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  • 151.
    Nilsson, Elin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Amini, Ahmad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Wretlind, Bengt
    Larsson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Pseudomonas aeruginosa infections are prevented in cystic fibrosis patients by avian antibodies binding Pseudomonas aeruginosa flagellin: Pseudomonas aeruginosa infections are prevented in cystic fibrosis patients by avian antibodies binding Pseudomonas aeruginosa flagellin2007In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 856, no 1-2, p. 75-80Article in journal (Refereed)
    Abstract [en]

    Pseudomonas aeruginosa (PA) is the main cause of morbidity and mortality in cystic fibrosis (CF) patients. CF patients with chronic PA infections have a more rapid deterioration of their lung function and the bacteria become impossible to eradicate from the lungs. Antibiotic resistance among PA strains in CF patients is steadily increasing. Specific chicken (IgY) antibodies against PA have been shown to have potential to prevent PA infections in CF. Anti-Pseudomonas IgY reduces PA adhesion to epithelia, but the mechanism has not been fully elucidated. To gain further insight into the prophylactic effect of these antibodies, the immunoreactivity was investigated by 2D electrophoresis of PA strains, immunoblotting and MALDI-TOF-MS. To confirm the identity of the proteins, the tryptic peptides were analyzed by MALDI-TOF-MS to accurately measure their monoisotopic masses as well as determine their amino acid sequences. In order to facilitate fragmentation of the peptides they were N-terminally or C-terminally labeled. Several strains were investigated and anti-Pseudomonas IgY was immunoreactive against all of these strains, which strengthens its potential as a prophylactic treatment against PA. Flagellin was identified as the major antigen. Flagellin is the main protein of the flagella and is crucial for establishing infections in hosts as well as being involved in PA chemotaxis, motility, adhesion and inflammation. Furthermore, secreted flagellin elicits an inflammatory response. In conclusion, anti-Pseudomonas IgY binds flagellin, which may prevent PA infections in CF patients by hindering host invasion.

  • 152.
    Nilsson, Lars B
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    The bioanalytical challenge of determining unbound concentration and protein binding for drugs2013In: Bioanalysis, ISSN 1757-6180, E-ISSN 1757-6199, Vol. 5, no 24, p. 3033-3050Article, review/survey (Refereed)
    Abstract [en]

    Knowledge regarding unbound concentrations is of vital importance when exploring the PK and PD of a drug. The accurate and reproducible determination of plasma protein binding and unbound concentrations for a compound/drug is a serious challenge for the bioanalytical laboratory. When the drug is in equilibrium with the binding protein(s), this equilibrium will shift when physiological conditions are not met. Furthermore, the true unbound fraction/concentration is unknown, and there are numerous publications in the scientific literature reporting and discussing data that have been produced without sufficient control of the parameters influencing the equilibrium. In this Review, different parameters affecting the equilibrium and analysis are discussed, together with suggestions on how to control these parameters in order to produce as trustworthy results for unbound concentrations/fractions as possible.

  • 153.
    Nilsson, Lars B.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Ahnoff, Martin
    Jonsson, Ove.
    Capillary microsampling in the regulatory environment: validation and use of bioanalytical capillary microsampling methods2013In: Bioanalysis, ISSN 1757-6180, E-ISSN 1757-6199, Vol. 5, no 6, p. 731-738Article in journal (Refereed)
    Abstract [en]

    Capillary microsampling (CMS) has recently been introduced as a response to the demands for more ethical use of lab. animals according to the 3R principles. In CMS, an exact vol. of the blood, plasma or other biofluid is collected in a capillary from which it is washed out, resulting in a dild. sample that can be handled using the existing equipment in the bioanal. lab. CMS differs from traditional large vol. sampling as the microsample is dild. before further handling and anal., and reanal. is performed using the dild. sample. This has some implications for the validation and this report is an attempt to clarify how to validate and use CMS methods in a regulatory environment. CMS also shows some distinct new opportunities: labile analytes can be immediately stabilized at sample collection and the addn. of the internal std. to the whole sample can improve anal. performance. The experiences from 5 years use of CMS of plasma and blood for detn. of drug exposure in animal studies are reviewed.

  • 154. Njobeh, P. B.
    et al.
    Dutton, M. F.
    Åberg, Annica Tevell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Haggblom, P.
    Estimation of multi-mycotoxin contamination in South African compound feeds2012In: Toxins, ISSN 2072-6651, E-ISSN 2072-6651, Vol. 4, no 10, p. 836-848Article in journal (Refereed)
    Abstract [en]

    A total of 92 commercial compound feeds from South Africa were investigated for various mycotoxins. The data reveal the highest incidence of feed contamination for fumonisins (FB) (range: 104-2999 μg/kg) followed by deoxynivalenol (DON) (range: 124-2352 μg/kg) and zearalenone (ZEA) (range: 30-610 μg/kg). The incidence of ochratoxin A (OTA) and aflatoxins (AF)-contaminated samples were generally low, i.e., 4% and 30% of samples with levels ranging between 6.4 and 17.1 μg/kg (mean: 9.9 μg/kg) for OTA and 0.2 to 71.8 μg/kg (mean: 9.0 μg/kg) for AF. No samples contained T-2 toxin or HT-2 toxin. However, all samples analyzed were contaminated with at least one mycotoxin with a majority containing several mycotoxins. In particular, 3 of 4 positive samples mainly cattle feeds that had relatively high contents of OTA (ranging from 7 to 17.1 μg/kg) also contained high amounts of AF (>27.5 μg/kg) together with FB, DON and ZEA. Apart from a few samples, the levels of mycotoxins may be regarded as safe for livestock production in South Africa. However, the persistent co-occurrence of mycotoxins in samples, especially those at high concentrations, i.e., AF and OTA, together with other mycotoxins studied, may elicit synergistic or additive effects in animals. This should raise concern as multiple contaminations may pose a risk to livestock production and health.

  • 155. Olsen, L.
    et al.
    Olsson, K.
    Hydbring-Sandberg, E.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Ingvast-Larsson, C.
    Methadone in healthy goats: Pharmacokinetics, behaviour and blood pressure2013In: Research in Veterinary Science, ISSN 0034-5288, E-ISSN 1532-2661, Vol. 95, no 1, p. 231-237Article in journal (Refereed)
    Abstract [en]

    The pharmacokinetics and effects of the opioid methadone on behaviour, arterial blood pressure, heart rate and haematocrit were studied in goats. Two goats received methadone (0.2 mg/kg) intravenously and the terminal half-life was 88 and 91 min, the volume of distribution 8.4 and 6.1 L/kg, and clearance 86 and 123 mL/min/kg. In a crossover study eight goats received methadone (0.6 mg/kg) or 0.15 M NaCl subcutaneously (SC). After SC administration bioavailability was complete and the terminal half-life was 215 +/- 84 min (mean +/- SD), T-max 31 +/- 15 min and C-max 45 +/- 11 ng/mL. Blood pressure and haematocrit increased while heart rate did not change. The goats did not ruminate and they climbed, scratched, gnawed and showed tail-flicking after SC methadone in contrast to NaCl administration. The use of methadone in goats may be restricted due to the inhibition of rumination and the rather short half-life.

  • 156. Olsen, Lena
    et al.
    Bremer, Hanna
    Olofsson, Karin
    Brojer, Johan
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Bergh, Anna
    Nostell, Katarina
    Brostrom, Hans
    Bengtsson, Bjorn
    Ingvast-Larsson, Carina
    Intramuscular administration of sodium benzylpenicillin in horses as an alternative to procaine benzylpenicillin2013In: Research in Veterinary Science, ISSN 0034-5288, E-ISSN 1532-2661, Vol. 95, no 1, p. 212-218Article in journal (Refereed)
    Abstract [en]

    The aim was to supply information about the possibility of replacing the procaine salt with the sodium salt for benzylpenicillin IM treatment in horse in order to diminish the risk for procaine adverse effects. In a crossover study eight horses were given 15 mg/kg sodium benzylpenicillin (Na-pc) twice daily or procaine benzylpenicillin (control) once daily IM for four days. The half-life of Na-pc was 1.9 h, peak concentration was 14,600 ng/mL reached after about 23 min. Trough plasma concentration Was 281 ng/mL and protein binding 62.8%. The fT > MIC for Staphylococcus aureus was 63% and 100% for Streptococcus equi subsp. equi and Streptococcus zooepidemicus, indicating an adequate antimicrobial therapy. However, Na-pc cannot be recommended from a welfare point of view since the horses showed more pain related behaviour and more pain and swelling compared to the control treatment.

