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  • 1.
    Elmsjö, Albert
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science.
    Haglöf, Jakob
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science.
    Engskog, Mikael K R
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science.
    Erngren, Ida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science.
    Nestor, Marika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Arvidsson, Torbjörn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science.
    Pettersson, Curt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science.
    Method selectivity evaluation using the co-feature ratio in LC/MS metabolomics: Comparison of HILIC stationary phase performance for the analysis of plasma, urine and cell extracts.2018In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1568, p. 49-56Article in journal (Refereed)
    Abstract [en]

    Evaluation of the chromatographic separation in metabolomics studies has primarily been done using preselected sets of standards or by counting the number of detected features. An alternative approach is to calculate each feature's co-feature ratio, which is a combined selectivity measurement for the separation (i.e. extent of co-elution) and the MS-signal (i.e. adduct formation and in-source fragmentation). The aim of this study was to demonstrate how the selectivity of different HILIC stationary phases can be evaluated using the co-feature ratio approach. The study was based on three sample types; plasma, urine and cell extracts. Samples were analyzed on an UHPLC-ESI-Q-ToF system using an amide, a bare silica and a sulfobetaine stationary phase. For each feature, a co-feature ratio was calculated and used for multivariate analysis of the selectivity differences between the three stationary phases. Unsupervised PCA models indicated that the co-feature ratios were highly dependent on type of stationary phase. For several metabolites a 15-30 fold difference in the co-feature ratio were observed between the stationary phases. Observed selectivity differences related primarily to the retention patterns of unwanted matrix components such as inorganic salts (detected as salt clusters), glycerophospholipids, and polyethylene glycols. These matrix components affected the signal intensity of co-eluting metabolites by interfering with the ionization efficiency and/or their adduct formation. Furthermore, the retention pattern of these matrix components had huge influence on the number of detected features. The co-feature ratio approach has successfully been applied for evaluation of the selectivity performance of three HILIC stationary phases. The co-feature ratio could therefore be used in metabolomics for developing selective methods fit for their purpose, thereby avoiding generic analytical approaches, which are often biased, as type and amount of interfering matrix components are metabolome dependent.

  • 2.
    Garg, Neeraj
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Chemical Biology for Biomarker Discovery. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hansson, Annelie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science. Nat Vet Inst SVA, Dept Chem Environm & Feed Hyg, SE-75189 Uppsala, Sweden..
    Knych, Heather K.
    Univ Calif Davis, Sch Vet Med, KL Maddy Equine Analyt Chem Lab, Davis, CA 95616 USA.;Univ Calif Davis, Sch Vet Med, Dept Vet Mol Biosci, Davis, CA 95616 USA..
    Stanley, Scott D.
    Univ Calif Davis, Sch Vet Med, KL Maddy Equine Analyt Chem Lab, Davis, CA 95616 USA.;Univ Calif Davis, Sch Vet Med, Dept Vet Mol Biosci, Davis, CA 95616 USA..
    Thevis, Mario
    German Sport Univ Cologne, Inst Biochem, D-50933 Cologne, Germany.;German Sport Univ Cologne, Ctr Prevent Doping Res, D-50933 Cologne, Germany..
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science. Nat Vet Inst SVA, Dept Chem Environm & Feed Hyg, SE-75189 Uppsala, Sweden..
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science. Nat Vet Inst SVA, Dept Chem Environm & Feed Hyg, SE-75189 Uppsala, Sweden..
    Globisch, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Chemical Biology for Biomarker Discovery. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Structural elucidation of major selective androgen receptor modulator (SARM) metabolites for doping control2018In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 16, no 5, p. 698-702Article in journal (Refereed)
    Abstract [en]

    Selective androgen receptor modulators (SARMs) are a class of androgen receptor drugs, which have a high potential to be performance enhancers in human and animal sports. Arylpropionamides are one of the major SARM classes and get rapidly metabolized significantly complicating simple detection of misconduct in blood or urine sample analysis. Specific drug-derived metabolites are required as references due to a short half-life of the parent compound but are generally lacking. The difficulty in metabolism studies is the determination of the correct regio and stereoselectivity during metabolic conversion processes. In this study, we have elucidated and verified the chemical structure of two major equine arylpropionamide-based SARM metabolites using a combination of chemical synthesis and liquid chromatography- mass spectrometry (LC-MS) analysis. These synthesized SARM-derived metabolites can readily be utilized as reference standards for routine mass spectrometry-based doping control analysis of at least three commonly used performance-enhancing drugs to unambigously identify misconduct.

  • 3.
    Haglind, Alfred
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science.
    Determination of the Environmental Fate of Drug Substances and the Matrix Effects of Complex Samples in SFC/ESI-MS2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Awareness of the potential problems caused by drug compounds in the environment has increased over the last decade, both among researchers and with the public. This thesis describes the development of analytical methods and their application to wetlands constructed for purification of wastewater from e.g. drug compounds. Different wetlands were investigated using microcosm-models, to determine their biodegradation. An enantioselective and sensitive SFC/ESI-QqQ method was developed and validated for the enantiomeric separation of atenolol, metoprolol, propranolol and metoprolol acid. It was applied measuring the enantiomeric fraction of the compounds in three different microcosm-models. The same microcosms were also used to investigate the transformation products formed in these wetlands. In this work, LC/ESI-QToF was used to identify the transformation products using standard references, the original compounds or analogs, comparing their accurate mass and product ions. One not previously observed major transformation product identified from propranolol were 1-naphthol. Several minor transformation products were also identified, showing how diverse the formation might be in wetlands.

    A second part compares the matrix effect of ESI/MS using SFC and reversed phase LC, utilizing general screening methods for drug compounds in plasma, horse urine and influent/effluent wastewater. These matrices are known to suffer from matrix effects when using the ESI-source, and if SFC would suffer less than LC it could be a great benefit. The matrix profiles showed that this is likely not the case: although SFC was affected by different interferences then LC. One example is the formation of clusters causing major ion suppression. This unique SFC-phenomenon was investigated further, showing that metal ions were separated and eluted at different retention times, forming clusters in the ion source between metal ions and the organic modifier and/or make-up solvent.

