycling of 4-hydroxy-17β-estradiol as analyzed by 32P-postlabeling. Evidence of high antioxidant activity of PC was also obtained based on ORAC assay (1556±79μmol of TE/g), as well as by 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)-, 2,2-diphenyl-1-picrylhydrazyl (DPPH)-, hydrogen peroxide (H2O2) scavenging and ferrous ion-chelating activities (IC50=1.1, 17.1, 24 and 45.4μg/ml, respectively). Further, PC exhibited strong anti-proliferative activity against the human lung, breast and cervical cancer cell lines. Together, these data suggest that PC can be isolated in its purified form by simple column chromatography, inhibits oxidative DNA damage and possesses high anti-proliferative activity.

Production of Bacillomycin- and Macrolactin-Type Antibiotics by Bacillus amyloliquefaciens NJN-6 for Suppressing Soilborne Plant Pathogens

Bacillus amyloliquefaciens strains have been used as biocontrol agents for the suppression of several soilborne plant pathogens. A clearer understanding of the antagonistic mechanisms of action of these bacteria will facilitate their use in the control of plant diseases. Antagonistic substances were isolated from the fermentation broth of B. amyloliquefaciens strain NJN-6 cultures. These compounds were preconcentrated using an XAD-16 column and were purified using reversed-phase high-performance liquid chromatography (RP-HPLC). Fractions were collected from the column and were analyzed, and two homologues of bacillomycin D [molecular weights of 1030 Da (C14) and 1044 Da (C15)] and three homologues of members of the macrolactin family, macrolactin A, 7-O-malonyl macrolactin A, and 7-O-succinyl macrolactin A (molecular weights of 402, 487, and 502 Da, respectively) were identified using HPLC/electrospray ionization mass spectrometry (ESI-MS) analysis. An antagonistic assay showed that bacillomycin D and macrolactin exhibited significant antagonistic effects against Fusarium oxysporum and Ralstonia solanacearum , respectively. A reliable method for the isolation and purification of bacillomycin D and macrolactin from bacterial broth cultures was developed. These data will help elucidate the mechanisms that B. amyloliquefaciens NJN-6 uses for the biocontrol of soilborne plant pathogens.

Characterisation of bound volatile compounds of a low flavour kiwifruit species: Actinidia eriantha

Aroma compounds in fruit are known to occur in free and glycosidically bound forms. The bound volatile fraction of a low flavour kiwifruit species, Actinidia eriantha, was studied. The fruit have a bland and grassy flavour. Glycosidic precursors were isolated from juice by adsorption onto an Amberlite XAD-2 column. After enzymatic hydrolysis with Rapidase AR2000, the released aglycones were analysed by GC-MS. Alcohols, terpenoids and phenolics were the most numerously represented compound classes. Alcohols, benzenoids and phenolics showed the highest concentrations. Major compounds were 2-phenylethanol, furfuryl alcohol, (Z)-3-hexen-1-ol, coniferyl alcohol, isoamyl alcohol and linolenic acid. Several of the bound compounds found, including linoleic, linolenic and benzoic acids and coniferyl alcohol, are precursors of odorous volatiles. Many compounds detected as bound volatiles have not been previously reported as free volatiles in A. eriantha. The bound volatile composition of A. eriantha also showed differences with those of other kiwifruit species.

Antioxidant Activity of Mulberry Fruit Extracts

Phenolic compounds were extracted from the fruits of Morus nigra and Morus alba using methanol and acetone. The sugar-free extracts (SFEs) were prepared using Amberlite XAD-16 column chromatography. All of the SFEs exhibited antioxidant potential as determined by ABTS (0.75–1.25 mmol Trolox/g), DPPH (2,2-diphenyl-1-picrylhydrazyl) (EC50 from 48 μg/mL to 79 μg/mL), and reducing power assays. However, a stronger activity was noted for the SFEs obtained from Morus nigra fruits. These extracts also possessed the highest contents of total phenolics: 164 mg/g (methanolic SFE) and 173 mg/g (acetonic SFE). The presence of phenolic acids and flavonoids in the extracts was confirmed using HPLC method and chlorogenic acid and rutin were found as the dominant phenolic constituents in the SFEs.