  • 157. Olsén, Lena
    et al.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Broström, Hans
    Olsson, Ulf
    Mazogi, Behnaz
    Sundqvist, Marie
    Tjälve, Hans
    Ingvast-Larsson, Carina
    Pharmacokinetics and effects of cetirizine in horses with insect bite hypersensitivity2011In: The Veterinary Journal, ISSN 0007-1935, E-ISSN 1879-3606, Vol. 187, no 3, p. 347-351Article in journal (Refereed)
    Abstract [en]

    Horses with insect bite hypersensitivity (IBH) have difficulty in completely avoiding allergens, so effective treatment options are required. A randomised, placebo controlled and double blinded field study was conducted to determine the pharmacokinetics and efficacy in reducing dermatitis of the antihistamine cetirizine given orally at 0.4mg/kg twice daily for 3weeks. The influence of protection blankets and stabling were also investigated. The estimated maximum plasma concentration (C(max)) and trough plasma concentration of cetirizine were 135ng/mL and 18ng/mL, respectively. There was no difference in dermatitis reduction between the treatment and placebo groups (P=0.77). The findings indicated that cetirizine was of no apparent benefit in treating IBH at the dose rate tested. The use of blankets and stabling were shown to have favourable influence on the dermatitis (P<0.05) and may be the preferred options to prevent this condition.

  • 158. Olsén, Lena
    et al.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Broström, Hans
    Tjälve, Hans
    Ingvast-Larsson, Carina
    Cetirizine in horses: Pharmacokinetics and pharmacodynamics following repeated oral administration2008In: The Veterinary Journal, ISSN 0007-1935, E-ISSN 1879-3606, Vol. 177, no 2, p. 242-249Article in journal (Refereed)
    Abstract [en]

    The pharmacokinetics of the histamine HI-antagonist cetirizine and its effect on histamine-induced cutaneous wheal formation were studied in six healthy horses following repeated oral administration. After three consecutive administrations of cetirizine (0.2 mg/kg body weight, bw) every 12 h, the trough plasma concentration of cetirizine was 16 +/- 4 ng/mL (mean +/- SD) and the wheal formation was inhibited by 45 +/- 23%. After four additional administrations of cetirizine (0.4 mg/kg bw) every 12 h, the trough plasma concentration was 48 +/- 15 ng/mL and the wheal formation was inhibited by 68 +/- 11%. The terminal half-life was about 5.8 h. A pharmacokinetic/ pharmacodynamic link model showed that the maximal inhibition of wheal formation was about 95% and the EC50 about 18 ng/mL. It is concluded that cetirizine in doses of 0.2-0.4 mg/kg bw administered at 12 h intervals exhibits favourable pharmacokinetic and pharmacodynamic properties without causing visible side effects, and the drug may therefore be a useful antihistamine in equine medicine.

  • 159.
    Paul, Prasanta
    et al.
    Univ Leuven, KU Leuven, Dept Pharmaceut & Pharmacol Sci, Leuven, Belgium..
    Duchateau, Tom
    Univ Leuven, KU Leuven, Dept Pharmaceut & Pharmacol Sci, Leuven, Belgium..
    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, Baarn, Netherlands..
    Adams, Erwin
    Univ Leuven, KU Leuven, Dept Pharmaceut & Pharmacol Sci, Leuven, Belgium..
    Van Schepdael, Ann
    Univ Leuven, KU Leuven, Dept Pharmaceut & Pharmacol Sci, Leuven, Belgium..
    Capillary electrophoresis with capacitively coupled contactless conductivity detection method development and validation for the determination of azithromycin, clarithromycin, and clindamycin2017In: Journal of Separation Science, ISSN 1615-9306, E-ISSN 1615-9314, Vol. 40, no 17, p. 3535-3544Article in journal (Refereed)
    Abstract [en]

    A capillary electrophoresis with capacitively coupled contactless conductivity detection based method for the assay of azithromycin, clarithromycin and clindamycin was optimized and validated in this study. A buffer solution of 20 mM 2-(N-morpholino) ethane sulfonic acid, 40 mM L-histidine and 0.6 mM cetyltrimethylammonium bromide (pH 6.39) was used for the electrophoresis. An uncoated, bare-fused silica capillary (total length 60 cm, effective length 32 cm, 75 mu m id) was used at 25 degrees C. The sample was injected hydrodynamically at 0.5 psi for 5 s. The electrophoresis was conducted at 30 kV in reverse polarity for 6 min with 3 and 2 min of in-between sodium hydroxide (0.1 M) and background electrolyte rinsing, respectively. Ammonium acetate was used as internal standard. This simple and robust method showed reasonable limit of detection and limit of quantitation for azithromycin (0.0125/0.03 mg/mL), clarithromycin (0.017/0.03 mg/mL), and clindamycin (0.038/0.06 mg/mL), with good selectivity, precision both intraday (relative standard deviation <= 1.0%) and interday (relative standard deviation < 3.7%), linearity (R-2 > 0.999) and recovery (99 - 101.7%). The method was successfully applied for the determination of azithromycin, clarithromycin and clindamycin in formulations.

  • 160.
    Persson, Anita M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Evaluation of a Miniaturized Rotating Disk Apparatus for In Vitro Dissolution Rate Measurements in Aqueous Media: Correlation of In Vitro Dissolution Rate with Apparent Solubility2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The general aim of this thesis was to evaluate a newly designed and constructed miniaturized rotating disk apparatus for in vitro dissolution rate measurements of different drug substances from all of the classes in the Biopharmaceutical Classification System (BCS). The new equipment is based on a low volume flow-through cell of Plexiglas, a gold plated magnetic bar and a special designed press. The disk of drug substance (approx. 5 mg) is placed eccentrically in the bar. Rotation speeds were set with a graded magnetic stirrer. An external HPLC pump delivered a continuous flow of aqueous medium to the flow-through cell during dissolution testing.

    A reversed phase high-performance liquid chromatography system using diode array detection (RP-HPLC-DAD) was coupled online to the new equipment. The injections from the miniaturized rotating disk outlet into the quantifying HPLC system were controlled by a six-position switching valve. The injection volumes from the valve and the autosampler, used for the external standards, were statistically evaluated to match each other volumetrically. No analyses were longer than three minutes, using isocratic mode.

    A traditional USP rotating disk apparatus was used as a reference system and the two instruments were shown to be statistically dissimilar in the numerical dissolution rate values probably due to different hydrodynamics, but had approximately the same precision/repeatability. When correlating the logarithmic values of the in vitro dissolution rate (G) with the apparent solubility (S), using shake-flask methodology in the solubility studies, the two apparatuses gave the same correlation patterns. Further correlation studies were done where the media components were altered by the use of different buffer species or additives into the buffers, such as inorganic salts.

    Chemometric tools, e.g. orthogonal partial least squares (OPLS), were used to better evaluate the most influential factors for G and S in different media. The most significant factor for a model basic drug substance (terfenadine) was pH, followed by the ionic strength (I) and added sodium chloride in one of the media. However, the surfactants in the Fasted State Simulated Intestinal Fluid (FaSSIF-V2) were found to be insignificant for G and S in this study (using a 95% confidence interval).

    The new miniaturized apparatus is a promising prototype for in vitro dissolution rate measurements both for early screening purposes and in dissolution testing during drug development, but needs further instrumental improvements.

    List of papers
    1. Design and Characterization of a New Miniaturized Rotating Disk Equipment for In Vitro Dissolution Rate Studies
    Open this publication in new window or tab >>Design and Characterization of a New Miniaturized Rotating Disk Equipment for In Vitro Dissolution Rate Studies
    Show others...
    2008 (English)In: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 97, no 8, p. 3344-3355Article in journal (Refereed) Published
    Abstract [en]

    A miniaturized apparatus for the determination of the apparent in vitro dissolution rate has been designed, constructed and characterized. The miniaturized apparatus was based on a low volume dissolution cell and a disk in a rotating magnetic bar. The disk tablet is pressed directly into the bar with a press designed and constructed for this purpose. It requires approximately 5 mg of substance. The disk was positioned eccentrically on the bar with an external flow of medium to increase the rate of solvent flow over the disk surface. Six different drug substances were used. The dissolution media were sodium phosphate buffer, pH 7.0, and ammonium acetate buffer, pH 6.8. All quantifications were made by integrating the dissolution cell with high-performance liquid chromatography (HPLC) using diode-array detection (DAD). The obtained results were compared with data from a conventional rotating disk equipment, where the disk was centrically mounted. The dissolution rates at 100 rpm seemed to be on an average of 2-3 times higher for the miniaturized apparatus (RSD 0.2-56%). The preliminary studies of this prototype indicate that the miniaturized rotating disk is a promising design for the qualitative estimation of dissolution rates of substances, for example during screening in early drug discovery.

