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ABSTRACT: Combining the most powerful separation techniques, i.e. liquid chromatography (LC) or capillary electrophoresis (CE) with a information rich detection system - the mass spectrometer or the nuclear magnetic resonance (NMR) spectrometer - has been pursued for more than three decades. This compilation shall provide an overview of the advantages and limitations of the LC-NMR hyphenation in the light of its most valued application-the unequivocal analyte identification. Especially the post LC trapping of analytes with an in-line solid phase extraction (SPE) device prior to transferring the analyte of interest to the NMR spectrometer (LC-SPE-NMR) proved to be a robust installation allowing a significant cut-down of the amount of analyte needed for the generation of high quality heteronuclear NMR shift correlation data. Different available technical realizations will be discussed and typical application examples from natural product research and from industrial settings will be given.
Journal of chromatography. A 05/2012; 1259:50-61. · 4.19 Impact Factor
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ABSTRACT: The levonorgestrel-releasing intrauterine system (LNG-IUS) is well accepted as an easy-to-use contraceptive with an excellent side-effect profile. It contains a reservoir of 52 mg of levonorgestrel (LNG) with continuous release of the steroid. Its contraceptive use is approved for 5 years. The aim of this study was to determine the plasma concentration of LNG and its variation with time in patients with in-dwelling LNG-IUS Mirena®.
In this study, we determined LNG plasma concentrations in 110 women with LNG-IUS at different time points of use. Time from insertion of the system in the study population ranged from 20 days to 11.1 years. Quantitative LNG levels were determined using a validated liquid chromatography-tandem mass spectrometry assay.
The mean±SD LNG plasma level in all women was 147±59 pg/mL. A highly significant negative correlation between LNG plasma level and LNG-IUS time of use could be demonstrated. In the first year of use, LNG plasma level was as high as 191±71 pg/mL, decreasing to 157±68 pg/mL in the second year and 134±41 pg/mL in the third year. Even after exceeding the recommended period of LNG-IUS use, systemic LNG concentrations were detectable: 133±38 pg/mL in the sixth year, 133±48 pg/mL in the seventh year and 117±45 pg/mL in the eighth year. Furthermore, a significant negative correlation between LNG plasma level and body mass index could be shown.
Systemic LNG concentrations can be found in all patients with LNG-IUS IUS. However, concentrations are much lower than in other forms of LNG application. Moreover, this study demonstrates that a systemic effect of LNG-IUS can also be found after the recommended contraceptive lifespan of 5 years.
Contraception 03/2012; 86(4):345-9. · 2.72 Impact Factor
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ABSTRACT: Co-administration of synthetic progestin containing hormonal contraceptives (HCs) and antiepileptic drugs (AEDs) is a common clinical situation which needs specific considerations due to drug interactions. Several studies have demonstrated that lamotrigine plasma levels are significantly decreased during co-medication with HCs, and that this interaction is associated with increased seizure frequency in most of the cases. Additionally, an increase in contraceptive failure and unintended pregnancy could be observed during co-medication. Hence, monitoring of progestin plasma levels in patients with AED co-medication is of interest. A rapid and reliable online solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry (online SPE-LC-MS/MS) method using gradient elution in the LC domain was established and validated for the simultaneous quantitative determination of gestodene, dienogest, drospirenone, etonogestrel, cyproterone acetate, and levonorgestrel in human plasma. The online SPE-LC-MS/MS method covered a quantification concentration range of 5-100 ng/ml for dienogest, 1-100 ng/ml for etonogestrel and 2-100 ng/ml for all other analytes. Stable isotope-labeled internal standards were used for analyte quantification based on selected reaction monitoring experiments. Inter- and intra-assay precision and accuracy were determined from quality control (QC) samples at the lower limits of quantification and at low, medium, and high concentration levels within the calibration range. Inter-assay reproducibility at the QC levels was better than 10% (relative standard deviation, RSD), accuracy at these levels ranged from -3.7% to 11.3%. Total extraction efficiency, tested at three concentrations, ranged from 92.5% to 106.4%. Matrix interferences were excluded by post-column infusion experiments. To prove the applicability of the assay in clinical cohorts, a sample set (n = 298) stemming from study patients under AED/oral HC co-medication was screened for progestin plasma levels. This method has to be considered a research-use-only assay and must not be used for diagnostic or therapeutic purposes, since it did not undergo formal performance evaluation in the sense of the IVD directive (98/79/EG) of the European Community.
