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  • 201.
    Sjögren, Erik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Halldin, M. M.
    Karolinska Inst, AlzeCure Fdn, Sci Pk, Huddinge, Sweden.
    Stålberg, O.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Sundgren-Andersson, A. K.
    AstraZeneca R&D, Global Med Dev, Gaithersburg, MD 20878 USA.
    Preclinical characterization of three transient receptor potential vanilloid receptor 1 antagonists for early use in human intradermal microdose analgesic studies2018In: European Journal of Pain, ISSN 1090-3801, E-ISSN 1532-2149, Vol. 22, no 5, p. 889-903Article in journal (Refereed)
    Abstract [en]

    Background

    The transient receptor potential vanilloid receptor 1 (TRPV1) is a nonselective cation channel involved in the mediation of peripheral pain to the central nervous system. As such, the TRPV1 is an accessible molecular target that lends itself well to the understanding of nociceptive signalling. This study encompasses preclinical investigations of three molecules with the prospect to establish them as suitable analgesic model compounds in human intradermal pain relief studies.

    Methods

    The inhibitory effectiveness was evaluated by means of invitro assays, TRPV1 expressing Chinese hamster ovary cells (CHO-K1) and rat dorsal root ganglion cultures in fluorescent imaging plate reader and whole cell patch clamp systems, as well as invivo by capsaicin-evoked pain-related behavioural response studies in rat. Secondary pharmacology, pharmacokinetics and preclinical safety were also assessed.

    Results

    In vitro, all three compounds were effective at inhibiting capsaicin-activated TRPV1. The concentration producing 50% inhibition (IC50) determined was in the range of 3-32nmol/L and 10-501nmol/L using CHO-K1 and dorsal root ganglion cultures, respectively. In vivo, all compounds showed dose-dependent reduction in capsaicin-evoked pain-related behavioural responses in rat. None of the three compounds displayed any significant activity on any of the secondary targets tested. The compounds were also shown to be safe from a toxicological, drug metabolism and pharmacokinetic perspective, for usage in microgram doses in the human skin.

    Conclusion

    The investigated model compounds displayed ideal compound characteristics as pharmacological and translational tools to address efficacy on the human native TRPV1 target in human skin insitu.

    Significance

    This work details the pharmaceutical work-up of three TRPV1-active investigational compounds, to obtain regulatory approval, for subsequent use in humans. This fast and cost-effective preclinical development path may impact research beyond the pain management area, as it allows human target engagement information gathering early in drug development.

  • 202.
    Sjögren, Erik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Lennernäs, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Effects of verapamil on the pharmacokinetics and hepatobiliary disposition of fexofenadine in pigs2014In: European Journal of Pharmaceutical Sciences, ISSN 0928-0987, E-ISSN 1879-0720, Vol. 57, p. 214-223Article in journal (Refereed)
    Abstract [en]

    The pharmacokinetics (PK) of fexofenadine (FEX) in pigs were investigated with the focus on exploring the interplay between hepatic transport and metabolism when administered intravenously (iv) alone or with verapamil. The in vivo pig model enabled simultaneous sampling from plasma (pre-liver, post-liver and peripheral), bile and urine. Each animal was administered FEX 35mg iv alone or with verapamil 35mg. Plasma, bile and urine were analyzed with liquid chromatography-tandem mass spectrometry. Non-compartmental analysis (NCA) was used to estimate traditional PK parameters. In addition, a physiologically based pharmacokinetic (PBPK) model consisting of 11 compartments (6 tissues +5 sample sites) was applied for mechanistic elucidation and estimation of individual PK parameters. FEX had a terminal half-life of 1.7h and a liver extraction of 3%. The fraction of the administered dose of unchanged FEX excreted into the bile was 25% and the bile exposure was more than 100 times higher than the portal vein total plasma exposure, indicating carrier-mediated (CM) disposition processes in the liver. 23% of the administered dose of FEX was excreted unchanged in the urine. An increase in FEX plasma exposure (+50%) and a decrease in renal clearance (-61%) were detected by NCA as a direct effect of concomitant administration of verapamil. However, analysis of the PBPK model also revealed that biliary clearance was significantly inhibited (-53%) by verapamil. In addition, PBPK analysis established that metabolism and CM uptake were important factors in the disposition of FEX in the liver. In conclusion, this study demonstrated that CM transport of FEX in both liver and kidneys was inhibited by a single dose of verapamil.

  • 203.
    Stubberud, Karin
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Studies of Micellar Electrokinetic Chromatography as an Analytical Technique in Pharmaceutical Analysis - an Industrial Perspective2002Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Studies have been performed to evaluate the use of micellar electrokinetic chromatography (MEKC), one mode of capillary electrophoresis (CE), as an analytical technique in industrial pharmaceutical analysis. The potential for using chemometrics for the optimisation of MEKC methods has also been studied as well as the possibilities of coupling MEKC with mass spectrometry (MS).

    Two methods were developed, one for the determination of ibuprofen and codeine and another for pilocarpine, together with their degradation products and impurities in both cases. MEKC was found to be the most suitable mode of CE for the methods. Both methods were optimised by means of experimental design. Valuable information was gathered and optimum conditions were defined which resulted in fast systems with baseline-separated peaks. The ibuprofen-codeine method was validated according to the recommended validation procedures of the International Conference of Harmonisation. The validation was performed on a commercially available tablet formulation to verify the suitability of the method, i.e. for quantification of the two main compounds and to determine the degradation products and impurities in area% of each main peak. The following parameters were determined: selectivity, linearity, accuracy, precision, detection limit, quantitation limit, robustness and range. The results confirm that the method is highly suitable for its intended purpose, i.e. as a routine method for assay and impurity determination. The MEKC method for ibuprofen-codeine was coupled to a mass spectrometer in order to evaluate the potential of partial filling (PF)-MEKC-MS for identification of impurities in pharmaceutical substances and products. The so-called partial-filling technique was used to prevent the non-volatile micelles from entering the MS and was shown to fulfil its purpose of providing detection limits of about 10 pg.

    The study clearly shows that micellar electrokinetic chromatography is well-suited as an analytical technique in industrial pharmaceutical analysis.

    List of papers
    1. Fractional Factorial Design Optimization of the Separation of Pilocarpine and its Degradation Products by Capillary Electrophoresis
    Open this publication in new window or tab >>Fractional Factorial Design Optimization of the Separation of Pilocarpine and its Degradation Products by Capillary Electrophoresis
    1997 (English)In: Journal of Chromatography B: Biomedical Sciences and Applications, ISSN 1387-2273, E-ISSN 1878-5603, Vol. 697, no 1-2, p. 207-215Article in journal (Refereed) Published
    Abstract [en]

    The separation of pilocarpine and its degradation products by micellar electrokinetic capillary chromatography (MECC) has been optimized by using fractional factorial design of the experiments. Critical parameters were identified in a screening design, and an optimization design was used to optimize the separation. The optimal separation method was based on a borate buffer with sodium dodecyl sulfate (SDS). It is concluded that by using fractional factorial design it is possible to improve the separation of pilocarpine, its trans epimer, isopilocarpine and their hydrolysis products, pilocarpic acid and isopilocarpic acid.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-89801 (URN)10.1016/S0378-4347(97)00108-4 (DOI)9342671 (PubMedID)
    Available from: 2002-04-15 Created: 2002-04-15 Last updated: 2017-12-14Bibliographically approved
    2. Separation of Ibuprofen, Codeine Phosphate, their Degradation Products and Impurities by Capillary Electrophoresis: I. Method Development and Optimization with Fractional factorial Design
    Open this publication in new window or tab >>Separation of Ibuprofen, Codeine Phosphate, their Degradation Products and Impurities by Capillary Electrophoresis: I. Method Development and Optimization with Fractional factorial Design
    1998 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 798, no 1-2, p. 307-314Article in journal (Refereed) Published
    Abstract [en]