    Place, publisher, year, edition, pages
    Wiley InterScience, 2008
    Keywords
    dissolution rate, in vitro models, physicochemical properties; analytical chemistry, HPLC (high-performance/pressure liquid chromatography), miniaturization
    National Category
    Medicinal Chemistry Medicinal Chemistry
    Research subject
    Analytical Pharmaceutical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-17637 (URN)10.1002/jps.21235 (DOI)000258081100034 ()
    Available from: 2010-01-11 Created: 2009-10-08 Last updated: 2018-01-12Bibliographically approved
    2. Correlation of in vitro dissolution rate and apparent solubility in buffered media using a miniaturized rotating disk equipment: Part I. Comparison with a traditional USP rotating disk apparatus
    Open this publication in new window or tab >>Correlation of in vitro dissolution rate and apparent solubility in buffered media using a miniaturized rotating disk equipment: Part I. Comparison with a traditional USP rotating disk apparatus
    2009 (English)In: Drug Discoveries & Therapeutics, ISSN 1881-7831, Vol. 3, no 3, p. 104-113Article in journal (Refereed) Published
    Abstract [en]

    A correlation of the logarithmic values of the in vitro dissolution rate, G, and the apparent solubility, S, was evaluated in phosphate and ammonium acetate buffer at an initial pH of 7. The dissolution rates were determined with a newly designed and build miniaturized rotating disk equipment, as well as with a traditional rotating disk apparatus. The two apparatuses gave the same correlation pattern of logG and logS. Thirteen diverse drug substances from all of the classes in the Biopharmaceutics Classification System (BCS) were used for the correlation in the phosphate buffer system, with the results from the miniaturized apparatus only. A coefficient of determination, R2, of 0.982 was found if bases formulated as hydrochloride salts were excluded in the correlation. The miniaturized equipment is used for rapid screening of the dissolution rate, approximately 10 min for one run, and consumes small amounts of substance (about 5 mg) and dissolution media. All quantifications were performed by using reversed phase high-performance liquid chromatography (RPHPLC) with a diode array detector (DAD), integrated with the miniaturized rotating disk equipment.

    Place, publisher, year, edition, pages
    IACMHR and SDU-DDSC, 2009
    Keywords
    dissolution rate, solubility, in vitro models, correlation, HPLC (high-performance liquid chromatography)
    National Category
    Medicinal Chemistry
    Research subject
    Analytical Pharmaceutical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-107740 (URN)
    Available from: 2010-01-11 Created: 2009-08-25 Last updated: 2018-01-13Bibliographically approved
    3. Correlation of in vitro dissolution rate and apparent solubility in buffered media using a miniaturized rotating disk equipment: Part II. Comparing different buffer media
    Open this publication in new window or tab >>Correlation of in vitro dissolution rate and apparent solubility in buffered media using a miniaturized rotating disk equipment: Part II. Comparing different buffer media
    2009 (English)In: Drug Discoveries & Therapeutics, ISSN 1881-7831, Vol. 3, no 3, p. 114-122Article in journal (Refereed) Published
    Abstract [en]

    A correlation of the logarithmic values of the in vitro dissolution rate, G, and apparent solubility, S, was made for seven different drug substances from all of the classes in the Biopharmaceutics Classification System (BCS), in four different phosphate buffers. The effect of inorganic salts added as sodium chloride, sodium nitrate, sodium phosphate and sodium sulfate in the buffer media was investigated for the correlation. Triethanolammonium acetate buffer was also included in the study of the correlation of logG vs. logS. The pH was 7.0 ± 0.1 in all of the buffers to mimic a pH condition in intestinal fluids. The dissolution rate was determined with a newly constructed miniaturized rotating disk equipment, which enables fast determinations and consumes only minute quantities of substance (about 5 mg). The solubility was determined by conventional shake-flask methodology, using 1.5 mL solution volumes. All quantifications were performed with reversed phase high-performance liquid chromatography (RP-HPLC) and diode array detection (DAD). The different inorganic anions seemed to affect the solubility more than the dissolution rate. The phosphate and nitrate ions decreased the solubility for amines compared to the chloride ion. The best correlations of logG and logS were however obtained with a triethanolammonium acetate buffer. The good correlation (R2 = 0.991) may be sufficient in initial screening of drug solubility, based on dissolution rates in aqueous buffer media.

    Place, publisher, year, edition, pages
    IACMHR and SDU-DDSC, 2009
    Keywords
    dissolution rate, solubility, in vitro models, correlation, HPLC (high-performance liquid chromatography)
    National Category
    Medicinal Chemistry
    Research subject
    Analytical Pharmaceutical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-107746 (URN)
    Available from: 2010-01-11 Created: 2009-08-25 Last updated: 2018-01-13Bibliographically approved
    4. Multivariate data analysis of factors affecting the in vitro dissolution rate and the apparent solubility for a model basic drug substance in aqueous media
    Open this publication in new window or tab >>Multivariate data analysis of factors affecting the in vitro dissolution rate and the apparent solubility for a model basic drug substance in aqueous media
    2010 (English)In: Pharmaceutical research, ISSN 0724-8741, E-ISSN 1573-904X, Vol. 27, no 7, p. 1309-1317Article in journal (Refereed) Published
    Abstract [en]

    Purpose. To evaluate the usefulness of a miniaturized rotating disk equipment for the determination of factors influencing the in vitro dissolution rate, G, of a model basic drug substance (terfenadine) in different aqueous media, using experimental design and multivariate data analysis. The apparent solubility, S, was included in the chemometric study.

    Methods. The dissolution rate was determined with a miniaturized rotating disk apparatus and the solubility by shake-flask methodology. Media were based on acetate, phosphate or maleate buffers. The later used in fasted state simulated intestinal fluid (FaSSIF-V2). The chemometric analyses included fractional factorial design, principal component analysis (PCA) and orthogonal partial least squares (OPLS). Quantifications were made with a RP-HPLC-DAD system.

    Results. The most influential factor for both G and S of terfenadine in the different media was pH. Apart from the ionic strength and sodium chloride concentration in the acetate medium, the effects of the other variables were insignificant implying no wetting effect of the surfactants.

    Conclusions. The miniaturized rotating disk equipment was suitable to use, in conjunction with the chemometric analyses, in the evaluation of the factors affecting the in vitro dissolution rate. The apparent solubility was found to be influenced by the same factors as G.

    Keywords
    dissolution media, dissolution rate, solubility, chemometrics, miniaturized rotating disk equipment
    National Category
    Medicinal Chemistry
    Research subject
    Analytical Pharmaceutical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-112089 (URN)10.1007/s11095-010-0111-0 (DOI)000278694400012 ()20358263 (PubMedID)
    Available from: 2010-01-08 Created: 2010-01-08 Last updated: 2018-01-12Bibliographically approved
  • 161.
    Persson, Anita M.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Baumann, Kajsa
    Inst för kemi, Göteborgs universitet.
    Sundelöf, Lars-Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Lindberg, Walter
    AstraZeneca, Mölndal.
    Sokolowski, Anders
    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.
    Design and Characterization of a New Miniaturized Rotating Disk Equipment for In Vitro Dissolution Rate Studies2008In: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 97, no 8, p. 3344-3355Article in journal (Refereed)
    Abstract [en]

    A miniaturized apparatus for the determination of the apparent in vitro dissolution rate has been designed, constructed and characterized. The miniaturized apparatus was based on a low volume dissolution cell and a disk in a rotating magnetic bar. The disk tablet is pressed directly into the bar with a press designed and constructed for this purpose. It requires approximately 5 mg of substance. The disk was positioned eccentrically on the bar with an external flow of medium to increase the rate of solvent flow over the disk surface. Six different drug substances were used. The dissolution media were sodium phosphate buffer, pH 7.0, and ammonium acetate buffer, pH 6.8. All quantifications were made by integrating the dissolution cell with high-performance liquid chromatography (HPLC) using diode-array detection (DAD). The obtained results were compared with data from a conventional rotating disk equipment, where the disk was centrically mounted. The dissolution rates at 100 rpm seemed to be on an average of 2-3 times higher for the miniaturized apparatus (RSD 0.2-56%). The preliminary studies of this prototype indicate that the miniaturized rotating disk is a promising design for the qualitative estimation of dissolution rates of substances, for example during screening in early drug discovery.