Analytical and Bioanalytical Chemistry 12/2011; 403(4):961-72. · 3.78 Impact Factor
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ABSTRACT: A selective and sensitive liquid chromatography-tandem mass spectrometry method for the determination of very low levonorgestrel (D-(-)-norgestrel) serum levels such as those found in patients using levonorgestrel-releasing intrauterine devices (IUDs) was developed. To achieve the sub-nanomolar sensitivity needed to measure such serum levels, a diethyl ether extraction sample preparation protocol was applied prior to the online solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) assay. Analyte quantification from the selected reaction monitoring experiments relied on the use of sixfold deuterated norgestrel as internal standard. The final method was linear up to 1.50 ng/ml with a lower limit of quantification (LLOQ) of 0.05 ng/ml. It was found to be precise and accurate with imprecision <8% and bias <6% assessed at three control levels. Total analyte recovery measured in patient pools at three concentration levels was found to exceed 92%. Matrix interferences were excluded by post-column analyte infusion experiments. As a proof of concept, a set of IUD patient serum samples was screened for their levonorgestrel content. A total of 97.5% (n = 94) of the samples did show serum levels exceeding the LLOQ, proving the applicability of the assay in relevant clinical cohorts. This method must not be used for diagnostic or therapeutic purposes, since it did not undergo formal performance evaluation in the sense of the in vitro diagnostic directive (98/79/EG) of the European community.
Analytical and Bioanalytical Chemistry 02/2011; 400(8):2655-62. · 3.78 Impact Factor
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ABSTRACT: The objective of this case-control study of 242 reproductive-age women was to determine the concentration of afamin in the serum and peritoneal fluid of women with and without endometriosis and to test afamin as a diagnostic marker of endometriosis. Afamin levels were altered significantly in the peritoneal fluid of women with endometriosis compared with disease-free controls, correlated with vitamin E levels, and are consistent with increased oxidative stress in the peritoneal cavity of women with endometriosis.
Fertility and sterility 12/2010; 94(7):2923-6. · 3.97 Impact Factor
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ABSTRACT: Fifteen prenylated or geranylated flavanones and flavanonols were isolated from the leaf extracts of different Glycosmis species collected in Thailand and Malaysia. All structures were elucidated by spectroscopic methods, especially 1D and 2D NMR. Six compounds were described for the first time and two were only known so far as synthetic products. The chemotaxonomic significance of flavanoid accumulation within the genus Glycosmis is highlighted.
Phytochemistry 07/2009; 70(8):1030-7. · 3.35 Impact Factor
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Claudia Hödl,
Katrin Raunegger,
Rainer Strommer,
Gerhard F Ecker,
Olaf Kunert,
Sonja Sturm, Christoph Seger,
Ernst Haslinger,
Rudolf Steiner,
Wolfgang S L Strauss,
Hans-Wolfgang Schramm
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ABSTRACT: A series of mifepristone derivatives with different "linker groups" in position 4' of the phenyl ring in the 11beta-position of the steroid scaffold (2-41) have been synthesized. Their antigestagenic activites were determined in a cell-based assay (alkali phosphatase assay in T47-D breast cancer cells) and compared with that of the parent compound mifepristone. SAR and QSAR studies reveal the influence of both lipophilicity and partial charge based van der Waals surface area descriptors on biological activity. Within the series of compounds described in this study, three mifepristone derivatives are identified with considerably high antigestagenic activity. These compounds are regarded as useful starting materials for the synthesis of either physiologically stable or cleavable progesterone receptor-binding conjugates for therapeutic or diagnostic purposes.
Journal of Medicinal Chemistry 03/2009; 52(5):1268-74. · 4.80 Impact Factor
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Ute Reisinger,
Stefan Schwaiger,
Iris Zeller,
Barbara Messner,
Robert Stigler,
Dominik Wiedemann,
Tobias Mayr, Christoph Seger,
Thomas Schachner,
Verena M Dirsch,
Angelika M Vollmar,
Johannes O Bonatti,
Hermann Stuppner,
Günther Laufer,
David Bernhard
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ABSTRACT: Despite the lower patency of venous compared with arterial coronary artery bypass grafts, approximately 50% of grafts used are saphenous vein conduits because of their easier accessibility. In a search for ways to increase venous graft patency, we applied the results of a previous pharmacological study screening for non-toxic compounds that inhibit intimal hyperplasia of saphenous vein conduits in organ cultures. Here we analyse the effects and mechanism of action of leoligin [(2S,3R,4R)-4-(3,4-dimethoxybenzyl)-2-(3,4-dimethoxyphenyl)tetrahydrofuran-3-yl]methyl (2Z)-2-methylbut-2-enoat, the major lignan from Edelweiss (Leontopodium alpinum Cass.).