    A capillary electrophoresis method has been developed and optimized for the separation of ibuprofen, codeine phosphate and their main degradation products and impurities. In the course of developing the method, it was found that micellar electrokinetic capillary chromatography was necessary for the separation of the eleven peaks. A fractional factorial design was used for the optimization of the experiments. Six process parameters were varied at two levels: the concentration of sodium dodecyl sulfate (SDS), the pH, the concentration of acetonitrile, the concentration of boric acid, the field strength and the temperature. All these factors had a significant effect on the migration time and resolution. The optimum conditions were found to be a borate buffer of 40 mM H3BO3 at pH 10 with the addition of 40 mM SDS and 9% acetonitrile, a field strength of 515 V/cm and a temperature of 25 degrees C. This resulted in baseline separation of the eleven peaks within 12 min.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-89802 (URN)10.1016/S0021-9673(97)00955-2 (DOI)9542142 (PubMedID)
    Available from: 2002-04-15 Created: 2002-04-15 Last updated: 2017-12-14Bibliographically approved
    3. Separation of Ibuprofen, Codeine Phosphate, their Degradation Products and Impurities by Capillary Electrophoresis: II. Validation
    Open this publication in new window or tab >>Separation of Ibuprofen, Codeine Phosphate, their Degradation Products and Impurities by Capillary Electrophoresis: II. Validation
    1998 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 826, no 1, p. 95-102Article in journal (Refereed) Published
    Abstract [en]

    A micellar electrokinetic chromatography method for the determination of ibuprofen and codeine phosphate hemihydrate and their degradation products and impurities in a commercial tablet formulation has been validated. The validation has been performed according to the International Conference of Harmonisation's guidance on the validation of analytical methods, and selectivity, linearity, accuracy, precision, detection limit, quantitation limit, robustness and range test were performed to determine the suitability of the method. It was possible to use the fractional factorial design model from the optimisation of the method to draw conclusions about its robustness. The results confirm that the method is highly suitable for its intended purpose.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-89803 (URN)10.1016/S0021-9673(98)00711-0 (DOI)9882139 (PubMedID)
    Available from: 2002-04-15 Created: 2002-04-15 Last updated: 2017-12-14Bibliographically approved
    4. Partial filling micellar electrokinetic chromatography optimisation studies of ibuprofen, codeine and degradation products, and coupling to mass spectrometry: Part I
    Open this publication in new window or tab >>Partial filling micellar electrokinetic chromatography optimisation studies of ibuprofen, codeine and degradation products, and coupling to mass spectrometry: Part I
    2002 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 23, no 4, p. 572-577Article in journal (Refereed) Published
    Abstract [en]

    Studies have been performed to evaluate whether an on-line partial filling-micellar electrokinetic chromatography (PF-MEKC) system could be applied to a recently developed MEKC method for the separation of ibuprofen, codeine and one of the degradation products. Attempts to couple the PF-MEKC system to MS have also been performed. SDS concentration, micellar zone length and concentration of acetonitrile in the buffer were optimized using factorial design. When a small micelle zone was injected directly after the sample introduction, the results improved markedly. The MS parameters have not been optimized, but the studies show promising results for the use of PF-MEKC-mass spectrometry for identification of the degradation products.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-89804 (URN)10.1002/1522-2683(200202)23:4<572::AID-ELPS572>3.0.CO;2-# (DOI)11870767 (PubMedID)
    Available from: 2002-04-15 Created: 2002-04-15 Last updated: 2017-12-14Bibliographically approved
    5. Partial filling micellar electrokinetic chromatography optimisation studies of ibuprofen, codeine and degradation products, and coupling to mass spectrometry: Part II
    Open this publication in new window or tab >>Partial filling micellar electrokinetic chromatography optimisation studies of ibuprofen, codeine and degradation products, and coupling to mass spectrometry: Part II
    2003 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 24, no 6, p. 1008-1015Article in journal (Refereed) Published
    Abstract [en]

    We describe the use of partial filling-micellar electrokinetic chromatography-mass spectrometry (PF-MEKC-MS) on the pharmaceutical ingredients ibuprofen and codeine phosphate as well as their degradation products and impurities. The study focuses on the change of the borate buffer to the volatile ammonium acetate and the optimization of critical MS parameters. The sensitivity of the method is also evaluated. The results are compared to an existing MEKC-UV method that is used for quantitative determination of the two main substances as well as for the analysis of the degradation products. It is concluded that the PF-MEKC-MS system is suitable for separation and identification.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-89805 (URN)10.1002/elps.200390116 (DOI)12658689 (PubMedID)
    Available from: 2002-04-15 Created: 2002-04-15 Last updated: 2017-12-14Bibliographically approved
  • 204.
    Stubberud, Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Callmer, Karin
    Westerlund, Douglas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Partial filling micellar electrokinetic chromatography optimisation studies of ibuprofen, codeine and degradation products, and coupling to mass spectrometry: Part II2003In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 24, no 6, p. 1008-1015Article in journal (Refereed)
    Abstract [en]

    We describe the use of partial filling-micellar electrokinetic chromatography-mass spectrometry (PF-MEKC-MS) on the pharmaceutical ingredients ibuprofen and codeine phosphate as well as their degradation products and impurities. The study focuses on the change of the borate buffer to the volatile ammonium acetate and the optimization of critical MS parameters. The sensitivity of the method is also evaluated. The results are compared to an existing MEKC-UV method that is used for quantitative determination of the two main substances as well as for the analysis of the degradation products. It is concluded that the PF-MEKC-MS system is suitable for separation and identification.

  • 205.
    Stubberud, Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Forsberg, Anna
    Callmer, Karin
    Westerlund, Douglas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Partial filling micellar electrokinetic chromatography optimisation studies of ibuprofen, codeine and degradation products, and coupling to mass spectrometry: Part I2002In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 23, no 4, p. 572-577Article in journal (Refereed)
    Abstract [en]

    Studies have been performed to evaluate whether an on-line partial filling-micellar electrokinetic chromatography (PF-MEKC) system could be applied to a recently developed MEKC method for the separation of ibuprofen, codeine and one of the degradation products. Attempts to couple the PF-MEKC system to MS have also been performed. SDS concentration, micellar zone length and concentration of acetonitrile in the buffer were optimized using factorial design. When a small micelle zone was injected directly after the sample introduction, the results improved markedly. The MS parameters have not been optimized, but the studies show promising results for the use of PF-MEKC-mass spectrometry for identification of the degradation products.

  • 206.
    Sundström, Ingela
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Liquid Chromatography-Mass Spectrometry as a Tool for Drug Metabolite Identification in Biological Fluids: With Application to Ketobemidone2007Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Electrospray ionization (ESI) mass spectrometry (MS) in combination with liquid chromatography (LC) is an excellent tool for the identification of drug metabolites. Utilizing this hyphenated technique in combination with proper sample pretreatment, the metabolic pathways of the analgesic drug ketobemidone were investigated in human urine and rat microdialysate from blood and brain. Two novel phase I metabolites (ketobemidone N-oxide and meta-hydroxymethoxyketobemidone) and three novel phase II metabolites (glucuronic acid conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone) were identified in human urine. Further, norketobemidone and ketobemidone N-oxide were identified in rat microdialysate from brain after regional distribution of ketobemidone in striatum. This indicates that the brain itself has the possibility to metabolize ketobemidone.

    Synthetic ketobemidone metabolites were used for comparison of retention times and tandem MS spectra with the possible metabolites recovered from the biological samples. The conjugated metabolites were identified by accurate mass measurements and tandem MS spectra of the aglycones. The accuracy of the estimated masses was better than 2.1 ppm for two out of three conjugates in presence of internal standard.

    On-line micro-SPE was successfully used for trapping and desalting of the microdialysates. The small SPE pre-column made it possible to inject approximately 100 times more sample on the analytical column compared to injection without pre-column. Selective trapping was demonstrated for the polar catechol amine metabolite, dihydroxyketobemidone, which forms covalent complexes with phenylboronic acid (PBA). A fluorinated silica type stationary phase was the only column out of several tested that was able to separate ketobemidone and all relevant phase I metabolites.

    Liquid chromatography and mass spectrometry are independently valuable tools in the field of analytical pharmaceutical chemistry. The present study showed that the combination of LC-MS, with its excellent selectivity and sensitivity, offers an outstanding tool in the qualitative analysis of drugs and metabolites in biological fluids.