  • 162.
    Persson, Anita M.
    et al.
    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.
    Sokolowski, Anders
    AS Consulting, Uppsala.
    Correlation of in vitro dissolution rate and apparent solubility in buffered media using a miniaturized rotating disk equipment: Part II. Comparing different buffer media2009In: Drug Discoveries & Therapeutics, ISSN 1881-7831, Vol. 3, no 3, p. 114-122Article in journal (Refereed)
    Abstract [en]

    A correlation of the logarithmic values of the in vitro dissolution rate, G, and apparent solubility, S, was made for seven different drug substances from all of the classes in the Biopharmaceutics Classification System (BCS), in four different phosphate buffers. The effect of inorganic salts added as sodium chloride, sodium nitrate, sodium phosphate and sodium sulfate in the buffer media was investigated for the correlation. Triethanolammonium acetate buffer was also included in the study of the correlation of logG vs. logS. The pH was 7.0 ± 0.1 in all of the buffers to mimic a pH condition in intestinal fluids. The dissolution rate was determined with a newly constructed miniaturized rotating disk equipment, which enables fast determinations and consumes only minute quantities of substance (about 5 mg). The solubility was determined by conventional shake-flask methodology, using 1.5 mL solution volumes. All quantifications were performed with reversed phase high-performance liquid chromatography (RP-HPLC) and diode array detection (DAD). The different inorganic anions seemed to affect the solubility more than the dissolution rate. The phosphate and nitrate ions decreased the solubility for amines compared to the chloride ion. The best correlations of logG and logS were however obtained with a triethanolammonium acetate buffer. The good correlation (R2 = 0.991) may be sufficient in initial screening of drug solubility, based on dissolution rates in aqueous buffer media.

  • 163.
    Persson, Anita M.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Sokolowski, Anders
    AS Consulting, Uppsala.
    Pettersson, Curt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Correlation of in vitro dissolution rate and apparent solubility in buffered media using a miniaturized rotating disk equipment: Part I. Comparison with a traditional USP rotating disk apparatus2009In: Drug Discoveries & Therapeutics, ISSN 1881-7831, Vol. 3, no 3, p. 104-113Article in journal (Refereed)
    Abstract [en]

    A correlation of the logarithmic values of the in vitro dissolution rate, G, and the apparent solubility, S, was evaluated in phosphate and ammonium acetate buffer at an initial pH of 7. The dissolution rates were determined with a newly designed and build miniaturized rotating disk equipment, as well as with a traditional rotating disk apparatus. The two apparatuses gave the same correlation pattern of logG and logS. Thirteen diverse drug substances from all of the classes in the Biopharmaceutics Classification System (BCS) were used for the correlation in the phosphate buffer system, with the results from the miniaturized apparatus only. A coefficient of determination, R2, of 0.982 was found if bases formulated as hydrochloride salts were excluded in the correlation. The miniaturized equipment is used for rapid screening of the dissolution rate, approximately 10 min for one run, and consumes small amounts of substance (about 5 mg) and dissolution media. All quantifications were performed by using reversed phase high-performance liquid chromatography (RPHPLC) with a diode array detector (DAD), integrated with the miniaturized rotating disk equipment.

  • 164.
    Persson, Anita Maria
    et al.
    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.
    Rosén, Josefin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Multivariate data analysis of factors affecting the in vitro dissolution rate and the apparent solubility for a model basic drug substance in aqueous media2010In: Pharmaceutical research, ISSN 0724-8741, E-ISSN 1573-904X, Vol. 27, no 7, p. 1309-1317Article in journal (Refereed)
    Abstract [en]

    Purpose. To evaluate the usefulness of a miniaturized rotating disk equipment for the determination of factors influencing the in vitro dissolution rate, G, of a model basic drug substance (terfenadine) in different aqueous media, using experimental design and multivariate data analysis. The apparent solubility, S, was included in the chemometric study.

    Methods. The dissolution rate was determined with a miniaturized rotating disk apparatus and the solubility by shake-flask methodology. Media were based on acetate, phosphate or maleate buffers. The later used in fasted state simulated intestinal fluid (FaSSIF-V2). The chemometric analyses included fractional factorial design, principal component analysis (PCA) and orthogonal partial least squares (OPLS). Quantifications were made with a RP-HPLC-DAD system.

    Results. The most influential factor for both G and S of terfenadine in the different media was pH. Apart from the ionic strength and sodium chloride concentration in the acetate medium, the effects of the other variables were insignificant implying no wetting effect of the surfactants.

    Conclusions. The miniaturized rotating disk equipment was suitable to use, in conjunction with the chemometric analyses, in the evaluation of the factors affecting the in vitro dissolution rate. The apparent solubility was found to be influenced by the same factors as G.

  • 165.
    Persson, Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Åström, Ove
    Fractional Factorial Design Optimization of the Separation of Pilocarpine and its Degradation Products by Capillary Electrophoresis1997In: 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)
    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.

  • 166.
    Persson, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Bioremediation optimization of diclofenac using Trametes versicolor with emphasis on analytical pharmaceutical chemistry2016Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Diclofenac has been reported to give adverse effects to water-living organisms at

    environmentally relevant concentrations and the removal rate of this substance in

    sewage treatment plants is poor, and therefor other techniques needs to be

    examined. The purpose of this study was to evaluate the ability of the white rot

    fungus Trametes versicolor to degrade diclofenac. An enzymatic assay was developed,

    where the activity of laccase was monitored. The concentration of diclofenac in

    E-flask and bioreactor experiments was measured using UHPLC-Q-Tof with a C18

    column. Three controls were used to fully investigate the observed diclofenac

    concentration decrease. Fungal mycelia were grown in nutrient solution and

    immobilized on polyurethane carriers. The initial concentration of diclofenac was 10

    mg/L and samples were taken at determined intervals during one week. The results

    showed a fast decrease in diclofenac concentration, and a clear difference could be

    seen between the active culture and the controls in the E-flask experiments. The

    E-flask experiments also had a high enzymatic activity throughout the week, while the

    enzymatic activity exponentially decreased in the bioreactor experiment. The

    enzymatic activity decrease corresponded well with the diclofenac concentration in

    the bioreactor, which was lower than the controls within the first four hours, and

    thereafter increased to the same level as the controls. Four degradation products of

    diclofenac were found in the E-flask experiments and the fungi was also capable to

    degrade other aromatic substances that originated from the carrier material.

  • 167.
    Persson-Stubberud, Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Åström, Ove
    Separation of Ibuprofen, Codeine Phosphate, their Degradation Products and Impurities by Capillary Electrophoresis: I. Method Development and Optimization with Fractional factorial Design1998In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 798, no 1-2, p. 307-314Article in journal (Refereed)
    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.

  • 168.
    Persson-Stubberud, Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Åström, Ove
    Separation of Ibuprofen, Codeine Phosphate, their Degradation Products and Impurities by Capillary Electrophoresis: II. Validation1998In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 826, no 1, p. 95-102Article in journal (Refereed)
    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.

  • 169.
    Pettersson, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Evaluating commercial MIP-columns for extraction of metoprolol with metabolites in environmental analysis2013Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    There is of great importance to have a well suited sample preparation method when studying pharmaceutical residues in the environment, due to low concentrations and often very complex matrices. A rather newly developed solid phase for extraction is the molecularly imprinted polymers (MIP), which consists of cross linked polymers with cavities formed after shape and chemical function of a molecule. The aim of this study was to investigate a commercial available molecularly imprinted polymer cartridge (Affinilute™ MIP) for β-blockers by the use of metoprolol and two of its major metabolites, α-hydroxymetoprolol and metoprolol acid in effluent wastewater. This is, to the best of the authours knowledge, the first time the Affinilute™ MIP for β-blockers have been investigated for metabolites. The solid phase extraction (SPE) method was optimized in regard to pH, volume of sample, volume of washing solvents and type of eluent. The extraction recoveries and matrix effects of the SPE method were evaluated for the three analytes. The developed SPE method with the Affinlute™ MIP was compared to an optimized SPE method with Oasis® HLB cartridges. According to this study the molecularly imprinted polymers showed rather good results for extraction of metoprolol and the metabolite α-hydroxymetoprolol, but the Oasis® HLB cartridge was more suitable for all three of the analytes. In previous studies the use of MIP-cartridges have shown to reduce matrix effects such as ion suppression, more efficient than the HLB-cartridges, but in this study ion enhancement was observed in both cases but with less impact using HLB-cartridges.