We found that leoligin potently inhibits vascular smooth muscle cell (SMC) proliferation by inducing cell cycle arrest in the G1-phase. Leoligin induced cell death neither in SMCs nor, more importantly, in endothelial cells. In a human saphenous vein organ culture model for graft disease, leoligin potently inhibited intimal hyperplasia, and even reversed graft disease in pre-damaged vessels. Furthermore, in an in vivo mouse model for venous bypass graft disease, leoligin potently inhibited intimal hyperplasia.
Our data suggest that leoligin might represent a novel non-toxic, non-thrombogenic, endothelial integrity preserving candidate drug for the treatment of vein graft disease.
Cardiovascular research 03/2009; 82(3):542-9. · 5.80 Impact Factor
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ABSTRACT: In the past few years, high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) has matured to a true alternative to antibody-based immunoassays in routine therapeutic drug monitoring. In transplantation medicine, mass spectrometry-based assessment of immunosuppressant drug levels is considered a gold standard diagnostic procedure. We describe a fast state-of-the-art routine online solid-phase extraction (SPE) HPLC-MS/MS analysis platform that allows monitoring of cyclosporine A, tacrolimus, sirolimus and everolimus from 50-microl aliquots of EDTA whole blood specimens within 3.4 min total analysis time. Sample purification is done by offline protein precipitation followed by two automated chromatographic separation steps. Mass spectrometry-based analyte quantification relies on selected reaction monitoring experiments. The assay underwent complete validation and performance evaluation studies and performs very well in several international proficiency testing schemes. In daily routine, it allows reporting of about 75 patient sample results per work shift with a typical total individual sample turnaround time of less than 3 h.
Nature Protocol 02/2009; 4(4):526-34. · 8.36 Impact Factor
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ABSTRACT: The immunosuppressive mammalian target of rapamycin inhibitor rapamycin is widely used in solid-organ transplantation, but the effect of rapamycin on kidney disease is controversial. This study evaluated the effect of rapamycin in the autologous phase of anti-glomerular basement membrane (anti-GBM) glomerulonephritis. Disease was induced by preimmunizing the animals with rabbit IgG 5 d before administration of rabbit anti-mouse GBM antiserum. When rapamycin was started on the day of immunization (group 1), mice were protected from glomerulonephritis, suggested by a dramatic decrease in albuminuria, influx of inflammatory cells, and Th1-cytokine expression in the kidneys. Activation of T cells and production of autologous mouse anti-rabbit IgG were also significantly reduced in rapamycin-treated animals. In contrast, when rapamycin was started 14 d after immunization (group 2), mice had a significant increase in albuminuria and renal infiltration of inflammatory cells compared with vehicle-treated animals, and there were no differences in T and B cell responses. A significant decrease in vascular endothelial growth factor-A and an increase in IL-6 were detected in kidneys of these rapamycin-treated mice. In conclusion, rapamycin has the potential to significantly reduce the B and T cell responses and thereby protect from glomerulonephritis when administered early in disease. Once disease is established, however, rapamycin seems to worsen glomerulonephritis by disturbing the endothelial cell/vascular endothelial growth factor system in the kidney.
Journal of the American Society of Nephrology 06/2008; 19(8):1520-9. · 9.66 Impact Factor
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ABSTRACT: Identification of putative biomarker molecules within the genus Corydalis (Papaveraceae) was pursued by combining conventional off-line sample enrichment with high-performance liquid chromatography-solid phase extraction-nuclear magnetic resonance (HPLC-SPE-NMR) based structure elucidation. Off-line reversed phase solid phase extraction (SPE) was used to enrich the desired analytes from a methanolic extract (93 mg dry weight) of a miniscule single tuber (233 mg dry weight) of C. solida. An aliquot of the SPE fraction (2.1 mg) was subjected to separation in the HPLC-SPE-NMR hyphenation. Chromatographic peaks bearing the metabolites under investigation were trapped in the SPE device in a single experiment and transferred to a 600 MHz NMR spectrometer equipped with a 30 microl cryofit insert fed into a 3 mm cryoprobe. Recorded homo- and heteronuclear 1D and 2D NMR data allowed the identification of the three analytes under investigation as protopine, allocryptopine, and N-methyl-laudanidinium acetate. The latter is a rare alkaloid, which has been isolated only once before.