    List of papers
    1. Identification of phase I and phase II metabolites of ketobemidone in patient urine using liquid chromatography-electrospray tandem mass spectrometry
    Open this publication in new window or tab >>Identification of phase I and phase II metabolites of ketobemidone in patient urine using liquid chromatography-electrospray tandem mass spectrometry
    2001 In: J. Chromatogr. B, ISSN 0378-4347, Vol. 763, no 1-2, p. 121-131Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-95951 (URN)
    Available from: 2007-05-10 Created: 2007-05-10Bibliographically approved
    2. Identification of glucuronide conjugates of ketobemidone and its phase I metabolites in human urine utilizing accurate mass and tandem time-of-flight mass spectrometry
    Open this publication in new window or tab >>Identification of glucuronide conjugates of ketobemidone and its phase I metabolites in human urine utilizing accurate mass and tandem time-of-flight mass spectrometry
    2002 In: J. Mass Spectrom., ISSN 1096-9888, Vol. 37, no 4, p. 414-420Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-95952 (URN)
    Available from: 2007-05-10 Created: 2007-05-10Bibliographically approved
    3. Identification of ketobemidone metabolites by capillary LC-MS/MS: Part I: Method development for analysis of in vivo micro-dialysates
    Open this publication in new window or tab >>Identification of ketobemidone metabolites by capillary LC-MS/MS: Part I: Method development for analysis of in vivo micro-dialysates
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-95953 (URN)
    Available from: 2007-05-10 Created: 2007-05-10 Last updated: 2010-01-13Bibliographically approved
    4. Identification of ketobemidone metabolites by capillary LC-MS/MS: Part II: Identification of ketobemidone metabolites in rat blood and brain in vivo microdialysates and urine
    Open this publication in new window or tab >>Identification of ketobemidone metabolites by capillary LC-MS/MS: Part II: Identification of ketobemidone metabolites in rat blood and brain in vivo microdialysates and urine
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-95954 (URN)
    Available from: 2007-05-10 Created: 2007-05-10 Last updated: 2010-01-13Bibliographically approved
  • 207.
    Sundström, Ingela
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Andrén, Per E.
    Westerlund, Douglas
    Identification of ketobemidone metabolites by capillary LC-MS/MS: Part I: Method development for analysis of in vivo micro-dialysatesManuscript (Other academic)
  • 208.
    Sundström, Ingela
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Andrén, Per E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Westerlund, Douglas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Method development for identification of ketobemidone metabolites in microdialysate samples by coupled-column capillary liquid chromatography-tandem mass spectrometry2008In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1189, no 1-2, p. 503-513Article in journal (Refereed)
    Abstract [en]

    Methodologies for identification of ketobemidone metabolites in microdialysate samples utilizing coupled-column capillary liquid chromatography-electrospray quadrupole time-of-flight tandem mass spectrometry are presented. Two different methods were developed to efficiently analyze the metabolites norketobemidone, ketobemidone N-oxide and hydroxyketobemidone, respectively. Both methods include on-line desalting and trapping of the analytes on micro-solid-phase extraction columns with different retention mechanisms. Norketobemidone and ketobemidone N-oxide were trapped on a C18 column and then eluted by back-flush followed by isocratic separation on a fluorinated reversed-phase type silica gel column (Fluofix). Retention equations are proposed for the chromatographic observations made on the Fluofix column. Hydroxyketobemidone was trapped on a phenylboronic acid column by complex formation at basic pH and then eluted at acidic pH directly into to the mass spectrometer. Oxidation of hydroxyketobemidone to its corresponding quinone was also observed. The methods were successfully used to analyze synthetic ketobemidone metabolites in dilute low-volume microdialysis samples

  • 209.
    Sundström, Ingela
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Arts, Joris
    Westerlund, Douglas
    Andrén, Per E.
    Identification of ketobemidone metabolites by capillary LC-MS/MS: Part II: Identification of ketobemidone metabolites in rat blood and brain in vivo microdialysates and urineManuscript (Other academic)
  • 210.
    Sundström, Ingela
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Arts, Joris
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Westerlund, Douglas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    In vivo investigation of brain and systemic ketobemidone metabolism2010In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 135, no 2, p. 405-413Article in journal (Refereed)
    Abstract [en]

    Ketobemidone metabolites have previously been identified in urine and plasma; here we show, for the first time, that norketobemidone and ketobemidone N-oxide are present in in vivo microdialysate from rat brain (striatum) after reverse microdialysis, suggesting striatal metabolism of ketobemidone. Ketobemidone metabolites were also identified in in vivo microdialysate samples from brain and blood, as well as in urine from rats, after subcutaneous administration of ketobemidone. Three Phase I metabolites (norketobemidone, ketobemidone N-oxide and hydroxymethoxyketobemidone) and three Phase II metabolites (glucuronic acid conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone) were identified in the microdialysates after subcutaneous administration. Coupled capillary liquid chromatography tandem mass spectrometry (LC-MS/MS) and SPE (boronate)-MS/MS were utilized for the analysis of the biological samples. The Phase I metabolites were identified by comparing the retention times and tandem mass spectra of the microdialysates with synthetic standards. The Phase II metabolites were identified by determination of exact masses and by comparing the tandem mass spectra of the microdialysates with those of synthetic standards for the aglycones. Hydroxyketobemidone, a catechol-type Phase I metabolite, was selectively isolated by solid-phase boronate-complexation but identified in urine alone. This work demonstrated that the in vivo microdialysis technique in combination with LC-MS/MS can be used to study the local metabolism of a drug in the brain.

  • 211.
    Sundström, Ingela
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Bondesson, Ulf
    Hedeland, Mikael
    Identification of phase I and phase II metabolites of ketobemidone in patient urine using liquid chromatography-electrospray tandem mass spectrometry2001In: J. Chromatogr. B, ISSN 0378-4347, Vol. 763, no 1-2, p. 121-131Article in journal (Refereed)
  • 212.
    Sundström, Ingela
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Bondesson, Ulf
    Andrén, Per E.
    Identification of glucuronide conjugates of ketobemidone and its phase I metabolites in human urine utilizing accurate mass and tandem time-of-flight mass spectrometry2002In: J. Mass Spectrom., ISSN 1096-9888, Vol. 37, no 4, p. 414-420Article in journal (Refereed)
  • 213.
    Svan, Alfred
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Arvidsson, Torbjorn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Jasper, Justin T.
    Sedlak, David L.
    Pettersson, Curt E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Rapid chiral separation of atenolol, metoprolol, propranolol and the zwitterionic metoprolol acid using supercritical fluid chromatography-tandem mass spectrometry - Application to wetland microcosms2015In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1409, p. 251-258Article in journal (Refereed)
    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.

  • 214.
    Svan, Alfred
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Natl Vet Inst SVA, Dept Chem Environm & Feed Hyg, SE-75189 Uppsala, Sweden..
    Arvidsson, Torbjörn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Med Prod Agcy, Box 26, SE-75103 Uppsala, Sweden..
    Jasper, Justin T.
    Univ Calif Berkeley, Dept Civil & Environm Engn, Berkeley, CA 94720 USA..
    Sedlak, David L.
    Univ Calif Berkeley, Dept Civil & Environm Engn, Berkeley, CA 94720 USA..
    Pettersson, Curt E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Identification of transformation products from -blocking agents formed in wetland microcosms using LC-Q-ToF2016In: Journal of Mass Spectrometry, ISSN 1076-5174, E-ISSN 1096-9888, Vol. 51, no 3, p. 207-218Article in journal (Refereed)
    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.

  • 215.
    Svan, Alfred
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Arvidsson, Torbjörn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Pettersson, Curt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Chiral separation of three beta-blocking pharmaceuticals and a major metabolite using SFC-MS2014Conference paper (Other academic)
  • 216.
    Svan, Alfred
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Statens veterinärmedicinska anstalt (SVA) Enhet för kemi, miljö och fodersäkerhet.
    Arvidsson, Torbjörn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Läkemedelsverket.
    Pettersson, Curt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Chiral separation of three β-blocking pharmaceuticals and a majormetabolite using SFC-MS2013Conference paper (Other academic)
  • 217.
    Svan, Alfred
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Arvidsson, Torbjörn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Pettersson, Curt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Matrix effects: Do they differ between SFC/ESI-MS and LC/ESI-MS2016Conference paper (Other academic)
    Abstract [en]

    Introduction (120 ord)

    Supercritical fluid chromatography (SFC) is increasingly used in many fields following the new generation of instruments available on the market, often combined with electrospray ionization mass spectrometry (ESI/MS). New methods are developed and validated, often for samples of complex matrices. Thus, measurements of the matrix effects are sometimes included. Comparisons between SFC/ESI/MS and LC/ESI/MS have previously been performed, often focusing on e.g. sensitivity and separation. However, a systematic and qualitative investigation of the matrix effects achieved using same ion source and mass spectrometer, but different types of chromatography, i.e. SFC or LC, is so far lacking.