    In conclusion, the first part of this thesis describes analytical methods for determination of drug compounds in the environment, using LC and SFC, connected to both high and low resolving MS. The second part focuses on fundamental analytical chemistry, comparing the matrix effects of SFC/ESI-MS with LC/ESI-MS, and investigates the cluster phenomena observed for samples containing alkali ions and an organic modifier in the mobile phase in SFC/ESI-MS.

    List of papers
    1. Rapid chiral separation of atenolol, metoprolol, propranolol and the zwitterionic metoprolol acid using supercritical fluid chromatography-tandem mass spectrometry - Application to wetland microcosms
    Open this publication in new window or tab >>Rapid chiral separation of atenolol, metoprolol, propranolol and the zwitterionic metoprolol acid using supercritical fluid chromatography-tandem mass spectrometry - Application to wetland microcosms
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    2015 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1409, p. 251-258Article in journal (Refereed) Published
    Abstract [en]

    A method for enantiomeric separation of the three beta-blocking agents atenolol, metoprolol, propranolol and the zwitterionic metoprolol acid, a major metabolite of both metoprolol and in environmental matrices also atenolol, has been developed. By use of supercritical fluid chromatography and the polysaccharide-based Chiralpak (R) IB-3, all four compounds were simultaneously enantiomerically separated (R-s >1.5) within 8 min. Detection was performed using tandem mass spectrometry, and to avoid isobaric interference between the co-eluting metoprolol and metoprolol acid, the achiral column Acquity (R) UPC2 BEH 2-EP was attached ahead of to the chiral column. Carbon dioxide with 18% methanol containing 0.5% (v/v) of the additives trifluoroacetic acid and ammonia in a 2:1 molar ratio were used as mobile phase. A post column make-up flow (0.3 mL/min) of methanol containing 0.1% (v/v) formic acid was used to enhance the positive electrospray ionization. Detection was carried out using a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode, using one transition per analyte and internal standard. The method was successfully applied for monitoring the enantiomeric fraction change over time in a laboratory scale wetland degradation study. It showed good precision, recovery, sensitivity and low effect of the sample matrix.

    Keywords
    Enantiomeric separation, SFC-MS, Metoprolol acid, Wetland microcosm, Chiralpak IB-3, UPC2
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-262417 (URN)10.1016/j.chroma.2015.07.075 (DOI)000359882600029 ()26228849 (PubMedID)
    Available from: 2015-09-18 Created: 2015-09-15 Last updated: 2018-03-15Bibliographically approved
    2. Identification of transformation products from -blocking agents formed in wetland microcosms using LC-Q-ToF
    Open this publication in new window or tab >>Identification of transformation products from -blocking agents formed in wetland microcosms using LC-Q-ToF
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    2016 (English)In: Journal of Mass Spectrometry, ISSN 1076-5174, E-ISSN 1096-9888, Vol. 51, no 3, p. 207-218Article in journal (Refereed) Published
    Abstract [en]

    Identification of degradation products from trace organic compounds, which may retain the biological activity of the parent compound, is an important step in understanding the long-term effects of these compounds on the environment. Constructed wetlands have been successfully utilized to remove contaminants from wastewater effluent, including pharmacologically active compounds. However, relatively little is known about the transformation products formed during wetland treatment. In this study, three different wetland microcosm treatments were used to determine the biotransformation products of the -adrenoreceptor antagonists atenolol, metoprolol and propranolol. LC/ESI-Q-ToF run in the MSE and MS/MS modes was used to identify and characterize the degradation products through the accurate masses of precursor and product ions. The results were compared with those of a reference standard when available. Several compounds not previously described as biotransformation products produced in wetlands were identified, including propranolol-O-sulfate, 1-naphthol and the human metabolite N-deaminated metoprolol. Transformation pathways were significantly affected by microcosm conditions and differed between compounds, despite the compounds' structural similarities. Altogether, a diverse range of transformation products in wetland microcosms were identified and elucidated using high resolving MS. This work shows that transformation products are not always easily predicted, nor formed via the same pathways even for structurally similar compounds.

    Keywords
    HRMS, wetland microcosms, transformation products, identification, LC-QToF, metoprolol, atenolol, propranolol
    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
    Identifiers
    urn:nbn:se:uu:diva-283784 (URN)10.1002/jms.3737 (DOI)000372280300003 ()26956388 (PubMedID)
    Available from: 2016-04-14 Created: 2016-04-14 Last updated: 2018-03-15Bibliographically approved
    3. The differences in matrix effect between supercritical fluid chromatography and reversed phase liquid chromatography coupled to ESI/MS
    Open this publication in new window or tab >>The differences in matrix effect between supercritical fluid chromatography and reversed phase liquid chromatography coupled to ESI/MS
    2018 (English)In: Analytica Chimica Acta, ISSN 0003-2670, E-ISSN 1873-4324, Vol. 1000, p. 163-171Article in journal (Refereed) Published
    Abstract [en]

    For many sample matrices, matrix effects are a troublesome phenomenon using the electrospray ionization source. The increasing use of supercritical fluid chromatography with CO2 in combination with the electrospray ionization source for MS detection is therefore raising questions: is the matrix effect behaving differently using SFC in comparison with reversed phase LC? This was investigated using urine, plasma, influent-and effluent-wastewater as sample matrices. The matrix effect was evaluated using the post-extraction addition method and through post-column infusions. Matrix effect profiles generated from the post-column infusions in combination with time of flight-MS detection provided the most valuable information for the study. The combination of both qualitative and semi-quantitative information with the ability to use HRMS-data for identifying interfering compounds from the same experiment was very useful, and has to the authors' knowledge not been used this way before. The results showed that both LC and SFC are affected by matrix effects, however differently depending on sample matrix. Generally, both suppressions and enhancements were seen, with a higher amount of enhancements for LC, where 65% of all compounds and all sample matrices were enhanced, compared to only 7% for SFC. Several interferences were tentatively identified, with phospholipids, creatinine, and metal ion clusters as examples of important interferences, with different impact depending on chromatographic technique. SFC needs a different strategy for limiting matrix interferences, owing to its almost reverse retention order compared to RPLC.