Journal of Chromatography 10/2007; 1163(1-2):138-44. · 4.53 Impact Factor
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ABSTRACT: In the presented study, Central European Corydalis species, namely C. cava, C. intermedia, C. pumila, and C. solida were investigated by nonaqueous capillary electrophoresis-electrospray ion trap mass spectrometry (NACE-ESI-MS) utilizing an electrolyte consisting of 50 mM ammonium acetate, 1 M acetic acid and 10% methanol in acetonitrile, applying a separation voltage of 30 and 20 kV for 1 s for injection. Isopropanol-water (1:1) was used as sheath liquid at a flow-rate of 3 microl/min. Peak assignment was assisted by multistage-ESI-mass spectrometry (ESI-MSn). The optimized method was fully validated (RSD inter- and intraday < 5%, LOD < 2.3 microg/ml, LOQ < 27.6 microg/ml, recovery rates > 98.8%) and subsequently applied for the qualitative and quantitative determination of isoquinoline alkaloids in single plant tubers (sample size < 0.5 g) of the four Central European Corydalis species, each of them showing a characteristic and unique alkaloid pattern. Application of a principal component analysis (PCA) to the complete dataset of 39 analytes and 79 samples allowed the identification of 8 analytes responsible for lot discrimination. Hierarchical cluster analysis and descriptive statistical methods were used to confirm the findings of the explorative PCA.
Journal of Chromatography 08/2007; 1159(1-2):42-50. · 4.53 Impact Factor
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ABSTRACT: Over the past years, metabolic profiling has been established as a comprehensive systems biology tool. Mass spectrometry or NMR spectroscopy-based technology platforms combined with unsupervised or supervised multivariate statistical methodologies allow a deep insight into the complex metabolite patterns of plant-derived samples. Within this review, we provide a thorough introduction to the analytical hard- and software requirements of metabolic profiling platforms. Methodological limitations are addressed, and the metabolic profiling workflow is exemplified by summarizing recent applications ranging from model systems to more applied topics.
Journal of Proteome Research 03/2007; 6(2):480-97. · 5.11 Impact Factor
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ABSTRACT: The absolute configuration of secondary hydroxy functions of seven natural occurring polyyne derivatives has been elucidated by the application of Mosher method of diastereomeric methoxy-2-trifluoromethyl-phenylacetyl (MTPA) ester formation. High-performance liquid chromatography with diode array detection (HPLC-DAD) of the reaction mixture using a water/acetonitrile gradient allowed monitoring the reaction progress. Coupling of high-performance liquid chromatography to solid-phase extraction combined with nuclear magnetic resonance (HPLC-SPE-NMR) was utilized to generate highly reproducible (1)H and (19)F NMR data needed as input for the absolute configuration determination based on the analysis of relative shift differences. Chromatographic peaks of reaction substrates and reaction products bearing less 10mug analyte were trapped on SPE cartridges with the aid of water as makeup solvent. Deuterated chloroform was used to elute and transfer the peak content from the SPE to the 60mul flow cell of a 500MHz NMR spectrometer. For each analyte (1)H NMR spectra were obtained within 15min. Additionally (19)F NMR spectra were recorded for selected analytes in the same timeframe. Based on the obtained NMR data, the absolute configuration of all polyynes under investigation was successfully designated.
Journal of Chromatography 01/2007; 1136(1):82-8. · 4.53 Impact Factor
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ABSTRACT: The analytical assessment of edelweiss (Leontopodium alpinum) herb extracts, used in traditional alpine medicine, has resulted in the development of a HPLC-PAD-MS method that allows baseline separation of almost all constituents. Peak assignment of 14 analytes was achieved by comparison of retention times, UV and mass spectra with those of reference compounds either commercially available (luteolin, apigenin and chlorogenic acid) or isolated from edelweiss plants by column chromatography. Ten of the isolated analytes were identified as the known natural products: quercetin-3-O-beta-D-glucoside, luteolin-7-O-beta-D-glucoside, luteolin-3'-O-beta-D-glucoside, luteolin-4'-O-beta-D-glucoside, apigenin-7-O-beta-D-glucoside, 6-hydroxy-luteolin-7-O-beta-D-glucoside, luteolin-7,4'-di-O-beta-D-glucoside, chrysoeriol-7-O-beta-D-glucoside, leontopodic acid and 3,5-dicaffeolyquinic acid. One analyte, 3,4,5-tri-(E)-caffeoly-D-glucaric acid proved to be a new natural product and was named leontopodic acid B. Structure elucidation was carried out by means of MS and NMR spectroscopy in all cases. The aerial plant parts of L. alpinum (capitula, inflorescence leaves, stems, stem leaves and leaves of the basal rosette) showed variable amounts of the above-mentioned constituents, although qualitative differences were not observable.