     

    Methods (120 ord)

    Effluent wastewater was collected and 100.0 ml was extracted using a generic SPE method (Oasis MCX), evaporated and redissolved in 1.0 ml of water:ACN (1:1). A 10 min gradient was developed for each technique, separating eight compounds chosen for being low molecular weight drugs with varying hydrophobicity and proteolytic properties. The mixture of compounds was then added to the ESI make-up solvent (MeOH) for the SFC/ESI/MS method. The extracted wastewater was injected without added compounds, and the SRM channel for each compound was monitored using tandem quadrupole MS (Quattro Micro, Waters®). The same interface and settings were used for LC, but an infusion of compounds was performed through a post-column syringe pump since no make-up flow was used.

     

    Preliminary data – Limit 300 words

    The retention times for the eight model compounds were evenly spread over the chromatographic time scale for both chromatographic methods. When applying the continuous compound infusion through the make-up solution (SFC/ESI/MS) or through a syringe pump (LC/ESI/MS), a stable signal was observed for all compounds. Injection of the extracted wastewater affected the signal in comparison with the blank. The difference in the signal profiles were however larger between LC and SFC than between blank and extracted wastewater. The two separation techniques also gave different levels of noise and variations in the SRM-channels for the different compounds, and occurred at differently time points during the gradients for both the techniques. In summary, the matrix effects seem to affect the detection differently depending on what chromatographic technique that is used. With increased knowledge about this, it could help future method development to minimize the matrix effects.    

     

    Novel aspect – Limit 20 words

    The first systematic comparison between SFC and LC in terms of matrix effects for ESI-MS/MS using qualitative methods (infusion).

  • 218.
    Svan, Alfred
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Natl Vet Inst SVA, Dept Chem Environm & Feed Hyg, SE-75189 Uppsala, Sweden..
    Arvidsson, Torbjörn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Med Prod Agcy, Box 26, SE-75103 Uppsala, Sweden..
    Pettersson, Curt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    The differences in matrix effect between supercritical fluid chromatography and reversed phase liquid chromatography coupled to ESI/MS2018In: Analytica Chimica Acta, ISSN 0003-2670, E-ISSN 1873-4324, Vol. 1000, p. 163-171Article in journal (Refereed)
    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.

  • 219.
    Svan, Alfred
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Arvidsson, Torbjörn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Pettersson, Curt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    The differences in matrix effects between supercritical fluid chromatography and reversed phase liquid chromatography coupled to ESI/MS analyzing blood plasma2017Conference paper (Other academic)
    Abstract [en]

    Introduction

    The increasing popularity of supercritical fluid chromatography (SFC) in combination with electrospray ionization mass spectrometry (ESI/MS) within several fields calls for a deeper knowledge regarding this combination of techniques. The ESI source is known for its sensitivity regarding matrix effects, often a factor controlled during method development and validation using LC. The different chemistry and chromatographic selectivity of LC and SFC give potentially different impact on the ionization process in ESI; however, this an area still not well studied.   

    Aim: To investigate how the matrix effects in ESI/MS differ for human plasma samples between SFC and reversed-phase LC, using generic screening conditions for both techniques, and a set of typical low molecular weight drug substances.

    Methods

    Pooled human plasma (500 µl) was precipitated using ice-cold acetonitrile (1000 µl). After mixing and centrifuging, 1200 µl of the supernatant were removed and evaporated at 40 ̊C. When dry, the samples were dissolved in 500µl water+0.1% FA (for LC) or acetonitrile:water 75:25 (for SFC). The samples were analyzed using SFC (Acquity UPC2, Waters®) and LC (Acquity UPLC, Waters®) and general screening conditions, using 10 min gradients. The same MS-system, a Q-ToF (Synapt G2-S, Waters®) acquiring in full scan mode, was used for detection with both separation techniques. The matrix effect was mainly evaluated using the Matrix Effect Profile, achieved from post-column compound infusions and injections of pretreated sample matrix and neat standards. From these data the average ME% was calculated for each data-point in the chromatogram, and through the full-scan mode using ToF, the compounds co-eluting with areas of suppression could be tentatively identified, suspected of creating the suppression. 

     

    Results and discussion The Matrix Effect Profile-evaluation of the experiments, combining qualitative and quantitative information with the added ability to use HRMS-data to identify interfering compounds from the same experiments were most useful for our aim. Phospholipids, creatinine, polyethylene glycol and cluster formations are examples of important interferences co-eluting with areas of ion suppression, but with different impact depending on chromatographic technique. The results also showed several areas of enhancement using LC, an effect not seen using SFC. 

  • 220.
    Sänger - van de Griend, Cari
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Revival of Capillary Electrophoretic Techniques in the Pharmaceutical Industry2012In: LC GC North America, ISSN 1527-5949, E-ISSN 1939-1889, Vol. 30, no 11, p. 954-971Article in journal (Refereed)
    Abstract [en]

    The article focuses on the integration of capillary electrophoresis (CE) techniques in the biopharmaceutical industry. It states that CE has been used by biotech and pharmaceutical industry in all phases of drug development. The article also discusses practical aspects of CE capillary, the use of CE as chiral separation tool, and types of CE such as capillary electrochromatography. INSETS: Capillary Electrophoresis;Electrophoresis in a Capillary: The Electroosmotic Flow.

  • 221.
    Sänger - van de Griend, Cari
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Ylva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Pettersson, Curt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Capillary Electrophoresis: an Attractive Technique for Chiral Separations2013In: Chromatography Today, ISSN 1752-8070, Vol. 6, no 2, p. 32-37Article, review/survey (Refereed)
  • 222.
    Tannergren, Christer
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Engman, Helena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Knutson, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry.
    Lennernäs, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    St John's wort decreases the bioavailability of R- and S-verapamil through induction of the first-pass metabolism2004In: Clinical Pharmacology and Therapeutics, ISSN 0009-9236, E-ISSN 1532-6535, Vol. 75, no 4, p. 298-309Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE:

    Our objective was to investigate the inducing effect of repeated oral administration of St John's wort on the jejunal transport and presystemic extraction of R- and S-verapamil in humans.

    METHODS:

    Jejunal single-pass perfusion experiments with 120-mg/L (244 micromol/L) R-/S-verapamil were performed in 8 healthy male volunteers for 100 minutes before and after 14 days of oral treatment with St John's wort (300 mg 3 times a day). The enantiomers of verapamil and the cytochrome P450 (CYP) 3A4-formed metabolite norverapamil in perfusate and plasma were quantified by chiral HPLC with fluorescence and tandem mass spectrometry detection, respectively.

    RESULTS:

    St John's wort did not affect the jejunal permeability or the fraction absorbed of either R- or S-verapamil. The values for area under the plasma concentration-time curve (AUC) for R- and S-verapamil decreased by 78% and 80%, respectively (P <.0001). The corresponding decreases in the maximum concentration were 76% and 78%, respectively (P <.0001), whereas the terminal half-life did not change significantly for any of the enantiomers. The AUC for R-verapamil was 6 times higher than that for S-verapamil in the control phase, and St John's wort did not change this ratio. The AUC values for R- and S-norverapamil decreased by 51% (P <.01) and 63% (P <.0001), respectively.

    CONCLUSIONS:

    Repeated administration of St John's wort significantly decreased the bioavailability of R- and S-verapamil. This effect is caused by induction of first-pass CYP3A4 metabolism, most likely in the gut, because the jejunal permeability and the terminal half-life were unchanged for both enantiomers.

  • 223.
    Telo, Jasmin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Conditioning of chromatographic systems prior to metabolomic studies: Investigation of the conditioning effect and the possibility to alter it2017Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The conditioning effect in metabolomic studies is the phenomenon of initial variation of analytical results in the first 5-10 injections of a biological sample in chromatographic systems. The deviation manifests itself as a drift in retention time, peak area and in multivariate analysis. It is a major quality assurance problem in the metabolomic field and if not accounted for would result in high analytical variance.

