Gedunin,ad-seco limonoid

Chloroform/Methanol extraction of the bark ofCedrela salvadorensis (Meliaceae), followed by chromatography afforded the crystalline C₂₈H₃₄O₇ terpene. The structure first proposed by spectroscopy is confirmed by this X-ray analysis as 7-acetyloxy-14,15:21,23-diepoxy-4,4,8-trimethyl-d-homo-24-nor-17-oxachola-1,20,22-triene-3,16,-dione. The title compound crystallized in the orthorhombic space groupP2₁2₁2₁ with unit cell parametersa=10.243(2),b=13.356(3) andc=18.058(4) Å. The molecule is comprised of a six-membered tetracyclic skeleton, with a circular arc overall conformation. No intermolecular hydrogen bonding is indicated.     

Anti-EPO and anti-NESP antibodies raised against synthetic peptides that reproduce the minimal amino acid sequence differences between EPO and NESP

Erythropoietin (EPO) is a hormone that regulates red blood cell production. Recombinant human EPO (rHuEPO) and NESP (novel erythropoiesis stimulating protein) have been produced for therapeutic purposes and also to improve sports performance. The primary sequences of rHuEPO and NESP differ by just five amino acids. Due to the high homology, no antibodies that are able to discriminate between both molecules have been obtained until now. The aim of the present work was to design synthetic peptides corresponding to the sequence that differs between EPO and NESP (87–90aa), that can then be used as immunogens to develop specific rabbit polyclonal antibodies for selectively detecting EPO and NESP. Three peptides were synthesized: EPO (81–95), NESP (81–95), and NESP (86–104), and these were coupled to KLH and OVA for immunization and screening purposes, respectively. The sera obtained were tested by ELISA on synthetic peptide–OVA conjugates and purified by immunoaffinity chromatography against the corresponding synthetic peptide. The specific purified antibodies were characterized by ELISA, SDS-PAGE, and isoelectric focusing, followed by western blot. Antisera raised against EPO (81–95) recognized rHuEPO but not NESP. In contrast, anti-NESP (84–106) sera gave a specific anti-NESP response only after immunoaffinity purification on a NESP (86–91) column. An efficient strategy for generating specific antibodies against EPO and NESP can be achieved by selecting suitable synthetic peptides. The antibodies obtained are able to differentiate between rHuEPO and NESP, and may be particularly useful for screening purposes in both therapeutic and antidoping contexts.     

Quantitation of 17beta-nandrolone metabolites in boar and horse urine by gas chromatography-mass spectrometry

A method to quantify metabolites of 17beta-nandrolone (17betaN) in boar and horse urine has been optimized and validated. Metabolites excreted in free form were extracted at pH 9.5 with tert-butylmethylether. The aqueous phases were applied to Sep Pak C18 cartridges and conjugated steroids were eluted with methanol. After evaporation to dryness, either enzymatic hydrolysis with beta-glucuronidase from Escherichia coli or solvolysis with a mixture of ethylacetate:methanol:concentrated sulphuric acid were applied to the extract. Deconjugated steroids were then extracted at alkaline pH with tert-butylmethylether. The dried organic extracts were derivatized with MSTFA:NH4I:2-mercaptoethanol to obtain the TMS derivatives, and were subjected to analysis by gas chromatography mass spectrometry (GC/MS). The procedure was validated in boar and horse urine for the following metabolites: norandrosterone, noretiocholanolone, norepiandrosterone, 5beta-estran-3alpha, 17beta-diol, 5alpha-estran-3beta, 17beta-diol, 5alpha-estran-3beta, 17alpha-diol, 17alpha-nandrolone, 17betaN, 5(10)-estrene-3alpha, 17alpha-diol, 17alpha-estradiol and 17beta-estradiol in the different metabolic fractions. Extraction recoveries were higher than 90% for all analytes in the free fraction, and better than 80% in the glucuronide and sulphate fractions, except for 17alpha-estradiol in the glucuronide fraction (74%), and 5alpha-estran-3beta, 17alpha-diol and 17betaN in the sulphate fraction (close to 70%). Limits of quantitation ranged from 0.05 to 2.1 ng mL(-1) in the free fraction, from 0.3 to 1.7 ng mL(-1) in the glucuronide fraction, and from 0.2 to 2.6 ng mL(-1) in the sulphate fraction. Intra- and inter-assay values for precision, measured as relative standard deviation, and accuracy, measured as relative standard error, were below 15% for most of the analytes and below 25%, for the rest of analytes. The method was applied to the analysis of urine samples collected after administration of 17betaN laureate to boars and horses, and its suitability for the quantitation of the metabolites in the three fractions has been demonstrated.     