The use of pretreated palm oil mill effluent for acetone–butanol–ethanol fermentation by Clostridium saccharoperbutylacetonicum N1-4

Palm oil mill effluent (POME) was used as an acetone–butanol–ethanol (ABE) fermentation medium using Clostridium saccharoperbutylacetonicum N1-4. Various pretreatment methods were applied on POME to increase the amount of fermentable sugars leading to enhanced ABE production. Sulfuric acid-treated POME (SA-POME) method was found to give the highest yield of total reducing sugars (glucose, cellobiose, xylose, and arabinose) as compared to other pretreatment methods. An increment in the concentration of H2SO4 from 1 to 2% resulted in the enhanced release of reducing sugars (18.3, 26.3 g/L, respectively). However, the treatment of POME with 3% H2SO4, decreased the reducing sugars to 21.6 g/L and consequently, the total ABE production was also reduced. The highest yield of ABE was observed from a culture grown with POME treated by 1% H2SO4. The total ABE production from 1, 2, and 3% SA-POME was obtained as 2.2, 0.45, and 0.41 g/L, respectively. Although, enzymatically treated POME (EH-POME) could produce 4.42 g/L glucose, sulfuric acid treatment (1%) was able to liberate only 1.76 g/L glucose, ABE production was higher when 1% SA-POME was used. Low yield of ABE from enzymatically treated POME can be attributed to the production of some inhibitors during hydrolysis of POME. When EH-POME was treated with XAD-4 resin to nullify the inhibitors, the production of ABE was increased to 4.29 g/L, and ABE yield was also increased to 0.29 g/g. In conclusion, enzymatic hydeolysis of POME followed by elution to XAD-4 column can be proposed as the best pretreatment method for highest productivity of ABE. It was found that addition of P2 medium to the POME hydrolysates was able to improve the production of butanol except in raw POME and sulfuric acid hydrolysates.

Presence of Caffeic Acid in Flaxseed Lignan Macromolecule

Phenolic compounds were extracted from defatted flaxseeds using ethanol-dioxane (1:1, v/v). The crude extract obtained was purified using Amberlite XAD-16 column chromatography with water and methanol as mobile phases. RP-HPLC and SE-HPLC showed a lignan macromolecule (LM) as a dominant phenolic compound in the purified extract. After the alkaline hydrolysis of LM caffeic acid glucoside (CaAG) was isolated using a semi-preparative HPLC and its structure was confirmed by LC-ESI-MS. In LM of the investigated flaxseed, one molecule of caffeic acid corresponded with five molecules of p-coumaric acid and two molecules of ferulic acid. The presence of caffeic acid in the lignan molecule might be very beneficial due to its high antioxidant activity.

Characterization of the Bound Volatile Extract from Baby Kiwi (Actinidia arguta)

The glycosidically bound volatile fraction of baby kiwi ( Actinidia arguta ) was studied. Glycosidic precursors were isolated from juice by adsorption onto an Amberlite XAD-2 column. After enzymatic hydrolysis with Rapidase AR2000, the released aglycones were analyzed by GC-MS. Alcohols, terpenoids, and benzenoids were the most abundant compound classes. Aromatic compounds and norisoprenoids showed the highest concentrations. Major compounds were 2,5-dimethyl-4-hydroxy-3(2H)-furanone (Furaneol), benzyl alcohol, 3-hydroxy-β-damascone, hexanal, and (Z)-3-hexen-1-ol. Precursors of aroma compounds including benzoic acid, cinnamic acid, and coniferyl alcohol were also found. Eugenol, raspberry ketone, and 4-vinylguaiacol were identified for the first time in the fruit of an Actinidia species. The high concentration of 2,5-dimethyl-4-hydroxy-3(2H)-furanone in bound form (95.36 μg/kg) is particularly interesting and justifies further investigation.