  • 170.
    Pierre, Pernilla Videhult
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery. Karolinska Inst, Div Audiol, Dept Clin Sci Intervent & Technol, Alfred Nobels Alle 10 Plan 5, SE-14183 Huddinge, Sweden.
    Haglöf, Jakob
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Linder, Birgitta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    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.
    Fransson, Anette E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Laurell, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Cisplatin-induced metabolome changes in serum: an experimental approach to identify markers for ototoxicity2017In: Acta Oto-Laryngologica, ISSN 0001-6489, E-ISSN 1651-2251, Vol. 137, no 10, p. 1024-1030Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Ototoxicity from treatment with the anticancer drug cisplatin remains a clinical problem. A wide range of intracellular targets of cisplatin has been found in vivo.

    AIM: To investigate cisplatin-induced change of the serum metabolite profile and its association with ototoxicity.

    MATERIAL AND METHODS: Guinea pigs (n = 14) were treated with cisplatin (8 mg/kg b.w., i.v.) 30 min after administration of the otoprotector candidate sodium thiosulfate (group STS; n = 7) or sodium chloride (group NaCl; n = 7). Ototoxicity was evaluated by ABR (3-30 kHz) before and 4 d after drug treatment, and by assessment of hair cell loss. A blood sample was drawn before and 4 d after drug treatment and the polar metabolome in serum was analyzed using LC-MS.

    RESULTS: Cisplatin-treatment caused significant threshold elevations and outer hair cell (OHC) loss in both groups. The ototoxicity was generally lower in group STS, but a significant difference was reached only at 30 kHz (p = .007). Cisplatin treatment altered the metabolite profile significantly and similarly in both groups. A significant inverse correlation was found between L-acetylcarnitine, N-acetylneuraminic acid, ceramide, and cysteinylserine and high frequency hearing loss in group NaCl. The implication of these correlations should be explored in targeted studies.

  • 171.
    Pierson, Johan
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Development of Methods for Protein and Peptide Analysis Applied in Neuroscience Utilizing Mass Spectrometry2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis describes the utilization of the matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and electrospray ionization (ESI) MS techniques for analysis of complex brain tissue samples.

    Direct molecular profiling of biological samples using MALDI MS is a powerful tool for identifying phenotypic markers. MALDI MS-profiling of proteins and peptides directly on brain tissue sections was used for the first time to study experimental models of Parkinson’s disease (PD). The mass spectrometer was used to map the peptide and protein expression directly on 12 µm tissue sections in mass-to-charge (m/z) values, providing the capability of mapping specific molecules of the original sample, that is, localization, intensity and m/z ratio. Several protein and peptide expression profile differences were found in the dopamine denervated brains when compared to the corresponding controls, for example, calmodulin, cytochrome c, cytochrome c oxidase, and the neuroimmunophilin protein FKBP-12. The increased expression of FKBP-12 from the profiling experiments was supported by mRNA expression analysis and two-dimensional gel electrophoresis separation analysis. Multiple genetic deficits have linked impaired ubiquitin-conjugation pathways to various forms of familiar PD. This study showed for the first time an increased level of unconjugated ubiquitin specifically in the dorsal striatum of the dopamine depleted PD brain. The strength of the MALDI MS-profiling technique is that a minimum of sample handling and manipulation is necessary pre-analysis. This ensures preservation of the spatial localization of the biomolecules in the tissue section.

    Biological liquid samples often contain high amounts of salt that is non-compatible with the ESI MS technique. A nano-flow capillary liquid chromatography (nanoLC) system coupled on-line with ESI-MS was used to study the metabolism of the peptide LVV-hemorphin-7 in the brain and blood using in vivo microdialysis. The microdialysis technique provides capabilities for very precise sampling in specific brain regions. The combination of on-line desalting and pre-concentration by nanoLC with ESI MS is a powerful tool to detect minute concentration of metabolic fragments and endogenous biomolecules.

    The utilization of mass spectrometry in neuroscience applications provides a uniquely advantageous tool for the analysis of complex biochemical events that underlie the pathological symptoms expressed in different disease states. Furthermore, the MALDI-MS profiling technique shows great potential for the future with regards to proteome analysis and drug discovery.

    List of papers
    1. In vivo processing of LVV-hemorphin-7 in rat brain and blood utilizing microdialysis combined with electrospray mass spectrometry
    Open this publication in new window or tab >>In vivo processing of LVV-hemorphin-7 in rat brain and blood utilizing microdialysis combined with electrospray mass spectrometry
    Show others...
    2003 In: Rapid Communications In Mass Spectrometry, Vol. 17, p. 838–844-Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-92417 (URN)
    Available from: 2004-11-09 Created: 2004-11-09Bibliographically approved
    2. Molecular Profiling of Experimental Parkinson’s Disease: Direct Analysis of Peptides and Proteins on Brain Tissue Sections by MALDI Mass Spectrometry
    Open this publication in new window or tab >>Molecular Profiling of Experimental Parkinson’s Disease: Direct Analysis of Peptides and Proteins on Brain Tissue Sections by MALDI Mass Spectrometry
    Show others...
    2004 In: Journal of Proteome Research, Vol. 3, p. 289- 295Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-92418 (URN)
    Available from: 2004-11-09 Created: 2004-11-09Bibliographically approved
    3. Increased Levels of Ubiquitin in the 6-OHDA-Lesioned Striatum of Rats
    Open this publication in new window or tab >>Increased Levels of Ubiquitin in the 6-OHDA-Lesioned Striatum of Rats
    2005 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 4, no 2, p. 223-226Article in journal (Refereed) Published
    Abstract [en]

    Multiple genetic deficits have linked impaired ubiquitin-conjugation pathways to various forms of familiar Parkinson's disease. We therefore examined the possible role of 6-hydroxydopamine, a dopaminergic neurotoxin used in Parkinson's disease experimental models, in causing protein degradation and its association with the ubiquitin proteasome system. Using unilaterally 6-hydroxydopamine-denervated rats and mass spectrometry profiling directly on brain tissue sections, we here report for the first time an increased level of unconjugated ubiquitin specifically in the dorsal striatum of the dopamine depleted hemisphere. No similar changes were found in the intact hemisphere or in the ventral striatum of the dopamine depleted hemisphere. The lesioning of the dopamine innervation to the striatum was confirmed by a strongly reduced dopamine transporter binding in the striatum, indicating an abundant loss of dopamine neurons. These results suggest that denervation of dopamine neurons per se is implicated in the regulation of ubiquitin pathways, at least in a classical animal model of Parkinson's disease. This study adds additional information regarding the involvement of the ubiquitin-proteasome system in Parkinson's disease.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-92419 (URN)10.1021/pr049836h (DOI)15822896 (PubMedID)
    Available from: 2004-11-09 Created: 2004-11-09 Last updated: 2017-12-14Bibliographically approved
    4. Increased striatal mRNA transcription and active protein expression of the immunophilin FKBP-12 in experimental Parkinson’s disease models
    Open this publication in new window or tab >>Increased striatal mRNA transcription and active protein expression of the immunophilin FKBP-12 in experimental Parkinson’s disease models
    Show others...
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-92420 (URN)
    Available from: 2004-11-09 Created: 2004-11-09 Last updated: 2010-01-13Bibliographically approved
  • 172.
    Pierson, Johan
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Norris, Jeremy L.
    Aerni, Hans-Rudolf
    Svenningsson, Per
    Caprioli, Richard M.
    Andrén, Per E.
    Molecular Profiling of Experimental Parkinson’s Disease: Direct Analysis of Peptides and Proteins on Brain Tissue Sections by MALDI Mass Spectrometry2004In: Journal of Proteome Research, Vol. 3, p. 289- 295Article in journal (Refereed)
  • 173.
    Pierson, Johan
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Sköld, Karl
    Svensson, Marcus
    Caprioli, Richard M.
    Svenningsson, Per
    Andrén, Per E.
    Increased striatal mRNA transcription and active protein expression of the immunophilin FKBP-12 in experimental Parkinson’s disease modelsManuscript (Other academic)
  • 174.
    Pierson, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Svenningsson, Per
    Caprioli, Richard M.
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Increased Levels of Ubiquitin in the 6-OHDA-Lesioned Striatum of Rats2005In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 4, no 2, p. 223-226Article in journal (Refereed)
    Abstract [en]