    The aim of this study was to investigate the conditioning effect and to gain further knowledge about it. The study was carried out on UPLC of hydrophilic liquid chromatography (HILIC) type coupled to quadrupole time of flight (QTOF) MS.

    A systematic study was designed to investigate the effects of the age of the analytical column. An investigation into certain matrix components as a possible cause of the conditioning effect was made. Different sample preparation methods were investigated.

    One result showed that no conditioning could be seen and the system appeared stable from the first injection. Differences in sample composition between samples with conditioning effect and samples without conditioning effect were investigated. No correlation between conditioning effect and levels of certain matrix compounds could be found. More studies of correlation between sample composition and the amount of conditioning occurring is needed.

    Some samples appear to have no retention time drift but have a significant drift in peak area and in multivariate analysis. This is an indication that the conditioning effect should be analysed in more ways than one before determining if a system is stable.

  • 224.
    Tevell Åberg, Annica
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Detection and Structure Elucidation of Drug Metabolites in Biological Samples using HPLC-MS/MS Techniques2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis describes the structure elucidation of drug metabolites in biological samples by the use of high performance liquid chromatography (HPLC) atmospheric pressure ionization (API) tandem mass spectrometry (MS/MS). Due to their different advantages, various mass analyzers have been used in the different experiments.

    The metabolism of clemastine, flutamide, and meloxicam were studied in vitro and/or in vivo in different species such as humans, dogs, and horses. Accurate mass measurements with the quadrupole-time of flight mass spectrometer and MSn data supplied by the ion trap instrument were useful in the structural investigation of the product ions of the drugs and their metabolites. Different scan modes of the triple quadrupole mass spectrometer resulted in great flexibility, selectivity, and sensitivity in the qualitative and semi-quantitative studies. Additionally, hydrogen/deuterium exchange and experiments with atmospheric pressure chemical ionization were conducted, and the fungus Cunninghamella elegans was utilized to produce amounts of drug metabolites sufficient for structural investigation.

    Six isomers of oxidized clemastine were detected and characterized in C. elegans incubations and their retention times and mass spectral data were compared to the metabolites detected in urine samples. Two of the metabolites were concluded to be diastereomeric N-oxides.

    In urine from horses treated with meloxicam, the peak of 5'-hydroxymethylmeloxicam resulted in much higher intensity than the parent drug or the other metabolites, and it was detectable for at least 14 days after the last dose in some of the horses. That is useful information in the development of analytical methods for the detection of prohibited use of meloxicam.

    A mercapturic acid conjugate of hydroxyflutamide was detected in urine from cancer patients, which indicated that a reactive metabolite was formed. This metabolite could be responsible for the adverse events reported for flutamide.

    The results from the four papers included in the thesis clearly demonstrate the usefulness and the flexibility of the HPLC-API-MS/MS technique.

    List of papers
    1. Identification of some new clemastine metabolites in dog, horse, and human urine with liquid chromatography/tandem mass spectrometry.
    Open this publication in new window or tab >>Identification of some new clemastine metabolites in dog, horse, and human urine with liquid chromatography/tandem mass spectrometry.
    2004 (English)In: Rapid Commun Mass Spectrom, ISSN 0951-4198, Vol. 18, no 19, p. 2267-72Article in journal (Other scientific) Published
    Identifiers
    urn:nbn:se:uu:diva-67268 (URN)15384147 (PubMedID)
    Available from: 2004-11-11 Created: 2004-11-11 Last updated: 2011-01-12
    2. Structure elucidation of N-oxidized clemastine metabolites by HPLC-MS-MS and the use of Cunninghamella elegans to facilitate drug metabolite identification
    Open this publication in new window or tab >>Structure elucidation of N-oxidized clemastine metabolites by HPLC-MS-MS and the use of Cunninghamella elegans to facilitate drug metabolite identification
    (English)Manuscript (Other (popular science, discussion, etc.))
    Keywords
    HPLC-MS/MS, hydrogen/deuterium exchange, N-oxidation, Clemastine, metabolites
    Identifiers
    urn:nbn:se:uu:diva-98343 (URN)
    Available from: 2009-02-19 Created: 2009-02-19 Last updated: 2010-01-14
    3. Flutamide metabolism in four different species in vitro and identification of flutamide metabolites in human patient urine by high performance liquid chromatography/tandem mass spectrometry.
    Open this publication in new window or tab >>Flutamide metabolism in four different species in vitro and identification of flutamide metabolites in human patient urine by high performance liquid chromatography/tandem mass spectrometry.
    Show others...
    2006 (English)In: Drug Metab Dispos, ISSN 0090-9556, Vol. 34, no 6, p. 984-92Article in journal (Refereed) Published
    Keywords
    Androgen Antagonists/*metabolism/therapeutic use/urine, Animals, Antineoplastic Agents; Hormonal/*metabolism/therapeutic use/urine, Biotransformation, Chromatography; Liquid, Comparative Study, Dogs, Flutamide/*analogs & derivatives/*metabolism/standards/therapeutic use/urine, Glucuronidase, Humans, In Vitro, Male, Microsomes; Liver/metabolism, Prostate/metabolism, Prostatic Neoplasms/drug therapy/*metabolism/urine, Rats, Spectrometry; Mass; Electrospray Ionization, Swine
    Identifiers
    urn:nbn:se:uu:diva-80893 (URN)16540588 (PubMedID)
    Available from: 2007-03-03 Created: 2007-03-03 Last updated: 2011-01-11
    4. A mass spectromeric study on meloxicam metabolism in horses and the fungus Cunninghamella elegans, and the relevance of this microbial system as a model of drug metabolism in the horse
    Open this publication in new window or tab >>A mass spectromeric study on meloxicam metabolism in horses and the fungus Cunninghamella elegans, and the relevance of this microbial system as a model of drug metabolism in the horse
    2009 (English)In: Journal of Mass Spectrometry, ISSN 1076-5174, E-ISSN 1096-9888, Vol. 44, no 7, p. 1026-1037Article in journal (Refereed) Published
    Abstract [en]

    This paper describes a study where the metabolism of the non-steroidal   anti-inflammatory drug meloxicam was investigated in six horses and in  the filamentous fungus Cunninghamella elegans. The metabolites identified were compared between the species, and then the fungus was  used to produce larger amounts of the metabolites for future use as   reference material. C. elegans proved to be a good model of phase I   meloxicam metabolism in horses since all four metabolites found were   the same in both species. Apart from the two main metabolites,   5'-hydroxymethylmeloxicam and 5'-carboxymeloxicam, a second isomer of   hydroxymeloxicam and dihydroxylated meloxicam were detected for the   first time in horse urine and the microbial incubations. Phase II   metabolites were not discovered in the C. elegans samples but   hydroxymeloxicam glucuronide was detected intact in horse urine for the   first time in this study. Urine from six horses was further analyzed in   a semi-quantitative sense and 5'-hydroxymethylmeloxicam gave peaks with   much higher intensity compared to the parent drug and the other   metabolites, and was detected for at least 14 days after the last given   dose in some of the horses. From the results presented in this article,   we suggest that analytical methods developed for the detection of   meloxicam in horse urine after prohibited use should focus on the   5'-hydroxymethyl metabolite and that C. elegans can be used to produce  large amounts of this metabolite for potential future use as a reference compound.