    Place, publisher, year, edition, pages
    ELSEVIER SCIENCE BV, 2018
    Keywords
    Matrix effects, Supercritical fluid chromatography, Electrospray ionization, Liquid chromatography, Ion enhancement, Ion suppression
    National Category
    Analytical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-338947 (URN)10.1016/j.aca.2017.10.014 (DOI)000418832900015 ()29289305 (PubMedID)
    Available from: 2018-01-25 Created: 2018-01-25 Last updated: 2018-03-15Bibliographically approved
    4. Major signal suppression from metal ion clusters in SFC/ESI-MS: Cause and Effects
    Open this publication in new window or tab >>Major signal suppression from metal ion clusters in SFC/ESI-MS: Cause and Effects
    2018 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 1084, p. 96-105Article in journal (Refereed) Published
    Abstract [en]

    The widening application area of SFC-MS with polar analytes and water-containing samples facilitates the use of quick and simple sample preparation techniques such as “dilute and shoot” and protein precipitation. This has also introduced new polar interfering components such as alkali metal ions naturally abundant in e.g. blood plasma and urine, which have shown to be retained using screening conditions in SFC/ESI-TOF-MS and causing areas of major ion suppression. Analytes co-eluting with these clusters will have a decreased signal intensity, which might have a major effect on both quantification and identification. When investigating the composition of the alkali metal clusters using accurate mass and isotopic pattern, it could be concluded that they were previously not described in the literature. Using NaCl and KCl standards and different chromatographic conditions, varying e.g. column and modifier, the clusters proved to be formed from the alkali metal ions in combination with the alcohol modifier and make-up solvent. Their compositions were [(XOCH3)n+X]+, [(XOH)n+X]+, [(X2CO3)n+X]+ and [(XOOCOCH3)n+X]+ for X= Na+ or K+ in ESI+. In ESI-, the clusters depended more on modifier, with [(XCl)n+Cl]- and [(XOCH3)n+OCH3]- mainly formed in pure methanol and [(XOOCH)n+OOCH]- when 20 mM NH4Fa was added.

    To prevent the formation of the clusters by avoiding methanol as modifier might be difficult, as this is a widely used modifier providing good solubility when analyzing polar compounds in SFC. A sample preparation with e.g. LLE would remove the alkali ions, however also introducing a time consuming and discriminating step into the method. Since the alkali metal ions were retained and affected by chromatographic adjustments as e.g. mobile phase modifications, a way to avoid them could therefore be chromatographic tuning, when analyzing samples containing them.

    Keywords
    SFC-MS, matrix effect, alkali metal, ion cluster, Supercritical fluid chromatography, ESI
    National Category
    Analytical Chemistry
    Research subject
    Analytical Pharmaceutical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-345978 (URN)10.1016/j.jchromb.2018.03.024 (DOI)000430524400012 ()29579734 (PubMedID)
    Available from: 2018-03-13 Created: 2018-03-13 Last updated: 2018-06-26Bibliographically approved
  • 4.
    Hansson, Annelie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science.
    Structural Determination of Drug Metabolites from Doping Classed Compounds Using Mass Spectrometry2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Doping control in equine sports is important for a fair competition, but also to ensure the integrity of the betting system, as well as for animal welfare reasons. To detect the use of illicit compounds, screening for the parent compound is common. However, by using a metabolite as the analytical target instead, the detection time can be prolonged. For some compounds, the use of a metabolite is a necessity since the parent drug may not be detected at all.

    The metabolites of the selective androgen receptor modulators (SARM) S1, S4 and S22 were investigated in horse urine and plasma. The unchanged parent compounds had the longest detection time in plasma, but were not detected at all in urine. Instead, the longest detection time was measured for the metabolites 2-amino-5-nitro-4-(trifluoromethyl)phenyl hydrogen sulfate (SARMs S1 and S4) and 2-amino-5-cyano-4-(trifluoromethyl)phenyl hydrogen sulfate (SARM S22). These metabolites were thus suggested as analytical targets for doping control in urine while the parent compounds were suggested for plasma samples. 2-amino-5-nitro-4-(trifluoromethyl)phenyl hydrogen sulfate could also be produced in large quantities by the fungus Cunninghamella elegans to potentially be used as reference compound.

    The horse metabolites of the SARM LGD-4033 were also studied in urine and plasma. The formate adduct of LGD-4033 had the longest detection time in plasma and in urine after hydrolysis with β-glucuronidase. In non-hydrolyzed urine, the glucuronidated LGD-4033 was detected instead.

    Different in vitro models were used to predict in vivo metabolites of roxadustat, a hypoxia-inducible factor stabilizer. Cunninghamella elegans was successful in producing more metabolites compared to human and equine liver microsomes and human hepatocytes.

    The metabolite detection and identification in all experiments were accomplished using a UHPLC-Q-TOF MS instrument, where the high-resolution MS data was vital in determining which metabolites were formed.

    The thesis shows the benefits of investigating the metabolites of doping substances to allow for a successful doping control method in horse urine and plasma by prolonging the detection time. It also highlights the usefulness of Cunninghamella elegans as an alternative to the more commonly used in vitro models for both predicting and producing metabolites.