    Multiple genetic deficits have linked impaired ubiquitin-conjugation pathways to various forms of familiar Parkinson's disease. We therefore examined the possible role of 6-hydroxydopamine, a dopaminergic neurotoxin used in Parkinson's disease experimental models, in causing protein degradation and its association with the ubiquitin proteasome system. Using unilaterally 6-hydroxydopamine-denervated rats and mass spectrometry profiling directly on brain tissue sections, we here report for the first time an increased level of unconjugated ubiquitin specifically in the dorsal striatum of the dopamine depleted hemisphere. No similar changes were found in the intact hemisphere or in the ventral striatum of the dopamine depleted hemisphere. The lesioning of the dopamine innervation to the striatum was confirmed by a strongly reduced dopamine transporter binding in the striatum, indicating an abundant loss of dopamine neurons. These results suggest that denervation of dopamine neurons per se is implicated in the regulation of ubiquitin pathways, at least in a classical animal model of Parkinson's disease. This study adds additional information regarding the involvement of the ubiquitin-proteasome system in Parkinson's disease.

  • 175.
    Prasanta, Paul
    et al.
    KU Leuven Univ Leuven, Pharmaceut Anal, Dept Pharmaceut & Pharmacol Sci, O&N2,PB 923,Herestr 49, B-3000 Leuven, Belgium.
    Van Laeken, Christophe
    KU Leuven Univ Leuven, Pharmaceut Anal, Dept Pharmaceut & Pharmacol Sci, O&N2,PB 923,Herestr 49, B-3000 Leuven, Belgium.
    Sänger-van de Griend, Cari
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Kantisto BV, Callenburglaan 22, NL-3742 MV Baarn, Netherlands.
    Adams, Erwin
    KU Leuven Univ Leuven, Pharmaceut Anal, Dept Pharmaceut & Pharmacol Sci, O&N2,PB 923,Herestr 49, B-3000 Leuven, Belgium.
    Van Schepdael, Ann
    KU Leuven Univ Leuven, Pharmaceut Anal, Dept Pharmaceut & Pharmacol Sci, O&N2,PB 923,Herestr 49, B-3000 Leuven, Belgium.
    CE-C4D method development and validation for the assay of ciprofloxacin2016In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 129, p. 1-8Article in journal (Refereed)
    Abstract [en]

    A capillary electrophoresis method with capacitively coupled contactless conductivity detection (CE-C(4)D) has been developed, optimized and validated for the determination of ciprofloxacin. Ciprofloxacin is a member of the fluoroquinolone antibiotics with a broad spectrum bactericidal activity and recommended for complicated respiratory infections, sexually transmitted diseases, tuberculosis, bacterial diarrhea etc. Method development was conducted with major focus on the quality by design (QbD) approach. During method development, multiple buffers were tried at different ionic strength. However, the optimized method finally involved a very simple background electrolyte, monosodium citrate at a concentration of 10mM without pH adjustment. The optimized CE-C(4)D method involved an uncoated fused silica capillary (59/39cm, 50μm i.d.) and hydrodynamic sample injection at a pressure of 0.5 p.s.i. for 5s. The actual separation was conducted for 10min at normal polarity with a voltage of 20kV corresponding to 5.9μA current. LiCl (1mg/mL) was used as an internal standard. The optimized method is robust and accurate (recovery >98%) which rendered the ciprofloxacin peak within five minutes with good linearity (R(2)>0.999) in the concentration range of 0.0126-0.8mg/mL. The repeatability is expressed by percentage relative standard deviation (%RSD) of the relative peak areas (RPA) and it showed good repeatability both intra-day (<3%) and inter-day (3.1%). This method, proven to be free of matrix interference, showed that the estimated percent content of ciprofloxacin (102%) was within the official requirements. Moreover, due to its ease of use and robustness, the method should also be applicable in less well controlled laboratory environments.

  • 176.
    Roslin, Sara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    De Rosa, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Deuther-Conrad, Winnie
    Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Research Site Leipzig, 04318 Leipzig, Germany.
    Eriksson, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Odell, Luke R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Antoni, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Brust, Peter
    Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Research Site Leipzig, 04318 Leipzig, Germany.
    Larhed, Mats
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Synthesis and In Vitro Evaluation of 5-Substituted Benzovesamicol Analogs containing N-Substituted Amides as Potential Positron Emission Tomography Tracers for the Vesicular Acetylcholine Transporter2017In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 25, no 19, p. 5095-5106Article in journal (Refereed)
    Abstract [en]

    Herein, new ligands for the vesicular acetylcholine transporter (VAChT), based on a benzovesamicol scaffold, are presented. VAChT is acknowledged as a marker for cholinergic neurons and a positron emission tomography tracer for VAChT could serve as a tool for quantitative analysis of cholinergic neuronal density. With an easily accessible triflate precursor, aminocarbonylations were utilized to evaluate the chemical space around the C5 position on the tetrahydronaphthol ring. Synthesized ligands were evaluated for their affinity and selectivity for VAChT. Small, preferably aromatic, N-substituents proved to be more potent than larger substituents. Of the fifteen compounds synthesized, benzyl derivatives (+/-)-7i and (+/-)-7l had the highest affinities for VAChT. Compound (+/-)-7i was chosen to investigate the importance of stereochemistry for binding to VAChT and selectivity toward the sigma(1) and sigma(2) receptors. Enantiomeric resolution gave (+/-)-7i and (-)-7i, and the eutomer showed seven times better affinity. Although racemate (+/-)-7i was initially promising, the affinity of (-)-7i for VAChT was not better than 56.7 nM which precludes further preclinical evaluation. However, the nanomolar binding together with the ready synthesis of [C-11]-(+/-)-7i shows that (-)-7i can serve as a scaffold for future optimizations to provide improved C-11-labelled VAChT PET tracers.

  • 177.
    Rosén, Johan
    et al.
    Natl Food Agcy, Box 622, SE-75126 Uppsala, Sweden..
    Westerberg, Erik
    Natl Food Agcy, Box 622, SE-75126 Uppsala, Sweden..
    Hellenäs, Karl-Erik
    Natl Food Agcy, Box 622, SE-75126 Uppsala, Sweden..
    Salomonsson, Matilda L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Natl Vet Inst SVA, Sect Chem Anal, Dept Chem Environm & Feed Hyg, SE-75189 Uppsala, Sweden..
    A new method for analysis of underivatized free beta-methylamino-alanine: Validation and method comparison2016In: Toxicon, ISSN 0041-0101, E-ISSN 1879-3150, Vol. 121, p. 105-108Article in journal (Refereed)
    Abstract [en]

    A new method was developed for analysis of free beta-Methylamino-alanine (BMAA) in biological matrices. The method is based on direct analysis of the underivatized molecule, using an amide column for separation by Hydrophilic Interaction Liquid Chromatography (HILIC) and detection by tandem mass spectrometry (MS/MS) using a deuterium labeled internal standard. The use of Ultra-High Performance Liquid Chromatography (UHPLC) combined with MS/MS detection allowed for high chromatographic resolution and a low limit of detection (0.025 mu g/g wet weight (ww) in mussels). The method was validated by analyzing spiked blank mussels from the Baltic Sea (0.15-4.4 mu g/g (ww), trueness 99%-105%, RSD 2%-8%). An inter-laboratory comparative analysis of extracts of mussel was performed. The mussels were extracted according to an established protocol for analysis of free BMAA, and the extracts were then analyzed in parallel by the new method and a validated procedure based on detection of BMAA derivatized with dansyl chloride. Both methods detected BMAA in similar concentrations. Thus, derivatization with dansyl chloride did not influence the results compared to direct detection. The new method presents an alternative to the commonly applied derivatization step, and is proved through validation and method comparison to reliably identify and quantify free BMAA at low concentration levels.