    Keywords
    Meloxicam, metabolism, mass spectrometry, horse, Cunninghamella elegans
    National Category
    Pharmaceutical Sciences
    Research subject
    Analytical Pharmaceutical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-98287 (URN)10.1002/jms.1575 (DOI)000268505500003 ()
    Available from: 2009-02-19 Created: 2009-02-18 Last updated: 2018-01-13Bibliographically approved
  • 225.
    Tevell Åberg, Annica
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Björnstad, Kristian
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Mass Spectrometric Detection of Protein-Based Toxins2013In: Biosecurity and bioterrorism, ISSN 1538-7135, E-ISSN 1557-850X, Vol. 11, no S1, p. S215-S226Article in journal (Refereed)
    Abstract [en]

    This review focuses on mass spectrometric detection of protein-based toxins, which are among the most toxic substances known. Special emphasis is given to the bacterial toxins botulinum neurotoxin from Clostridium botulinum and anthrax toxins from Bacillus anthracis as well as the plant toxin ricin produced by Ricinus communis. A common feature, apart from their extreme toxicity, is that they are composed of 2 polypeptide chains, one of which is responsible for cell uptake and another that has enzymatic function with the ability to destroy basic cellular functions. These toxins pose a threat, both regarding natural spread and from a terrorism perspective. In order for public health and emergency response officials to take appropriate action in case of an outbreak, whether natural or intentional, there is a need for fast and reliable detection methods. Traditionally, large molecules like proteins have been detected using immunological techniques. Although sensitive, these methods suffer from some drawbacks, such as the risk of false-positives due to cross-reactions and detection of inactive toxin. This article describes recently developed instrumental methods based on mass spectrometry for the reliable detection of botulinum neurotoxins, anthrax toxins, and ricin. Unequivocal identification of a protein toxin can be carried out by mass spectrometry-based amino acid sequencing. Furthermore, in combination with antibody affinity preconcentration and biochemical tests with mass spectrometric detection demonstrating the toxin's enzymatic activity, very powerful analytical methods have been described. In conclusion, the advent of sensitive, easily operated mass spectrometers provides new possibilities for the detection of protein-based toxins.

  • 226.
    Tevell Åberg, Annica
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Characterization of C- and N-oxidized Clemastine Metabolites using LC-MSn2008Conference paper (Other academic)
  • 227.
    Tevell Åberg, Annica
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Löfgren, Helena
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Structural elucidation of N-oxidized clemastine metabolites by liquid chromatography/tandem mass spectrometry and the use of Cunninghamella elegans to facilitate drug metabolite identification2010In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 24, no 10, p. 1447-1456Article in journal (Refereed)
    Abstract [en]

    Cunninghamella elegans is a filamentous fungus that has been shown to biotransform drugs into the same metabolites as mammals. In this paper we describe the use of C. elegans to aid the identification of clemastine metabolites since high concentrations of the metabolites were produced and MS(n) experiments were facilitated. The combination of liquid chromatography and tandem mass spectrometry with two different ionization techniques and hydrogen/deuterium exchange were used for structural elucidation of the clemastine metabolites. Norclemastine, four isomers of hydroxylated clemastine, and two N-oxide metabolites were described for the first time in C. elegans incubations. The N-oxidations were confirmed by hydrogen/deuterium exchange and deoxygenation (-16 Da) upon atmospheric pressure chemical ionization mass spectrometry. By MS(n) fragmentation it was concluded that two of the hydroxylated metabolites were oxidized on the methylpyrridyl moiety, one on the aromatic ring with the chloro substituent, and one on the aromatic ring without the chlorine.

  • 228.
    Tevell Åberg, Annica
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Löfgren, Helena
    Department of Chemistry, National Veterinary Institute.
    Bondesson, Ulf
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Structure elucidation of N-oxidized clemastine metabolites by HPLC-MS-MS and the use of Cunninghamella elegans to facilitate drug metabolite identificationManuscript (Other (popular science, discussion, etc.))
  • 229.
    Tevell Åberg, Annica
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Olsson, Charlotte
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    A mass spectromeric study on meloxicam metabolism in horses and the fungus Cunninghamella elegans, and the relevance of this microbial system as a model of drug metabolism in the horse2009In: Journal of Mass Spectrometry, ISSN 1076-5174, E-ISSN 1096-9888, Vol. 44, no 7, p. 1026-1037Article in journal (Refereed)
    Abstract [en]

    This paper describes a study where the metabolism of the non-steroidal   anti-inflammatory drug meloxicam was investigated in six horses and in  the filamentous fungus Cunninghamella elegans. The metabolites identified were compared between the species, and then the fungus was  used to produce larger amounts of the metabolites for future use as   reference material. C. elegans proved to be a good model of phase I   meloxicam metabolism in horses since all four metabolites found were   the same in both species. Apart from the two main metabolites,   5'-hydroxymethylmeloxicam and 5'-carboxymeloxicam, a second isomer of   hydroxymeloxicam and dihydroxylated meloxicam were detected for the   first time in horse urine and the microbial incubations. Phase II   metabolites were not discovered in the C. elegans samples but   hydroxymeloxicam glucuronide was detected intact in horse urine for the   first time in this study. Urine from six horses was further analyzed in   a semi-quantitative sense and 5'-hydroxymethylmeloxicam gave peaks with   much higher intensity compared to the parent drug and the other   metabolites, and was detected for at least 14 days after the last given   dose in some of the horses. From the results presented in this article,   we suggest that analytical methods developed for the detection of   meloxicam in horse urine after prohibited use should focus on the   5'-hydroxymethyl metabolite and that C. elegans can be used to produce  large amounts of this metabolite for potential future use as a reference compound.

  • 230.
    Tevell Åberg, Annica
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Solyakov, Alexey
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Development and in-house validation of an LC-MS/MS method for the quantification of the mycotoxins deoxynivalenol, zearalenone, T-2 and HT-2 toxin, ochratoxin A and fumonisin B1 and B2 in vegetable animal feed2013In: Food additives & contaminants. Part A, Chemistry, analysis, control, exposure & risk assessment, ISSN 1944-0049, Vol. 30, no 3, p. 541-549Article in journal (Refereed)
    Abstract [en]

    Animal feed can be contaminated with various mycotoxins. To ensure animal health and safe food and feed production, the European Commission has recommended increased monitoring of the co-occurrence of deoxynivalenol, zearalenone, ochratoxin A, fumonisin B(1) and B(2), T-2 and HT-2 toxin in feed. Thus, there is a need for an analytical method that enables their simultaneous detection and quantification. This paper describes the development and in-house validation of such a method, in which the mycotoxins were extracted from spiked and naturally contaminated cereal-based compound feed, corn and wheat. The extracts were divided into two aliquots where one was diluted and then analysed directly and the other was cleaned by using MultiSep(®)226 and then diluted and analysed. Separation and detection was achieved with LC-ESI-MS/MS by using a triple quadrupole instrument in the SRM mode. The precision (in terms of intra-day repeatability and inter-day reproducibility), accuracy, linearity, apparent recovery and expanded measurement uncertainty in feed, corn and wheat were evaluated. The LODs ranged from 1.0 to 72 μg/kg, and the LOQs ranged from 2.5 to 115 μg/kg. The apparent recovery was higher than 86% for all the mycotoxins, and the precision was better than that defined by the Horwitz equation for all concentrations. Proficiency test materials were analysed to assess the accuracy of the method, and the results were satisfactory for all seven mycotoxins. The method will be used to monitor the occurrence of these mycotoxins in products intended for animal feeding in Sweden.

  • 231.
    Thevis, M.
    et al.
    German Sport Univ Cologne, Ctr Prevent Doping Res, Inst Biochem, Sportpk Mungersdorf 6, D-50933 Cologne, Germany.;European Monitoring Ctr Emerging Doping Agents Eu, Cologne, Germany..
    Machnik, M.
    German Sport Univ Cologne, Ctr Prevent Doping Res, Inst Biochem, Sportpk Mungersdorf 6, D-50933 Cologne, Germany..
    Schenk, I.
    German Sport Univ Cologne, Ctr Prevent Doping Res, Inst Biochem, Sportpk Mungersdorf 6, D-50933 Cologne, Germany..
    Krug, O.
    German Sport Univ Cologne, Ctr Prevent Doping Res, Inst Biochem, Sportpk Mungersdorf 6, D-50933 Cologne, Germany.;European Monitoring Ctr Emerging Doping Agents Eu, Cologne, Germany..
    Piper, T.
    German Sport Univ Cologne, Ctr Prevent Doping Res, Inst Biochem, Sportpk Mungersdorf 6, D-50933 Cologne, Germany..
    Schaenzer, W.
    German Sport Univ Cologne, Ctr Prevent Doping Res, Inst Biochem, Sportpk Mungersdorf 6, D-50933 Cologne, Germany..
    Duee, M.
    Deutsch Reiterliche Vereinigung eV FN, D-48231 Warendorf, Germany..
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Natl 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 Pharmaceutical Chemistry. Natl Vet Inst SVA, Dept Chem Environm & Feed Hyg, SE-75189 Uppsala, Sweden..
    Nickel in equine sports drug testing - pilot study results on urinary nickel concentrations2016In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 30, no 7, p. 982-984Article in journal (Refereed)
    Abstract [en]