Phytochemical Analysis 10/2006; 17(5):291-8. · 2.63 Impact Factor
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ABSTRACT: A novel high yield isolation procedure for lipophilic cyclic peptide derivatives is presented. Destruxin (dtx) A, B, D, E, and E-diol retrieval from Metarhizium anisopliae culture broth was achieved with a three-step purification protocol. After liquid-liquid extraction column chromatography over Sephadex LH-20 served as enrichment step. High-speed counter-current chromatography (HSCCC) was used for the final purification. Within the first chromatographic step dtx D and dtx E-diol were separated in purities exceeding 90%. The separation of dtx A, B, and E was achieved from an enriched Sephadex LH-20 fraction by a HSCCC protocol using light petroleum-ethyl acetate-methanol-water = 2:5:2:5 (v/v) as eluent system. These derivatives were obtained in purities above 98% and total yields exceeding 40%.
Journal of Chromatography 07/2006; 1117(1):67-73. · 4.53 Impact Factor
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ABSTRACT: Looking for acetylcholinesterase (AChE) inhibiting compounds within the plant kingdom, we came across the triterpene alpha-onocerin, which has recently been described as the active principle (IC(50) of 5.2 microM) of Lycopodium clavatum L. In order to discover related terpenoid structures with similar AChE inhibitory activity, we investigated the roots of Ononis spinosa L. using Ellman's reagent in a microplate assay. No inhibitory effect could be measured, not even with the isolated alpha-onocerin (1), which is in contrast to previous findings. Bioassay-guided fractionation of L. clavatum resulted in the isolation of lyclavatol (2), showing a weak, but dose-dependent inhibitory effect on AChE. (1)H- and (13)C NMR shift assignments for 1 and 2 are presented and discussed.
Planta Medica 12/2005; 71(11):1040-3. · 2.15 Impact Factor
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ABSTRACT: A high-performance liquid chromatography-diode array detection (HPLC-DAD) assay is described for the detection and quantification of the secreted Beauveria brongniartii metabolite oosporein from potato tubers. Analyte recovery was achieved with a Britton-Robinson buffer system at pH 5.5 diluted with methanol 3:7 (v/v) (BR5.5-MeOH). An internal standard protocol using 2-iodobenzoic acid was established to minimize analytical error. The resulting assay, using a binary solvent gradient with acidic modifiers and detecting the metabolite at 287 nm, showed a limit of detection (LOD) of 2.4 mg oosporein/kg potato tubers. The oosporein content of potato tuber samples obtained from a field trial using the biological pest control B. brongniartii formulation Melocont-Pilzgerste in up to five-fold higher doses (250 kg Melocont-Pilzgerste/ha) as recommended per year was found to be below the established LOD.
Journal of Chromatography 11/2005; 1092(2):254-7. · 4.53 Impact Factor
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ABSTRACT: 1H and 13C NMR signal assignments derived from 2D NMR experiment based correlations are presented for 22-deoxocucurbitacin D and cucurbitacin D. Both derivatives have been isolated from Ecballium elaterium L. (Cucurbitaceae).
Monatshefte fuer Chemie/Chemical Monthly 08/2005; 136(9):1645-1649. · 1.53 Impact Factor
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ABSTRACT: 2D NMR-derived 1H and 13C NMR signal assignments of six structurally closely related cucurbitacin derivatives are presented. The investigated 2-O-beta-D-glucopyranosylcucurbitacins I, J, K, and L were obtained from Citrullus colocynthis (L.) Schrader whereas the aglyca cucurbitacin E and I were isolated from Ecballium elaterium L.
Magnetic Resonance in Chemistry 07/2005; 43(6):489-91. · 1.44 Impact Factor