    List of papers
    1. Characterization of equine urinary metabolites of selective androgen receptor modulators (SARMs) S1, S4 and S22 for doping control purposes
    Open this publication in new window or tab >>Characterization of equine urinary metabolites of selective androgen receptor modulators (SARMs) S1, S4 and S22 for doping control purposes
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    2015 (English)In: Drug Testing and Analysis, ISSN 1942-7603, E-ISSN 1942-7611, Vol. 7, no 8, p. 673-683Article in journal (Refereed) Published
    Abstract [en]

    Selective androgen receptor modulators, SARMs, constitute a class of compounds with anabolic properties but with few androgenic side-effects. This makes them possible substances of abuse and the World Anti-Doping Agency (WADA) has banned the entire class of substances. There have been several cases of illicit use of aryl propionamide SARMs in human sports and in 2013, 13 cases were reported. These substances have been found to be extensively metabolized in humans, making detection of metabolites necessary for doping control. SARMs are also of great interest to equine doping control, but the in vivo metabolite pattern and thus possible analytical targets have not been previously studied in this species. In this study, the urinary metabolites of the SARMs S1, S4, and S22 in horses were studied after intravenous injection, using ultra high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QToF-MS). Eight different metabolites were found for SARM S1, nine for SARM S4, and seven for SARM S22. The equine urinary metabolite profiles differed significantly from those of humans. The parent compounds were only detected for SARMs S4 and S22 and only at the first sampling time point at 3h post administration, making them unsuitable as target compounds. For all three SARMs tested, the metabolite yielding the highest response had undergone amide hydrolysis, hydroxylation and sulfonation. The resulting phase II metabolites (4-nitro-3-trifluoro-methyl-phenylamine sulfate for SARMs S1 and S4 and 4-cyano-3-trifluoro-methyl-phenylamine sulfate for SARM S22) are proposed as analytical targets for use in equine doping control.

    Keywords
    selective androgen receptor modulators, SARM, metabolite, equine, horse
    National Category
    Medicinal Chemistry
    Identifiers
    urn:nbn:se:uu:diva-261969 (URN)10.1002/dta.1768 (DOI)000359603700003 ()25560998 (PubMedID)
    Available from: 2015-09-08 Created: 2015-09-07 Last updated: 2018-03-14Bibliographically approved
    2. Investigation of the selective androgen receptor modulators S1, S4 and S22 and their metabolites in equine plasma using high-resolution mass spectrometry
    Open this publication in new window or tab >>Investigation of the selective androgen receptor modulators S1, S4 and S22 and their metabolites in equine plasma using high-resolution mass spectrometry
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    2016 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 30, no 7, p. 833-842Article in journal (Refereed) Published
    Abstract [en]

    RationaleSelective androgen receptor modulators (SARMs) are prohibited in sports due to their performance enhancing ability. It is important to investigate the metabolism to determine appropriate targets for doping control. This is the first study where the equine metabolites of SARMs S1, S4 (Andarine) and S22 (Ostarine) have been studied in plasma. MethodsEach SARM was administered to three horses as an intravenous bolus dose and plasma samples were collected. The samples were pretreated with protein precipitation using cold acetonitrile before separation by liquid chromatography. The mass spectrometric analysis was performed using negative electrospray, quadrupole time-of-flight mass spectrometry operated in MSE mode and triple-quadrupole mass spectrometry operated in selected reaction monitoring mode. For the quantification of SARM S1, a deuterated analogue was used as internal standard. ResultsThe numbers of observed metabolites were eight, nine and four for the SARMs S1, S4 and S22, respectively. The major metabolite was formed by the same metabolic reactions for all three SARMs, namely amide hydrolysis, hydroxylation and sulfonation. The values of the determined maximum plasma concentrations were in the range of 97-170 ng/mL for SARM S1, 95-115 ng/mL for SARM S4 and 92-147 ng/mL for SARM S22 and the compounds could be detected for 96 h, 12 h and 18 h, respectively. ConclusionsThe maximum plasma concentration of SARMs S1, S4 and S22 was measured in the first sample (5 min) after administration and they were eliminated fast from plasma. The proposed targets to be used in equine doping control are the parent compounds for all three SARMs, but with the metabolite yielding the highest response as a complementary target. 

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-286622 (URN)10.1002/rcm.7512 (DOI)000372508100006 ()26969924 (PubMedID)
    Available from: 2016-04-28 Created: 2016-04-21 Last updated: 2018-03-14Bibliographically approved
    3. Equine in vivo-derived metabolites of the SARM LGD-4033 and comparison with human and fungal metabolites.
    Open this publication in new window or tab >>Equine in vivo-derived metabolites of the SARM LGD-4033 and comparison with human and fungal metabolites.
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    2018 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 1074-1075, p. 91-98Article in journal (Refereed) Published
    Abstract [en]

    LGD-4033 has been found in human doping control samples and has the potential for illicit use in racehorses as well. It belongs to the pharmacological class of selective androgen receptor modulators (SARMs) and can stimulate muscle growth, much like anabolic steroids. However, SARMs have shown superior side effect profiles compared to anabolic steroids, which arguably makes them attractive for use by individuals seeking an unfair advantage over their competitors. The purpose of this study was to investigate the metabolites formed from LGD-4033 in the horse in order to find suitable analytical targets for doping controls. LGD-4033 was administered to three horses after which plasma and urine samples were collected and analyzed for metabolites using ultra high performance liquid chromatography coupled to a high resolution mass spectrometer. In horse urine, eight metabolites, both phase I and phase II, were observed most of which had not been described in other metabolic systems. Six of these were also detected in plasma. The parent compound was detected in plasma, but not in non-hydrolyzed urine. The longest detection times were observed for unchanged LGD-4033 in plasma and in urine hydrolyzed with β-glucuronidase and is thus suggested as the analytical target for doping control in the horse. The metabolite profile determined in the horse samples was also compared to those of human urine and fungal incubate from Cunninghamella elegans. The main human metabolite, dihydroxylated LGD-4033, was detected in the horse samples and was also produced by the fungus. However, it was a not a major metabolite for horse and fungus, which highlights the importance of performing metabolism studies in the species of interest.