Abstract 3711: Enhanced bioactivity of punicalagins by polymeric implants against benzo[a]pyrene-induced DNA adducts in vivo

Abstract Ellagitannins are the most abundant polyphenols present in pomegranate (Punica granatum) peel and black raspberry and contribute greatly towards reported biological properties. In this study, we report on i) the bulk isolation of punicalagins from pomegranate peel, and ii) various chemopreventive/chemotherapeutic activities. The pre-enriched pomegranate peel powder was extracted repeatedly with water. The pooled extract was concentrated using rotovapor and then further purified by an amberlite XAD-16 column. Punicalagins were eluted using a gradient of methanol. Fractions eluted with 20-25% methanol yielded 1.2 g light brown powder from a total of 40 g extract. This fraction was identified as punicalagins by HPLC using reference compounds and confirmed by LC/MS analysis. The punicalagins (40 µM) were found to inhibit oxidative DNA products (70% inhibition; p=0.0017) from Cu2+-catalyzed redox cycling of 4-hydroxy-17ß-estradiol as analyzed by 32P-postlabeling. Evidence of high antioxidant activity of punicalagins was also obtained based on ORAC assay (1556 ± 79 μmol of TE/mg), as well as by 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS)- and 2,2-diphenyl-1-picrylhydrazyl (DPPH)-scavenging, and ferrous ions-chelating activities (IC50 1.2 µg/ml, 19 µg/ml, and 46.5 µg/ml respectively). We further investigated the effect of punicalagins on benzo[a]pyrene (BP)-induced DNA adducts in vitro and in vivo: Incubation of BP (1 µM) with rat liver microsomes, appropriate co-factors and DNA in the presence of vehicle or punicalagins (1-40 µM) showed dose-dependent inhibition of the resultant DNA adducts, with essentially complete (97%) inhibition at 40 µM. To determine its efficacy in vivo, female S/D rats were treated with punicalagins via diet (1,500 ppm) or by subcutaneous polymeric implants (two 2-cm implants; 20% drug load) and then challenged with BP by subcutaneous polymeric implants (2-cm; 10% load) and euthanized after 1 week. Analysis of the lung DNA by 32P-postlabeling showed significant (60%; p = 0.029) inhibition of DNA adducts by punicalagins administered by the implants; the dietary administration of punicalagins also showed modest but statistically insignificant inhibition (34%). Measurement of the residual compound in the implants recovered from the animals showed that the total dose administered by the implants was 40-fold lower than the oral route. Together, our data show that punicalagins isolated from pomegranate peel had significant antioxidant activity and was highly effective against BP-induced DNA adducts in vivo only when administered by sustained-release polymeric implants (Supported from the USPHS grants CA-118114 and CA-125152 and the Duggan Endowment). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3711. doi:10.1158/1538-7445.AM2011-3711

Antioxidant and Anti-inflammatory Activities of Polyphenolics from Southeastern U.S. Range Blackberry Cultivars

The antioxidant and topical anti-inflammatory activities of low and high molecular weight phenolic fractions (LMPF and HMPF, respectively) isolated from three blackberry cultivars (i.e., Navaho, Kiowa, and Ouachita), bred to tolerate the warm and humid climatic conditions of the southeastern United States, were investigated by the in vitro ferric reducing antioxidant power (FRAP) assay and an in vivo mouse ear edema model. Seventy percent (v/v) acidified acetone was employed to extract phenolics from the Georgia-grown blackberry cultivars, which were subsequently cleaned up on an Amberlite XAD-16 column and then further fractionated with Sephadex LH-20 to LMPF and HMPF. The anti-inflammatory response from topical application of solutions of the LMPF and HMPF as well as indomethacin, a potent nonsteroidal anti-inflammatory drug, was assessed in the TPA mouse ear model. All treatments significantly (P < 0.05) reduced TPA-induced irritation injury. Furthermore, mouse ear myeloperoxidase (MPO) activity, an indicator of polymorphonuclear leukocyte infiltration, was assessed and found to be significantly (P < 0.05) reduced after topical application of indomethacin and all blackberry preparations. Correlation coefficients of 0.925 and 0.923 (P < 0.01) were determined when the anti-inflammatory activities of the blackberry fractions were compared to their total phenolics contents and antioxidant activities (i.e., FRAP values), respectively.