Precise mass measurement of a double-stranded 500 base-pair (309 kDa) polymerase chain reaction product by negative ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) has been used to determine the mass of a double-stranded 500 base-pair (bp) polymerase chain reaction (PCR) product with an average theoretical mass of the blunt-ended (i.e. unadenylated) species of 308 859.35 Da. The PCR product was generated from the linearized bacteriophage Lambda genome which is a double-stranded template. Utilization of ethanol precipitation in tandem with a rapid microdialysis step to purify and desalt the PCR product was crucial to obtain a precise mass measurement. The PCR product (0.8 pmol/μL) was electrosprayed from a solution containing 75% acetonitrile, 25 mM piperidine, and 25 mM imidazole and was infused at a rate of 200 nL/min. The average molecular mass and the corresponding precision were determined using the charge-states ranging from 172 to 235 net negative charges. The experimental mass and corresponding precision (reported as the 95% confidence interval of the mean) was 309 406 +/- 27 Da (87 ppm). The mass accuracy was compromised due to the fact that the PCR generates multiple products when using Taq polymerase due to the non-template directed 3'-adenylation. This results in a mixture of three PCR products with nearly identical mass (i.e. blunt-ended, mono-adenylated and di-adenylated) with unknown relative abundances that were not resolved in the spectrum. Thus, the experimental mass will be a weighted average of the three species which, under our experimental conditions, reflects a nearly equal concentration of the mono- and di-adenylated species. This report demonstrates that precise mass measurements of PCR products up to 309 kDa (500 bp) can be routinely obtained by ESI-FTICR requiring low femtomole amounts. Copyright 1999 John Wiley & Sons, Ltd.     

Determination of the antibacterial drug sulfamethoxazole in pharmaceutical preparations containing trimethoprim by spectrofluorimetry after derivatization with fluorescamine

An analytical method was developed for the determination of sulfamethoxazole (SMZ) in the presence of trimethoprim (TMP) by normal fluorescence. When both analytes are present a selective derivatization with fluorescamine of SMZ gives an intense fluorescent derivative with no interference from the other compound. The reaction was optimized to obtain the best analytical performance. The detection limit and the lower limit of quantitation of SMZ in the reaction medium was 5.2 ng/mL. The intra-day precision (relative standard deviation) was 1.51% for a 100 ng/mL SMZ standard solution and the inter-day precision over 7 days for a 100 ng/mL solution in the presence of 20 ng/mL TMP solution was 2.5%. The method has been applied to three pharmaceutical preparations containing both compounds, without any separation steps required.     

Electrochemical synthesis of PEDOT derivatives bearing imidazolium‐ionic liquid moieties

Novel poly(3,4‐ethylenedioxythiophene) (PEDOT) polymers bearing imidazolium‐ionic liquid moieties were synthesized by electrochemical polymerizations. For this purpose, new functional monomers were synthesized having an 3,4‐ethylenedioxythiophene (EDOT) unit and an imidazolium‐ionic liquid with different anions such as tetrafluoroborate (BF), bis(trifluoromethane)sulfonimide ((CF₃SO₂)₂N^?), and hexafluorophosphate (PF). Next, polymer films were obtained by electrochemical synthesis in dicholoromethane solutions. Obtained polymers were characterized, revealing the characteristics of PEDOT in terms of electrochemical and spectroelectrochemical properties, FTIR, ¹H NMR, and AFM microscopy. Interestingly, the hydrophobic character of electropolymerized films could be modified depending on the anion type. The hydrophobicity followed the trend PF > (CF₃SO₂)₂N^? > BF > pure PEDOT as determined by water contact angle measurements. Furthermore, the polymers could be dissolved in a range of polar organic solvents such as dimethylformamide, propylene carbonate, and dimethyl sulfoxide making these polymers interesting candidates for wet processing methods. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 3010–3021, 2009     

Extraction of arsenic as the diethyl dithiophosphate complex with supercritical fluid and quantitation by cathodic stripping voltammetry