    Keywords
    Doping, LGD-4033, Horse, Mass Spectrometry, Metabolite, SARM, Selective Androgen Receptor Modulator
    National Category
    Medicinal Chemistry
    Identifiers
    urn:nbn:se:uu:diva-344303 (URN)10.1016/j.jchromb.2017.12.010 (DOI)000425204900013 ()29334634 (PubMedID)
    Available from: 2018-03-06 Created: 2018-03-06 Last updated: 2018-05-07Bibliographically approved
    4. Investigation of the metabolites of the HIF stabilizer FG-4592 (roxadustat) in five different in vitro models and in a human doping control sample using high resolution mass spectrometry
    Open this publication in new window or tab >>Investigation of the metabolites of the HIF stabilizer FG-4592 (roxadustat) in five different in vitro models and in a human doping control sample using high resolution mass spectrometry
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    2017 (English)In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 134, p. 228-236Article in journal (Refereed) Published
    Abstract [en]

    FG-4592 is a hypoxia-inducible factor (HIF) stabilizer, which can increase the number of red blood cells in the body. It has not been approved by regulatory authorities, but is available for purchase on the Internet. Due to its ability to improve the oxygen transportation mechanism in the body, FG-4592 is of interest for doping control laboratories, but prior to this study, little information about its metabolism was available. In this study, the metabolism of FG-4592 was investigated in a human doping control sample and in five in vitro models: human hepatocytes and liver microsomes, equine liver microsomes and S9 fraction and the fungus Cunninghamella elegans. By using liquid chromatography coupled to a Q-TOF mass spectrometer operated in MSE and MSMS modes, twelve different metabolites were observed for FG-4592. One monohydroxylated metabolite was detected in both the human and equine liver microsome incubations. For the fungus Cunninghamella elegans eleven different metabolites were observed of which the identical monohydroxylated metabolite had the highest response. This rich metabolic profile and the higher levels of metabolites produced by Cunninghamella elegans demonstrates its usefulness as a metabolite producing medium. In the doping control urine sample, one metabolite, which was the result of a direct glucuronidation, was observed. No metabolites were detected in neither the human hepatocyte nor in the equine liver S9 fraction incubates.

    Keywords
    FG-4592, Drug metabolism, High resolution mass spectrometry
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-317588 (URN)10.1016/j.jpba.2016.11.041 (DOI)000392909900029 ()27918992 (PubMedID)
    Available from: 2017-03-24 Created: 2017-03-24 Last updated: 2018-03-14Bibliographically approved
  • 5.
    Hansson, Annelie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science.
    Knych, Heather
    Stanley, Scott
    Berndtson, Emma
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science. Natl Vet Inst SVA, Dept Chem Environm & Feed Hyg, SE-75651 Uppsala, Sweden.
    Jackson, Liora
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science. Natl Vet Inst SVA, Dept Chem Environm & Feed Hyg, SE-75651 Uppsala, Sweden.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science. Natl Vet Inst SVA, Dept Chem Environm & Feed Hyg, SE-75651 Uppsala, Sweden.
    Thevis, Mario
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science. Natl Vet Inst SVA, Dept Chem Environm & Feed Hyg, SE-75651 Uppsala, Sweden.
    Equine in vivo-derived metabolites of the SARM LGD-4033 and comparison with human and fungal metabolites.2018In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 1074-1075, p. 91-98Article in journal (Refereed)
    Abstract [en]

    LGD-4033 has been found in human doping control samples and has the potential for illicit use in racehorses as well. It belongs to the pharmacological class of selective androgen receptor modulators (SARMs) and can stimulate muscle growth, much like anabolic steroids. However, SARMs have shown superior side effect profiles compared to anabolic steroids, which arguably makes them attractive for use by individuals seeking an unfair advantage over their competitors. The purpose of this study was to investigate the metabolites formed from LGD-4033 in the horse in order to find suitable analytical targets for doping controls. LGD-4033 was administered to three horses after which plasma and urine samples were collected and analyzed for metabolites using ultra high performance liquid chromatography coupled to a high resolution mass spectrometer. In horse urine, eight metabolites, both phase I and phase II, were observed most of which had not been described in other metabolic systems. Six of these were also detected in plasma. The parent compound was detected in plasma, but not in non-hydrolyzed urine. The longest detection times were observed for unchanged LGD-4033 in plasma and in urine hydrolyzed with β-glucuronidase and is thus suggested as the analytical target for doping control in the horse. The metabolite profile determined in the horse samples was also compared to those of human urine and fungal incubate from Cunninghamella elegans. The main human metabolite, dihydroxylated LGD-4033, was detected in the horse samples and was also produced by the fungus. However, it was a not a major metabolite for horse and fungus, which highlights the importance of performing metabolism studies in the species of interest.

  • 6.
    Henrohn, Dan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Björkstrand, Kristoffer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Lundberg, Jon O
    Granstam, Sven-Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Baron, Tomasz
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Ingimarsdóttir, Inga J
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Hedenström, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Malinovschi, Andrei
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Wernroth, Mona-Lisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Jansson, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science.
    Wikström, Gerhard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Effects of Oral Supplementation With Nitrate-Rich Beetroot Juice in Patients With Pulmonary Arterial Hypertension-Results From BEET-PAH, an Exploratory Randomized, Double-Blind, Placebo-Controlled, Crossover Study.2018In: Journal of Cardiac Failure, ISSN 1071-9164, E-ISSN 1532-8414, Vol. 24, no 10, p. 640-653, article id S1071-9164(18)31003-0Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: The nitrate-nitrite-nitric oxide (NO) pathway may represent a potential therapeutic target in patients with pulmonary arterial hypertension (PAH). We explored the effects of dietary nitrate supplementation, with the use of nitrate-rich beetroot juice (BRJ), in patients with PAH.

    METHODS AND RESULTS: We prospectively studied 15 patients with PAH in an exploratory randomized, double-blind, placebo-controlled, crossover trial. The patients received nitrate-rich beetroot juice (∼16 mmol nitrate per day) and placebo in 2 treatment periods of 7 days each. The assessments included; exhaled NO and NO flow-independent parameters (alveolar NO and bronchial NO flux), plasma and salivary nitrate and nitrite, biomarkers and metabolites of the NO-system, N-terminal pro-B-type natriuretic peptide, echocardiography, ergospirometry, diffusing capacity of the lung for carbon monoxide, and the 6-minute walk test. Compared with placebo ingestion of BRJ resulted in increases in; fractional exhaled NO at all flow-rates, alveolar NO concentrations and bronchial NO flux, and plasma and salivary levels of nitrate and nitrite. Plasma ornithine levels decreased and indices of relative arginine availability increased after BRJ compared to placebo. A decrease in breathing frequency was observed during ergospirometry after BRJ. A tendency for an improvement in right ventricular function was observed after ingestion of BRJ. In addition a tendency for an increase in the peak power output to peak oxygen consumption ratio (W peak/VO2 peak) was observed, which became significant in patients reaching an increase of plasma nitrite >30% (responders).