Identification of free and conjugated metabolites of mesocarb in human urine by LC-MS/MS

The objective of the present study was to investigate mesocarb metabolism in humans. Samples obtained after administration of mesocarb to healthy volunteers were studied. The samples were extracted at alkaline pH using ethyl acetate and salting-out effect to recover metabolites excreted free and conjugated with sulfate. A complementary procedure was applied to recover conjugates with glucuronic acid or with sulfate consisting of the extraction of the urines with XAD-2 columns previously conditioned with methanol and deionized water; the columns were then washed with water and finally eluted with methanol. In both cases, the dried extracts were reconstituted and analyzed by ultra-performance liquid chromatography-tandem mass spectrometry. Chromatographic separation was carried out using a C(18) column (100 mm x 2.1 mm i.d., 1.7 microm particle size) and a mobile phase consisting of water and acetonitrile with 0.01% formic acid with gradient elution. The chromatographic system was coupled to a mass spectrometer with an electrospray ionization source working in positive mode. Metabolic experiments were performed in multiple-reaction monitoring mode by monitoring one transition for each potential mesocarb metabolite. Mesocarb and 19 metabolites were identified in human urine, including mono-, di-, and trihydroxylated metabolites excreted free as well as conjugated with sulfate or glucuronic acid. All metabolites were detected up to 48 h after administration. The structures of most metabolites were proposed based on data from reference standards available and molecular mass and product ion mass spectra of the peaks detected. The direct detection of mesocarb metabolites conjugated with sulfate and glucuronic acid without previous hydrolysis has been described for the first time. Finally, a screening method to detect the administration of mesocarb in routine antidoping control analyses was proposed and validated based on the detection of the main mesocarb metabolites in human urine (p-hydroxymesocarb and p-hydroxymesocarb sulfate). After analysis of several blank urines, the method demonstrated to be specific. Extraction recoveries of 100.3 +/- 0.8 and 105.9 +/- 10.8 (n = 4), and limits of detection of 0.5 and 0.1 ng mL(-1) were obtained for p-hydroxymesocarb sulfate and p-hydroxymesocarb, respectively. The intra- and inter-assay precisions were estimated at two concentration levels, 50 and 250 ng mL(-1), and relative standard deviations were lower than 15% in all cases (n = 4).

Free and bound volatile compounds in juice and peel of Jincheng oranges

Free and glycosidically bound volatile compounds in juice and peel of Jincheng oranges were investigated. Solid phase microextraction (SPME) was used for extraction of free volatile compounds. Bound volatile compounds in juice were isolated by using Amberlite XAD-2 column adsorption and methanol elution. Subsequently, they were hydrolyzed by almond β-glucosidase. Acid hydrolysis was also applied on the release of the bound fraction in juice. Both free and bound volatile compounds were then determined by GC-MS. A total of 26 and 40 free volatile compounds were identified in juice and peel, respectively. Terpenes followed by aldehydes were the major free volatile compounds both in juice and peel. Eleven and ten aglycons hydrolyzed by enzyme were identified in juice and peel, respectively. Benzenic compounds and hydroxyl esters were the main bound volatile compounds in juice, while C13-norisoprenoid and monoterpenes were the most abundant bound volatile compounds in peel. 3-Oxo-α-ionol (41%) predominated in bound fraction of peel. Five aglycons hydrolyzed by acid were found in juice. p-Vinylguaiacol and eugenol were the two compounds found both after enzymatic and acid hydrolysis. Rhamnose, xylose, and glucose were determined as sugar moieties in juice.