The separation of arsenic based on in situ chelation with ammonium diethyl dithiophosphate (ADDTP) has been carried out using methanol-modified supercritical CO₂. Aliquots of extract were added to an electroanalytical cell and arsenic was determined by square wave cathodic stripping voltammetry (SWCSV) at a hanging mercury drop electrode (HMDE). Quantitative extractions of As(DDTP)₃ were achieved when the experiments were carried out at a pressure of 2500 psi, a temperature of 90 °C, 2.0 mL of methanol, 20.0 min of static extraction and 5.0 min of dynamic extraction in the presence of 18 mg of ADDTP. Analysis of arsenic was made using 150 mg L⁻¹ of Cu(II) in 1 M HCl solution as supporting electrolyte in the presence of ADDTP as ligand. Preconcentration was carried out by deposition at a potential of −0.50 V and the intermetallic compound Cu_xAs_y was reduced at a potential of −0.77 to −0.82 V, depending on ligand concentration. The results showed that the presence of ligand plays an important role, increasing the method's sensitivity and preventing the oxidation of As(III). The calibration graph of the As(DDTP)₃ solution was linear from 0.8 to 12.5 μg L⁻¹ of arsenic (LOD 0.5 μg L⁻¹, R = 0.9992, t_acc = 60 s). The method was validated using carrot pulp spiked with arsenic solution. This method was applied to the determination of arsenic in samples of carrots, beets and irrigation water. Arsenic in beets was: skin 4.10 ± 0.18 mg kg⁻¹; pulp 3.83 ± 0.19 mg kg⁻¹ and juice 0.71 ± 0.09 mg L⁻¹; arsenic in carrots was: skin 2.15 ± 0.09 mg kg⁻¹; pulp 0.59 ± 0.11 mg kg⁻¹ and juice 0.71 ± 0.03 mg L⁻¹. Arsenic in water were: Chiu-Chiu 0.08 mg L⁻¹, Inacaliri 1.12 mg L⁻¹, and Salado river 0.17 ± 0.07 mg L⁻¹.     

Assessing the instability of the isoelectric focusing patterns of erythropoietin in urine

IEF can be used to differentiate human urinary erythropoietin (uEPO), recombinant human erythropoietin or epoetin (rEPO) and darbepoetin (novel erythropoiesis stimulating protein (NESP)). This is the basis of the method currently used to detect misuse of rEPO and NESP by elite athletes. Recently, an unknown activity has been attributed to some urine samples (denominated 'unstable' urine by the World Anti-Doping Agency; WADA). This activity has shown to give rise to artefactual profiles for both rEPO and NESP when incubated with such urine and, thus, raised concerns with respect to doping control. We have evaluated which charges produce the characteristic IEF profiles of uEPO, rEPO and NESP and how these profiles respond to distinct enzymatic reactions. From sialidase digestions it became evident that only uEPO contains charges different from sialic acid, and a comparison of all substances after complete de-N-glycosylation localized these charges in the carbohydrate moiety. Partial desialylation, or digestion with arylsulfatase from Helix pomatia yielded profiles for recombinants species similar to those observed for unstable urine samples. The contributions from our studies to the anti-doping problem include: (i) protocols that may corroborate the potential misuse of rEPO or NESP based on the particular enzymatic activity of an arylsulfatase preparation, or a broad-specificity sialidase; (ii) assurance that the instability observed in some urine samples may only result from false-negatives, but not from false-positive testing; and (iii) a simple remedy to prevent an unstable urine from altering the IEF profile by adding selective competitive substrates.     

Graphene functionalisation with a conjugated poly(fluorene) by click coupling: striking electronic properties in solution

Graphene flakes covalently modified with a conjugated polymer, poly[(9,9-dihexylfluorene)-co-alt-(9,9-bis-(6-azidohexyl)fluorene)] (PFA), were efficiently synthesised by a Cu-catalysed Huisgen 1,3-dipolar cycloaddition between alkyne-modified graphene and an azide-functionalised polymer. Two approaches for the modification of graphene with alkyne groups were investigated (coupling with a diazonium salt generated in situ or an amidation reaction) and the optimum conditions determined. The success of the click-coupling approach was confirmed by FTIR, (1)H?NMR, Raman, and X-ray photoelectron spectroscopy (XPS). The absorption and emission spectra of the click product show a strong solvent dependency.     

Anomalous high-pressure Jahn-Teller behavior in CuWO4

High-pressure optical-absorption measurements performed in CuWO(4) up to 20 GPa provide experimental evidence of the persistence of the Jahn-Teller (JT) distortion in the whole pressure range both in the low-pressure triclinic and in the high-pressure monoclinic phase. The electron-lattice couplings associated with the e(g)(E?e) and t(2g)(T?e) orbitals of Cu(2+) in CuWO(4) are obtained from correlations between the JT distortion of the CuO(6) octahedron and the associated structure of Cu(2+) d-electronic levels. This distortion and its associated JT energy (E(JT)) decrease upon compression in both phases. However, both the distortion and associated E(JT) increase sharply at the phase-transition pressure (P(PT)=9.9 GPa), and we estimate that the JT distortion persists for a wide pressure range not being suppressed up to 37 GPa. These results shed light on the transition mechanism of multiferroic CuWO(4), suggesting that the pressure-induced structural phase transition is a way to minimize the distortive effects associated with the toughness of the JT distortion.