    CONCLUSIONS: BRJ administered for 1 week increases pulmonary NO production and the relative arginine bioavailability in patients with PAH, compared with placebo. An increase in the W peak/VO2 peak ratio was observed after BRJ ingestion in plasma nitrite responders. These findings indicate that supplementation with inorganic nitrate increase NO synthase-independent NO production from the nitrate-nitrite-NO pathway.

  • 7.
    Lundgren, Jakob
    et al.
    Lund Univ, Dept Clin Sci Lund, Cardiol, Lund, Sweden; Skåne Univ Hosp, Sect Heart Failure & Valvular Dis, Hemodynam Lab, Heart & Lung Clin, Lund, Sweden.
    Sandqvist, Anna
    Umeå Univ, Dept Pharmacol & Clin Neurosci, Clin Pharmacol, Umeå, Sweden.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science. Natl Vet Inst (SVA), Dept Chem Environm & Feed Hyg, Uppsala, Sweden.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science. Natl Vet Inst (SVA), Dept Chem Environm & Feed Hyg, Uppsala, Sweden.
    Wikström, Gerhard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cardiology.
    Rådegran, Göran
    Lund Univ, Dept Clin Sci Lund, Cardiol, Lund, Sweden; Skåne Univ Hosp, Sect Heart Failure & Valvular Dis, Hemodynam Lab, Heart & Lung Clin, Lund, Sweden.
    Alterations in plasma L-arginine and methylarginines in heart failure and after heart transplantation2018In: Scandinavian Cardiovascular Journal, ISSN 1401-7431, E-ISSN 1651-2006, Vol. 52, no 4, p. 196-204Article in journal (Refereed)
    Abstract [en]

    Objective: Endothelial function, including the nitric oxide (NO)-pathway, has previously been extensively investigated in heart failure (HF). In contrast, studies are lacking on the NO pathway after heart transplantation (HT). We therefore investigated substances in the NO pathway prior to and after HT in relation to hemodynamic parameters.

    Design: 12 patients (median age 50.0 yrs, 2 females), heart transplanted between June 2012 and February 2014, evaluated at our hemodynamic lab, at rest, prior to HT, as well as four weeks and six months after HT were included. All patients had normal left ventricular function post-operatively and none had post-operative pulmonary hypertension or acute cellular rejection requiring therapy at the evaluations. Plasma concentrations of ADMA, SDMA, L-Arginine, L-Ornithine and L-Citrulline were analyzed at each evaluation.

    Results: In comparison to controls, the plasma L-Arginine concentration was low and ADMA high in HF patients, resulting in low L-Arginine/ADMA-ratio pre-HT. Already four weeks after HT L-Arginine was normalized whereas ADMA remained high. Consequently the L-Arginine/ADMA-ratio improved, but did not normalize. The biomarkers remained unchanged at the six-month evaluation and the L-Arginine/ADMA-ratio correlated inversely to pulmonary vascular resistance (PVR) six months post-HT.

    Conclusions: Plasma L-Arginine concentrations normalize after HT. However, as ADMA is unchanged, the L-Arginine/ADMA-ratio remained low and correlated inversely to PVR. Together these findings suggest that (i) the L-Arginine/ADMA-ratio may be an indicator of pulmonary vascular tone after HT, and that (ii) NO-dependent endothelial function is partly restored after HT. Considering the good postoperative outcome, the biomarker levels may be considered “normal” after HT.

  • 8. Paul, Prasanta
    et al.
    Sänger - van de Griend, Cari
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science. Kantisto BV, Calenburglaan, Netherlands.
    Adams, Erwin
    Van Schepdael, Ann
    A simple, low-cost and robust capillary zone electrophoresis method with capacitively coupled contactless conductivity detection for the routine determination of four selected penicillins in money-constrained laboratories.2018In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 39, no 20, p. 2521-2529Article in journal (Refereed)
    Abstract [en]

    A simple and robust capillary zone electrophoresis method was developed and validated for the determination of amoxicillin and clavulanate, ampicillin, phenoxymethyl penicillin (Pen V) as well as flucloxacillin. Capacitively coupled contactless conductivity detection was employed as detection mode that makes CE a simple and economic tool for money-constrained laboratories. The developed method is straightforward and user-friendly. It offers good sensitivity and sufficient selectivity for the routine assay of the selected penicillins. The repeatabilities were <1.9% RSD for relative peak areas and <1% RSD for migration times for all the analytes. The method showed good linearity (R2  > 0.995) within the 80-120% range of the target concentration (0.5 mg/mL) for each antibiotic. The accuracy of the method, evaluated by standard fortification at three levels, was good for all the analytes. An extended robustness study was performed by varying ±10% of the optimum value of TRIS concentration, l-histidine concentration and temperature in a full factorial design at two levels. This was to evaluate larger than usual variability of factors on the assay value, in order to better cover potential global variance in lab conditions and equipment. Finally, the method was applied for the determination of percent (%) content of all antibiotics in available formulations.