  • 178. Rundlöf, Torgny
    et al.
    Mathiasson, Marie
    Bekiroglu, Somer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hakkarainen, Birgit
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Bowden, Tim
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
    Arvidsson, Torbjörn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Survey and qualification of internal standards for quantification by 1H NMR spectroscopy2010In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 52, no 5, p. 645-651Article in journal (Refereed)
    Abstract [en]

    In quantitative NMR (qNMR) selection of an appropriate internal standard proves to be crucial. In this study, 25 candidate compounds considered to be potent internal standards were investigated with respect to the ability of providing unique signal chemical shifts, purity, solubility, and ease of use. The 1H chemical shift (δ) values, assignments, multiplicities and number of protons (for each signal), appropriateness (as to be used as internal standards) in four different deuterated solvents (D2O, DMSO-d6, CD3OD, CDCl3) were studied. Taking into account the properties of these 25 internal standards, the most versatile eight compounds (2,4,6-triiodophenol, 1,3,5-trichloro-2-nitrobenzene, 3,4,5-trichloropyridine, dimethyl terephthalate, 1,4-dinitrobenzene, 2,3,5-triiodobenzoic acid, maleic acid and fumaric acid) were qualified using both differential scanning calorimetry (DSC) and NMR spectroscopy employing highly pure acetanilide as the reference standard. The data from these two methods were compared as well as utilized in the quality assessment of the compounds as internal standards. Finally, the selected internal standards were tested and evaluated in a real case of quantitative NMR analysis of a paracetamol pharmaceutical product.

  • 179. Rundlöf, Torgny
    et al.
    McEwen, Ian
    Johansson, Monika
    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.
    Use and qualification of primary and secondary standards employed in quantitative H-1 NMR spectroscopy of pharmaceuticals2014In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 93, no SI, p. 111-117Article in journal (Refereed)
    Abstract [en]

    Standards are required in quantitative NMR (qNMR) to obtain accurate and precise results. In this study acetanilide was established and used as a primary standard. Six other chemicals were selected as secondary standards: 3,4,5-trichloropyridine, dimethylterephthalate, maleic acid, 3-sulfolene, 1,4-bis(trimethylsilyl)benzene, and 1,3,5-trimethoxybenzene. The secondary standards were quantified using the primary standard acetanilide. A protocol for qualification and periodic checks of these secondary standards was developed, and used for evaluation of the stability of the compounds. Periodic monitoring of purity was performed for several years. The purity was higher than 99% for all secondary standards. All standards maintained the initial purity during the time period of monitoring, with very small variations in purity (0.3-0.4%). The selected secondary standards were shown to be suitable qNMR standards and that periodic requalification of the standards by qNMR ensures reliable analytical results. These standards have been used in our laboratory for compliance testing of pharmaceutical active substances and approved medicinal products as well as for analysis of suspected illegal medicines. In total more than 1000 samples have been tested using both internal and external standardization and examples are given.

  • 180.
    Rydevik, Axel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Drug Metabolites Formed by Cunninghamella Fungi: Mass Spectrometric Characterization and Production for use in Doping Control2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis describes the in vitro production of drug metabolites using fungi of the Cunninghamella species. The metabolites were characterized with mainly liquid chromatography-mass spectrometry using ion-trap and quadrupole-time-of-flight instruments. A fungal in vitro model has several advantages e.g., it is easily up-scaled and ethical problems associated with animal-based models are avoided.

    The metabolism of bupivacaine and the selective androgen receptor modulators (SARMs) S1, S4 and S24 by the fungi Cunninghamella elegans and Cunninghamella blakesleeana was investigated. The detected metabolites were compared with those formed in vitro and in vivo by human and horse and most phase I metabolites formed by mammals were also formed by the fungi. The higher levels of bupivacaine metabolites in the fungal samples allowed an extensive mass spectrometric structural characterization which shows that the fungi are relevant metabolic models.

    Glucuronides are important drug metabolites but they are difficult to synthesize. The discovery that the fungus Cunninghamella elegans formed large amounts of glucosides led to the idea that they could be used to form glucuronides. A new concept was developed where a fungal incubate containing a SARM S1 glucoside was mixed with the free radical tetramethylpiperidinyl-1-oxy (TEMPO), sodium bromide and sodium hypochlorite which produced a glucuronide. Isolation and characterization by nuclear magnetic resonance spectroscopy proved that the new method could produce glucuronides for use as reference material.

    An investigation of reactive metabolite formation of the drugs paracetamol, mefenamic acid and diclofenac by the fungus Cunninghamella elegans was performed. It was demonstrated for the first time that the fungus could produce glutathione, glutathione ethyl-ester, cysteine and N-acetylcysteine conjugates that are indicative of a preceding formation of reactive intermediates. A comparison with conjugates formed by human liver microsomes showed that both models formed identical metabolites.

    The presented investigations prove that Cunninghamella fungi are relevant drug metabolism models. They show that the fungi to a large extent forms the same metabolites as mammals and that they can produce metabolites for use as reference material in, e.g. doping control. It was also demonstrated that the fungal model can be used in the important assessment of drug toxicity.

    List of papers
    1. Structural elucidation of phase I and II metabolites of bupivacaine in horse urine and fungi of the Cunninghamella species using liquid chromatography/multi-stage mass spectrometry
    Open this publication in new window or tab >>Structural elucidation of phase I and II metabolites of bupivacaine in horse urine and fungi of the Cunninghamella species using liquid chromatography/multi-stage mass spectrometry
    2012 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 26, no 11, p. 1338-1346Article in journal (Refereed) Published
    Abstract [en]

    RATIONALE: Bupivacaine is a local anaesthetic prohibited in equine sports. It is highly metabolized in the horse but a thorough description of its metabolite profile is lacking. An administration study should find appropriate analytical targets for doping control. Furthermore, knowledge of an in vitro system for production of metabolites would be beneficial.

    METHODS: Marcain® (bupivacaine hydrochloride) was administered subcutaneously to a horse and urine samples were collected. In vitro metabolic systems consisting of the fungi Cunninghamella elegans and Cunninghamella blakesleeana were incubated with bupivacaine and bupivacaine-d9. Samples were analyzed directly after dilution or cleaned up using liquid-liquid extraction. Separation was achieved with liquid chromatography. Mass spectrometric analysis was performed using positive electrospray ionization with both a tandem quadrupole and an ion trap instrument using MSn and hydrogen/deuterium exchange.

    RESULTS: In horse urine, seven phase I metabolites were found: 3'- and 4'-hydroxybupivacaine, N-desbutylbupivacaine, two aliphatically hydroxylated metabolites, one N-oxide, and dihydroxybupivacaine. Sulfated hydroxybupivacaine and glucuronides of 3'- and 4'-hydroxybupivacaine and of dihydroxybupivacaine were also detected. All these metabolites were previously undescribed in the horse, except for 3'-hydroxybupivacaine. 3'- and 4'-Hydroxybupivacaine were designated as appropriate targets for doping control. Interestingly, all the equine phase I metabolites were also detected in the samples from C. elegans and C. blakesleeana.

    CONCLUSIONS: The qualitative aspects of the metabolism of bupivacaine in the horse have been investigated with many novel metabolites described. The fungi C. elegans and C. blakesleeana have proven to be relevant models for mammalian metabolism of bupivacaine and they may in the future be used to produce analytical reference materials.

    National Category
    Medicinal Chemistry Analytical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-173545 (URN)10.1002/rcm.6225 (DOI)000303597100009 ()
    Available from: 2012-04-26 Created: 2012-04-26 Last updated: 2018-01-12Bibliographically approved
    2. The fungus Cunninghamella elegans can produce human and equine metabolites of selective androgen receptor modulators (SARMs)
    Open this publication in new window or tab >>The fungus Cunninghamella elegans can produce human and equine metabolites of selective androgen receptor modulators (SARMs)
    Show others...
    2013 (English)In: Xenobiotica, ISSN 0049-8254, E-ISSN 1366-5928, Vol. 43, no 5, p. 409-420Article in journal (Refereed) Published
    Abstract [en]

    1. Selective androgen receptor modulators (SARMs) are a group of substances that have potential to be used as doping agents in sports. Being a relatively new group not available on the open market means that no reference materials are commercially available for the main metabolites. In the presented study, the in vitro metabolism of SARMs by the fungus Cunninghamella elegans has been investigated with the purpose of finding out if it can produce relevant human and equine metabolites.

    2. Three different SARMs, S1, S4 and S24, were incubated for 5 days with C. elegans. The samples were analysed both with and without sample pretreatment using ultra performance liquid chromatography coupled to high resolution mass spectrometry.

    3. All the important phase I and some phase II metabolites from human and horse were formed by the fungus. They were formed through reactions such as hydroxylation, deacetylation, O-dephenylation, nitro-reduction, acetylation and sulfonation.