    RationaleThe issue of illicit performance enhancement spans human and animal sport in presumably equal measure, with prohibited substances and methods of doping conveying both ways. Due to the proven capability of unbound ionic cobalt (Co2+) to stimulate erythropoiesis in humans, both human and equine anti-doping regulations have listed cobalt as a banned substance, and in particular in horse drug testing, thresholds for cobalt concentrations applying to plasma and urine have been suggested or established. Recent reports about the finding of substantial amounts of undeclared nickel in arguably licit performance- and recovery-supporting products raised the question whether the ionic species of this transition metal (Ni2+), which exhibits similar prolyl hydroxylase inhibiting properties to Co2+, has been considered as a substitute for cobalt in doping regimens. MethodsTherefore, a pilot study with 200 routine post-competition doping control horse urine samples collected from animals participating in equestrian, gallop, and trotting in Europe was conducted to provide a first dataset on equine urinary Ni2+ concentrations. All specimens were analyzed by conventional inductively coupled plasma mass spectrometry (ICP-MS) to yield quantitative data for soluble nickel. ResultsConcentrations ranging from below the assay's limit of quantification (LOQ, 0.5 ng/mL) up to 33.4 ng/mL with a mean value ( standard deviation) of 6.1 (+/- 5.1) ng/mL were determined for the total nickel content. ConclusionsIn horses, nickel is considered a micronutrient and feed supplements containing nickel are available; hence, follow-up studies are deemed warranted to consolidate potential future threshold levels concerning urine and blood nickel concentrations in horses using larger sets of samples for both matrices and to provide in-depth insights by conducting elimination studies with soluble Ni2+-salt species.

  • 232. Thevis, Mario
    et al.
    Lagojda, Andreas
    Kuehne, Dirk
    Thomas, Andreas
    Dib, Josef
    Hansson, Annelie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Wigger, Tina
    Karst, Uwe
    Schaenzer, Wilhelm
    Characterization of a non-approved selective androgen receptor modulator drug candidate sold via the Internet and identification of in vitro generated phase-I metabolites for human sports drug testing2015In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 29, no 11, p. 991-999Article in journal (Refereed)
    Abstract [en]

    RATIONALE: Potentially performance-enhancing agents, particularly anabolic agents, are advertised and distributed by Internet-based suppliers to a substantial extent. Among these anabolic agents, a substance referred to as LGD-4033 has been made available, comprising the core structure of a class of selective androgen receptor modulators (SARMs). METHODS: In order to provide comprehensive analytical data for doping controls, the substance was obtained and characterized by nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography/electrospray ionization high resolution/high accuracy tandem mass spectrometry (LC/ESI-HRMS). Following the identification of 4-(2-(2,2,2-trifluoro-1-hydroxyethyl) pyrrolidin-1-yl)-2-(trifluoromethyl) benzonitrile, the substance was subjected to in vitro metabolism studies employing human liver microsomes and Cunninghamella elegans (C. elegans) preparations as well as electrochemical metabolism simulations. RESULTS: By means of LC/ESI-HRMS, five main phase-I metabolites were identified as products of liver microsomal preparations including three monohydroxylated and two bishydroxylated species. The two most abundant metabolites (one mono-and one bishydroxylated product) were structurally confirmed by LC/ESI-HRMS and NMR. Comparing the metabolic conversion of 4-(2-(2,2,2-trifluoro-1-hydroxyethyl) pyrrolidin-1-yl)-2-(trifluoromethyl) benzonitrile observed in human liver microsomes with C. elegans and electrochemically derived metabolites, one monohydroxylated product was found to be predominantly formed in all three methodologies. CONCLUSIONS: The implementation of the intact SARM-like compound and its presumed urinary phase-I metabolites into routine doping controls is suggested to expand and complement existing sports drug testing methods.

  • 233. Thevis, Mario
    et al.
    Lagojda, Andreas
    Thomas, Andreas
    Dib, Josef
    Hansson, Annelie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Wigger, Tina
    Karst, Uwe
    Schänzer, Wilhelm
    Charactierization of a selective androgen receptor modulator drug candidate and identification of in vitro generated metabolites for sports drug testing2015Conference paper (Other academic)
  • 234.
    Thorén, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    The investigation of the biotransformation products formed by Cunninghamella elegans for different classes of drugs by the use of UPLC Q-TOF MS2015Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The fungus Cunninghamella elegans has in many studies shown to have abiotransformation similar to the metabolism of mammals. If the biotransformation isgeneral, it enables the production of metabolites by the fungus and the use asreference material. The purpose of the project were to examine whether themetabolic process of C. elegans is general, with respect to the formation ofglucosides, and can be applied to different classes of drugs. During the project, theanalyses were performed on a UPLC Q-TOF, run in both MSE and MSMS mode. Themobile phase used consisted of MeOH and 0.1 % formic acid in MQ water. Toincrease the concentration of possible glucosides, the samples were subjected to anacidic or alkaline SPE. Glucosides were detected in the fungal incubates of diclofenac,buprenorphine, norbuprenorphine and oxazepam. For diclofenac, besides twodifferent glucosides (diclofenac glucoside and hydroxylated diclofenac glucoside), ahydroxylated metabolite and a hydroxylated metabolite conjugated with sulfate werediscovered. In the samples containing buprenorphine, the phase I metabolitenorbuprenorphine was also encountered. Further, in the fungal incubates ofdexamethasone a defluorinated metabolite was identified, which is a metabolicpathway never before described for C. elegans.ISSN: 1650

  • 235. Thörn, H
    et al.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Knutson, L
    Yasin, M
    Dickinson, P
    Lennernäs, H
    Different effects of ketoconazole on the stereoselective first-pass metabolism of R/S-verapamil in the intestine and the liver2009Conference paper (Other academic)
  • 236.
    Thörn, Helena Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Knutson, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Yasin, Mohammed
    Dickinson, Paul
    Lennernäs, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Different effects of ketoconazole on the stereoselective first-pass metabolism of R/S-verapamil in the intestine and the liver: important for the mechanistic understanding of first-pass drug-drug interactions2009In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 37, no 11, p. 2186-2196Article in journal (Refereed)
    Abstract [en]

    In this acute study a pig jejunal intestinal perfusion model with multiple plasma sampling sites and three different administration routes was used to investigate the quantitative contribution of the intestine versus liver to the first-pass extraction of each enantiomer of verapamil (VER). A subclinical dose of ketoconazole (8 mg) was coadministered in the perfusion solution to selectively inhibit gut wall CYP3A. Both enantiomers of VER and its main metabolite norverapamil (NOR) as well as the inhibitor ketoconazole were quantified in all plasma compartments by liquid chromatography-tandem mass spectrometry. The overall first-pass metabolic extraction of VER and the metabolite NOR was shown to be stereoselective with the S-isomer being more extensively extracted. For VER the ratio of R- enantiomer to S-enantiomer was greater in the hepatic vein than in the portal vein (approximately 2.2 versus 1.4), indicating that the stereoselective metabolism of VER in pigs mainly occurs on the first pass through the liver and not in the intestine. Ketoconazole increased the area under the curve from time 0 to 6 h and C(max) of R- and S-VER at least 3-fold in the portal vein, most likely explained by inhibition of gut wall metabolism. Conversely, hepatic extraction was increased because the effect of gut wall metabolism was not observed at the peripheral sampling sites. In conclusion, this study provided novel and more direct information on the contribution of the intestine and the liver, respectively, to the overall first-pass extraction of racemic VER.