  • 9.
    Paul, Prasanta
    et al.
    KU Leuven Univ Leuven, Dept Pharmaceut & Pharmacol Sci, Pharmaceut Anal, O&N2, PB 923,Herestr 49, B-3000 Leuven, Belgium..
    Sänger - van de Griend, Cari
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science. Kantisto BV, Callenburglaan 22, NL-3742 MV Baarn, Netherlands..
    Adams, Erwin
    KU Leuven Univ Leuven, Dept Pharmaceut & Pharmacol Sci, Pharmaceut Anal, O&N2, PB 923,Herestr 49, B-3000 Leuven, Belgium..
    Van Schepdael, Ann
    KU Leuven Univ Leuven, Dept Pharmaceut & Pharmacol Sci, Pharmaceut Anal, O&N2, PB 923,Herestr 49, B-3000 Leuven, Belgium..
    Recent advances in the capillary electrophoresis analysis of antibiotics with capacitively coupled contactless conductivity detection2018In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 158, p. 405-415Article, review/survey (Refereed)
    Abstract [en]

    This review describes briefly the high rate of counterfeiting of antimicrobial drugs with focus upon its immediate health consequences. The major part of this review encompasses accounts of the improvements achieved in the domain of miniaturization of capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-(CD)-D-4). The application of this principle into the development of portable devices as well as its application to counter the health-system-crippling phenomenon of counterfeit antibiotic formulations, are discussed in the context of developing countries. 

  • 10.
    Roos, Carl
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Dahlgren, David
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Sjögren, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Sjöblom, Markus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science.
    Lennernäs, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Effects of absorption-modifying excipients on jejunal drug absorption in simulated fasted and fed luminal conditionsIn: European journal of pharmaceutics and biopharmaceutics, ISSN 0939-6411, E-ISSN 1873-3441Article in journal (Refereed)
    Abstract [en]

    The pharmaceutical industry, prescribers, and patients have all traditionally preferred oral administration of drug products. In recent years there has been an increase in drug candidates with low solubility and/or low permeability, which may limit the use of oral administration. To increase the possibility of oral administration for the poorly permeating drugs, the use of absorption modifying excipients (AMEs) has been proposed, with the aim of increasing the fraction of dose absorbed. The effects of AMEs have previously been investigated in various animal models, including the single-pass intestinal perfusion (SPIP) in rats. To further improve the biorelevance and the in vivo predictiveness of the SPIP model, four compounds (atenolol, enalaprilat, ketoprofen, metoprolol) were perfused in fasted or fed state simulated intestinal fluid (FaSSIF or FeSSIF) together with the AMEs N-acetyl-cysteine, caprate, or sodium dodecyl sulphate. For the poorly permeating compounds enalaprilat and atenolol, the flux was increased the most by the addition of SDS in both FaSSIF and FeSSIF. For ketoprofen, the flux decreased in the presence of all AMEs in at least one of the perfusion media. The flux of metoprolol was not affected by any of the excipients. The changes in magnitude in the compounds’ absorptions were in general smaller in FeSSIF than in FaSSIF, possibly due to differences in colloidal structures present in FeSSIF that made the AMEs less available. The results in FeSSIF were similar to those from bolus-dosing in rat, which further suggests that the effect of AMEs on permeability is strongly affected by interactions between AMEs and colloidal structures in the intestinal lumen. The results suggest that, when investigating the effects of AMEs, the biorelevance of the SPIP method can be increased by the addition of intraluminal constituents to the perfusate.

  • 11.
    Roos, Carl
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Dahlgren, David
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Sjögren, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Sjöblom, Markus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science.
    Lennernäs, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Jejunal absorption of aprepitant from nanosuspensions: Role of particle size, prandial state and mucus layer.2018In: European journal of pharmaceutics and biopharmaceutics, ISSN 0939-6411, E-ISSN 1873-3441, Vol. 132, p. 222-230, article id S0939-6411(18)30760-4Article in journal (Refereed)
    Abstract [en]

    The number of highly lipophilic active pharmaceutical ingredients (APIs) in pharmaceutical development has been constantly increasing over recent decades. These APIs often have inherent issues with solubility and dissolution, limiting their oral bioavailability. Traditionally, a reduction in particle size to the micrometer range has been used to improve dissolution. More recently, size reduction to the nanometer range has been introduced, which further increases the dissolution rate, but may also involve other mechanisms for increasing bioavailability. The effect of particle size on the absorption of aprepitant was investigated using the single-pass intestinal perfusion (SPIP) model in the rat jejunum. Phosphate buffer, fasted-state simulated intestinal fluid (FaSSIF), and fed-state simulated intestinal fluid (FeSSIF) were used as perfusion media to increase understanding of the processes involved and the effects of colloidal structures. The role of mucus on intestinal absorption was investigated by adding the mucolytic agent N-acetyl-cysteine (NAC). The absorption of aprepitant from the nanosuspensions was similar with all perfusion media (buffer = FaSSIF = FeSSIF), whereas food had a pronounced effect on absorption from the microsuspensions (FeSSIF > FaSSIF > buffer). The colloidal structures hence contributed to absorption from the microsuspensions. Partitioning of aprepitant from the nanosuspension into the colloidal structures decreased the amount of nanoparticles available, which offset the effect of food. The appearance flux of aprepitant in blood was non-significantly decreased for nanosuspensions of aprepitant with NAC versus without NAC in buffer (ratio of 2:1), indicating that particle deposition in the mucus may have been decreased as the layer thinned, with subsequently reduced intestinal absorption. The study also showed that the SPIP model is suitable for investigating detailed absorption mechanisms using complex perfusion media, which increase the biorelevance of the model.

  • 12.
    Sandqvist, Anna
    et al.
    Umea Univ, Clin Pharmacol, Dept Pharmacol & Clin Neurosci, S-90187 Umea, Sweden..
    Schneede, Jörn
    Umea Univ, Clin Pharmacol, Dept Pharmacol & Clin Neurosci, S-90187 Umea, Sweden..
    Kylhammar, David
    Lund Univ, Dept Clin Sci Lund, Cardiol, Lund, Sweden.;Skane Univ Hosp, Sect Heart Failure & Valvular Dis, Lund, Sweden..
    Henrohn, D
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cardiology.
    Lundgren, Jakob
    Lund Univ, Dept Clin Sci Lund, Cardiol, Lund, Sweden.;Skane Univ Hosp, Sect Heart Failure & Valvular Dis, Lund, Sweden..
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science. Natl Vet Inst SVA, Dept Chem, Uppsala, Sweden..
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science. Natl Vet Inst SVA, Dept Chem, Uppsala, Sweden..
    Rådegran, Göran
    Lund Univ, Dept Clin Sci Lund, Cardiol, Lund, Sweden.;Skane Univ Hosp, Sect Heart Failure & Valvular Dis, Lund, Sweden..
    Wikström, Gerhard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cardiology. Uppsala Univ, Dept Med Sci, Cardiol, Uppsala, Sweden..
    Plasma l-arginine levels distinguish pulmonary arterial hypertension from left ventricular systolic dysfunction2018In: Heart and Vessels, ISSN 0910-8327, E-ISSN 1615-2573, Vol. 33, no 3, p. 255-263Article in journal (Refereed)
    Abstract [en]

    Pulmonary arterial hypertension (PAH) is a life-threatening condition, characterized by an imbalance of vasoactive substances and remodeling of pulmonary vasculature. Nitric oxide, formed from l-arginine, is essential for homeostasis and smooth muscle cell relaxation in PAH. Our aim was to compare plasma concentrations of l-arginine, asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) in PAH compared to left ventricular systolic dysfunction (LVSD) and healthy subjects. This was an observational, multicenter study comparing 21 patients with PAH to 14 patients with LVSD and 27 healthy subjects. Physical examinations were obtained and blood samples were collected. Plasma levels of ADMA, SDMA, l-arginine, l-ornithine, and l-citrulline were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasma levels of ADMA and SDMA were higher, whereas l-arginine and l-arginine/ADMA ratio were lower in PAH patients compared to healthy subjects (p < 0.001). Patients with PAH also had lower levels of l-arginine than patients with LVSD (p < 0.05). l-Arginine correlated to 6 min walking distance (6MWD) (r (s) = 0.58, p = 0.006) and l-arginine/ADMA correlated to WHO functional class (r (s) = -0.46, p = 0.043) in PAH. In conclusion, l-arginine levels were significantly lower in treatment na < ve PAH patients compared to patients with LVSD. Furthermore, l-arginine correlated with 6MWD in PAH. l-arginine may provide useful information in differentiating PAH from LVSD.

  • 13.
    Yao, Han
    et al.
    Univ Ghent, Dept Pharmaceut Anal, Fac Pharmaceut Sci, Drug Qual & Registrat DruQuaR Grp, Ottergemsesteenweg 460, B-9000 Ghent, Belgium.
    Vandenbossche, Jana
    Univ Ghent, Dept Pharmaceut Anal, Fac Pharmaceut Sci, Drug Qual & Registrat DruQuaR Grp, Ottergemsesteenweg 460, B-9000 Ghent, Belgium.
    Sänger - van de Griend, Cari
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Science.
    Janssens, Yorick
    Univ Ghent, Dept Pharmaceut Anal, Fac Pharmaceut Sci, Drug Qual & Registrat DruQuaR Grp, Ottergemsesteenweg 460, B-9000 Ghent, Belgium.
    Fernandez, Cristina Soto
    Univ Ghent, Dept Pharmaceut Anal, Fac Pharmaceut Sci, Drug Qual & Registrat DruQuaR Grp, Ottergemsesteenweg 460, B-9000 Ghent, Belgium.
    Xu, Xiaolong
    Univ Ghent, Dept Pharmaceut Anal, Fac Pharmaceut Sci, Drug Qual & Registrat DruQuaR Grp, Ottergemsesteenweg 460, B-9000 Ghent, Belgium.
    Wynendaele, Evelien
    Univ Ghent, Dept Pharmaceut Anal, Fac Pharmaceut Sci, Drug Qual & Registrat DruQuaR Grp, Ottergemsesteenweg 460, B-9000 Ghent, Belgium.
    Somsen, Govert Willem
    Vrije Univ Amsterdam, AIMMS Res Grp BioMol Anal, Div BioAnalyt Chem, De Boelelaan 1085, NL-1081 HV Amsterdam, Netherlands.
    Haselberg, Rob
    Vrije Univ Amsterdam, AIMMS Res Grp BioMol Anal, Div BioAnalyt Chem, De Boelelaan 1085, NL-1081 HV Amsterdam, Netherlands.
    De Spiegeleer, Bart
    Univ Ghent, Dept Pharmaceut Anal, Fac Pharmaceut Sci, Drug Qual & Registrat DruQuaR Grp, Ottergemsesteenweg 460, B-9000 Ghent, Belgium.
    Development of a capillary zone electrophoresis method to quantify E. coli L-asparaginase and its acidic variants2018In: Talanta: The International Journal of Pure and Applied Analytical Chemistry, ISSN 0039-9140, E-ISSN 1873-3573, Vol. 182, p. 83-91Article in journal (Refereed)
    Abstract [en]

    A capillary zone electrophoresis (CZE) method with UV detection was developed for the quantification of the E.coli L-asparaginase (L-ASNase) and its acidic variants. During the initial method development, a variety of experimental conditions were screened. Subsequently, a Design of Experiments (DoE) was used to optimize the pH and concentration of the selected background electrolyte (BGE) containing both TRIS and boric acid. Optimization was performed taking into account both the separation efficiency of L-ASNase and its acidic variants as well as overall method robustness. A repeatable separation between E.coli L-ASNase and its acidic variants was achieved on a bare fused silica capillary in combination with a BGE consisting of both 400 mM TRIS and boric acid. The method was validated for linearity, accuracy, precision, LOD, LOQ and robustness. The recovery for L-ASNase was 97.9-104.4% with a precision RSD of 1.5-3.2%, while the recovery of impurities was 92.1-109.8% with a RSD of 1.7-4.6%. The quantification limit was 1.9% (m/m). Moreover, the CZE-UV method was applied to determine the degradation rate in the presence of ammonium bicarbonate, confirming the suitability of the method. The degraded, partially charged L-ASNase was evaluated for its in-vitro enzymatic activity showing an insignificant different enzyme activity compared to the unmodified sample.

1 - 13 of 13
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