  • 237. Tretzel, Laura
    et al.
    Thomas, Andreas
    Piper, Thomas
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Geyer, Hans
    Schänzer, Wilhelm
    Thevis, Mario
    Fully automated determination of nicotine and its major metabolites in whole blood by means of a DBS online-SPE LC-HR-MS/MS approach for sports drug testing2016In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 123, p. 132-140Article in journal (Refereed)
    Abstract [en]

    Dried blood spots (DBS) represent a sample matrix collected under minimal-invasive, straightforward and robust conditions. DBS specimens have been shown to provide appropriate test material for different analytical disciplines, e.g., preclinical drug development, therapeutic drug monitoring, forensic toxicology and diagnostic analysis of metabolic disorders in newborns. However, the sample preparation has occasionally been reported as laborious and time consuming. In order to minimize the manual workload and to substantiate the suitability of DBS for high sample-throughput, the automation of sample preparation processes is of paramount interest. In the current study, the development and validation of a fully automated DBS extraction method coupled to online solid-phase extraction using the example of nicotine, its major metabolites nornicotine, cotinine and trans-3′-hydroxycotinine and the tobacco alkaloids anabasine and anatabine is presented, based on the rationale that the use of nicotine-containing products for performance-enhancing purposes has been monitored by the World Anti-Doping Agency (WADA) for several years. Automation-derived DBS sample extracts were directed online to liquid chromatography high resolution/high mass accuracy tandem mass spectrometry, and target analytes were determined with support of four deuterated internal standards. Validation of the method yielded precise (CV <7.5% for intraday and <12.3% for interday measurements) and linear (r2 > 0.998) results. The limit of detection was established at 5 ng mL−1 for all studied compounds, the extraction recovery ranged from 25 to 44%, and no matrix effects were observed. To exemplify the applicability of the DBS online-SPE LC–MS/MS approach for sports drug testing purposes, the method was applied to authentic DBS samples obtained from smokers, snus users, and e-cigarette users. Statistical evaluation of the obtained results indicated differences in metabolic behavior depending on the route of administration (inhalative versus buccal absorption) in terms of the ratio of nicotine and nornicotine.

  • 238.
    Tyrefors, Niklas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Michelsen, Peter
    Grubb, Anders
    Two new types of assays to determine protein concentrations in biological fluids using mass spectrometry of intact proteins with cystatin C in spinal fluid as an example2014In: Scandinavian Journal of Clinical and Laboratory Investigation, ISSN 0036-5513, E-ISSN 1502-7686, Vol. 74, no 6, p. 546-554Article in journal (Refereed)
    Abstract [en]

    There is no reference method that is generally acknowledged to be unbiased for the determination of the concentration of any protein in biological fluids. This is probably because mass spectrometry (MS) methods acknowledged as reference methods for determination of low molecular mass substances in biological fluids, e.g. creatinine, have been difficult to adapt for proteins. Here we suggest two tentative MS methods, which might be used as reference methods for the determination of protein concentrations in biological fluids. One is based upon the addition to the fluid of a non-proteome reference protein, very similar to the one to be measured, and analyzing the ratio between the corresponding peaks in a selected ion monitoring (SIM) chromatogram. We call this method LC-MS-NPRP (NPRP, Non-Proteome Reference Protein). The other method is based upon the classical standard addition assay for low molecular mass substances. The results of these assays for cystatin C in spinal fluid were compared to those obtained by an immunoassay. Both methods indicated lower concentration than the immunoassay. This was found to be due to the presence of a significant fraction of monohydroxylated cystatin C in spinal fluid. It turned out that the sum of the unhydroxylated and hydroxylated cystatin C concentrations, determined by either of the two MS methods, were close to the results obtained by the immunoassay. These MS-based methods analyze intact proteins and therefore seem more suitable for the determination of protein concentrations in biological fluids than other MS-based methods requiring proteolytic degradation with its inherent lack of precision.

  • 239.
    van Tricht, Ewoud
    et al.
    Janssen Vaccines & Prevent BV, Pharmaceut & Analyt Dev, Newtonweg 1, NL-2333 CP Leiden, Netherlands..
    Geurink, Lars
    Janssen Vaccines & Prevent BV, Pharmaceut & Analyt Dev, Newtonweg 1, NL-2333 CP Leiden, Netherlands..
    Backus, Harold
    Janssen Vaccines & Prevent BV, Pharmaceut & Analyt Dev, Newtonweg 1, NL-2333 CP Leiden, Netherlands..
    Germano, Marta
    Janssen Vaccines & Prevent BV, Pharmaceut & Analyt Dev, Newtonweg 1, NL-2333 CP Leiden, Netherlands..
    Somsen, Govert W.
    Vrije Univ Amsterdam, AIMMS Div BioMol Anal, Fac Sci, De Boelelaan 1083, NL-1081 HV Amsterdam, Netherlands..
    Sänger - van de Griend, Cari
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Kantisto BV, Callenburglaan 22, NL-3742 MV Baarn, Netherlands.; Univ Tasmania, Sch Phys Sci Chem, ACROSS, Hobart, Tas, Australia..
    One single, fast and robust capillary electrophoresis method for the direct quantification of intact adenovirus particles in upstream and downstream processing samples2017In: Talanta: The International Journal of Pure and Applied Analytical Chemistry, ISSN 0039-9140, E-ISSN 1873-3573, Vol. 166, p. 8-14Article in journal (Refereed)
    Abstract [en]

    During development of adenovirus-based vaccines, samples have to be analyzed in order to either monitor the production process or control the quality and safety of the product. An important quality attribute is the total concentration of intact adenoviruses, which currently is determined by quantitative polymerase chain reaction (qPCR) or anion exchange-HPLC. Capillary Electrophoresis (CE) was evaluated as alternative to the current methods with the aim to have one single method that allows reliable and fast quantification of adenovirus particles throughout the full process. Intact adenoviruses samples from downstream processing and upstream processing were analyzed directly by CE with UV-detection at 214 nm. Only the samples with high amounts of DNA required a simple sample pretreatment by benzonase. Adenovirus particles were separated from matrix components such as cell debris, residual cell DNA, and/or proteins on a PVA-coated capillary using a BGE consisting of 125 mM Tris, 338 mM tricine and 0.2% v/v polysorbate-20 at pH 7.7. Full factorial design of experiments was used for method optimization as part of the analytical quality by design (AQbD) method development approach. The method was validated for the quantification of adenoviruses on five representative samples from the manufacturing process in the range of 0.5 x10(11)-1.5 x10(11) adenovirus particles per ml (similar to 80 to 250 pmo1/1). The CE method showed intermediate precision of 7.8% RSD on concentration and an accuracy (spiked recovery) of 95-110%. CE proved highly useful for process development support and is being implemented for in-process control testing for adenovirus vaccine manufacturing.

  • 240. van Tricht, Ewoud
    et al.
    Geurink, Lars
    Pajic, Bojana
    Nijenhuis, Johan
    Backus, Harold
    Germano, Marta
    Somsen, Govert W
    Sänger - van de Griend, Cari
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    New capillary gel electrophoresis method for fast and accurate identification and quantification of multiple viral proteins in influenza vaccines2015In: Talanta: The International Journal of Pure and Applied Analytical Chemistry, ISSN 0039-9140, E-ISSN 1873-3573, Vol. 144, p. 1030-1035Article in journal (Refereed)
    Abstract [en]

    Current methods for the identification and/or quantification of viral proteins in influenza virus and virosome samples suffer from long analysis times, limited protein coverage and/or low accuracy and precision. We studied and optimized capillary gel electrophoresis (CGE) in order to achieve faster and enhanced characterization and quantification of viral proteins. Sample preparation as well the composition of the gel buffer was investigated in order to achieve adequate protein separation in relatively short times. The total sample preparation (reduction and deglycosylation) could be carried out efficiently within two hours. Hydrodynamic injection, separation voltage, and capillary temperature were optimized in full factorial design. The final method was validated and showed good performance for hemagglutinin fragment 1 (HA1), hemagglutinin fragment 2 (HA2), matrix protein (M) and nucleoprotein (NP). The CGE method allowed identification of different virus strains based on their specific protein profile. B/Brisbane inactivated virus and virosome samples could be analyzed within one day. The CGE results (titers) were comparable to single radial immune-diffusion (SRID), but the method has the advantage of a much faster time to results. CGE analysis of A/Christchurch from upstream process demonstrated the applicability of the method to samples of high complexity. The CGE method could be used in the same analyte concentration range as the RP-HPLC method, but showed better precision and accuracy. Overall, the total analysis time for the CGE method was much shorter, allowing analysis of 100 samples in 4 days instead of 10 days for SRID.

  • 241. Videhult Pierre, Pernilla
    et al.
    Haglöf, Jakob
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Linder, Birgitta
    Engskog, Mikael K R
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Arvidsson, Torbjörn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Pettersson, Curt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Laurell, Göran
    Ototoxicity of Cisplatin Correlates with Changes in Blood Antioxidant Status2014Conference paper (Other academic)
2345 201 - 241 of 241
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