    4. The study showed that the fungus produced relevant metabolites of the SARMs and thus can be used to mimic mammalian metabolism. Furthermore, it has the potential to be used for future production of reference material.

    National Category
    Pharmaceutical Sciences Medicinal Chemistry Analytical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-196318 (URN)10.3109/00498254.2012.729102 (DOI)000316952100002 ()
    Note

    MEDLINE AN 2013135005(Journal; Article; (JOURNAL ARTICLE))

    Available from: 2013-03-07 Created: 2013-03-07 Last updated: 2018-01-11Bibliographically approved
    3. Mass spectrometric characterization of glucuronides formed by a new concept, combining Cunninghamella elegans with TEMPO
    Open this publication in new window or tab >>Mass spectrometric characterization of glucuronides formed by a new concept, combining Cunninghamella elegans with TEMPO
    2013 (English)In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 84, p. 278-284Article in journal (Refereed) Published
    Abstract [en]

    A new concept for the production of drug glucuronides is presented and the products formed were characterized using ultra high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). Glucuronic acid conjugates are important phase II metabolites of a wide range of drugs. There is a lack of commercially available glucuronides and classic synthetic methods are tedious and expensive. Thus, new methods of glucuronide synthesis are needed. Selective androgen receptor modulators (SARMs) of the aryl propionamide class were used as model compounds and were incubated with the fungus Cunninghamella elegans which was previously known to conjugate drugs with glucose. The resulting glucoside metabolites were then oxidized with tetramethylpiperidinyl-1-oxy (TEMPO). UPLC-HRMS analysis showed that the peaks corresponding to the glucosides had disappeared after the reaction and were replaced by peaks with m/z consistent with the corresponding glucuronic acid conjugates. The MS/MS spectra of the reaction products were investigated and the observed fragment ion pattern corroborated the suggested structural change. A comparison in terms of retention times and product ion spectra between the glucuronides formed by the new method and those produced by liver microsomes indicated that the conjugates from the two different sources were identical, thus demonstrating the human relevance of the presented technique. Furthermore, the glucuronides formed by the presented method were readily hydrolyzed by β-glucuronidase which further gave evidence as to the fact that they were of β configuration. The investigated method was easy to perform, required a low input of work and had a low cost.

    Keywords
    High resolution mass spectrometry, UPLC, Glucuronides, SARM, TEMPO
    National Category
    Analytical Chemistry
    Research subject
    Analytical Pharmaceutical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-205709 (URN)10.1016/j.jpba.2013.06.012 (DOI)000322752800040 ()23867089 (PubMedID)
    Available from: 2013-08-22 Created: 2013-08-22 Last updated: 2017-12-06Bibliographically approved
    4. Isolation and characterization of a beta-glucuronide of hydroxylated SARM S1 produced using a combination of biotransformation and chemical oxidation
    Open this publication in new window or tab >>Isolation and characterization of a beta-glucuronide of hydroxylated SARM S1 produced using a combination of biotransformation and chemical oxidation
    Show others...
    2014 (English)In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 98, p. 36-39Article in journal (Refereed) Published
    Abstract [en]

    In this study, using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, it has been confirmed that biotransformation with the fungus Cunninghamella elegans combined with chemical oxidation with the free radical tetramethylpiperidinyl-1-oxy (TEMPO) can produce drug glucuronides of beta-configuration. Glucuronic acid conjugates are a common type of metabolites formed by the human body. The detection of such conjugates in doping control and other kinds of forensic analysis would be beneficial owing to a decrease in analysis time as hydrolysis can be omitted. However the commercial availability of reference standards for drug glucuronides is poor. The selective androgen receptor modulator (SARM) SARM Si was incubated with the fungus C elegans. The sample was treated with the free radical TEMPO oxidizing agent and was thereafter purified by SPE. A glucuronic acid conjugate was isolated using a fraction collector connected to an ultra high performance liquid chromatographic (UHPLC) system. The isolated compound was characterized by NMR spectroscopy and mass spectrometry and its structure was confirmed as a glucuronic acid beta-conjugate of hydroxylated SARM Si bearing the glucuronide moiety on carbon C-10.

    National Category
    Medicinal Chemistry Analytical Chemistry
    Research subject
    Analytical Pharmaceutical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-220769 (URN)10.1016/j.jpba.2014.05.001 (DOI)000339859800005 ()
    Available from: 2014-03-20 Created: 2014-03-20 Last updated: 2018-01-11Bibliographically approved
    5. A novel trapping system for the detection of reactive drug metabolites using the fungus Cunninghamella elegans and high resolution mass spectrometry
    Open this publication in new window or tab >>A novel trapping system for the detection of reactive drug metabolites using the fungus Cunninghamella elegans and high resolution mass spectrometry
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Medicinal Chemistry Pharmaceutical Sciences Analytical Chemistry
    Research subject
    Analytical Pharmaceutical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-220770 (URN)
    Available from: 2014-03-20 Created: 2014-03-20 Last updated: 2018-01-11
  • 181.
    Rydevik, Axel
    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.
    A mass spectrometric study of bupivacaine metabolites found in fungi and horse2012Conference paper (Other academic)
  • 182.
    Rydevik, Axel
    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.
    Mikael, Hedeland
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Structural elucidation of phase I and II metabolites of bupivacaine in horse urine and fungi of the Cunninghamella species using liquid chromatography/multi-stage mass spectrometry2012In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 26, no 11, p. 1338-1346Article in journal (Refereed)
    Abstract [en]

    RATIONALE: Bupivacaine is a local anaesthetic prohibited in equine sports. It is highly metabolized in the horse but a thorough description of its metabolite profile is lacking. An administration study should find appropriate analytical targets for doping control. Furthermore, knowledge of an in vitro system for production of metabolites would be beneficial.

    METHODS: Marcain® (bupivacaine hydrochloride) was administered subcutaneously to a horse and urine samples were collected. In vitro metabolic systems consisting of the fungi Cunninghamella elegans and Cunninghamella blakesleeana were incubated with bupivacaine and bupivacaine-d9. Samples were analyzed directly after dilution or cleaned up using liquid-liquid extraction. Separation was achieved with liquid chromatography. Mass spectrometric analysis was performed using positive electrospray ionization with both a tandem quadrupole and an ion trap instrument using MSn and hydrogen/deuterium exchange.

    RESULTS: In horse urine, seven phase I metabolites were found: 3'- and 4'-hydroxybupivacaine, N-desbutylbupivacaine, two aliphatically hydroxylated metabolites, one N-oxide, and dihydroxybupivacaine. Sulfated hydroxybupivacaine and glucuronides of 3'- and 4'-hydroxybupivacaine and of dihydroxybupivacaine were also detected. All these metabolites were previously undescribed in the horse, except for 3'-hydroxybupivacaine. 3'- and 4'-Hydroxybupivacaine were designated as appropriate targets for doping control. Interestingly, all the equine phase I metabolites were also detected in the samples from C. elegans and C. blakesleeana.

    CONCLUSIONS: The qualitative aspects of the metabolism of bupivacaine in the horse have been investigated with many novel metabolites described. The fungi C. elegans and C. blakesleeana have proven to be relevant models for mammalian metabolism of bupivacaine and they may in the future be used to produce analytical reference materials.

  • 183.
    Rydevik, Axel
    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.
    Thevis, Mario
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Drug glucuronides synthesized using a new concept combining Cunninghamella elegans with TEMPO2013Conference paper (Other academic)
  • 184.
    Rydevik, Axel
    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.
    Thevis, Mario
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Mass spectrometric characterization of glucuronides formed by a new concept, combining Cunninghamella elegans with TEMPO2013In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 84, p. 278-284Article in journal (Refereed)
    Abstract [en]

    A new concept for the production of drug glucuronides is presented and the products formed were characterized using ultra high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). Glucuronic acid conjugates are important phase II metabolites of a wide range of drugs. There is a lack of commercially available glucuronides and classic synthetic methods are tedious and expensive. Thus, new methods of glucuronide synthesis are needed. Selective androgen receptor modulators (SARMs) of the aryl propionamide class were used as model compounds and were incubated with the fungus Cunninghamella elegans which was previously known to conjugate drugs with glucose. The resulting glucoside metabolites were then oxidized with tetramethylpiperidinyl-1-oxy (TEMPO). UPLC-HRMS analysis showed that the peaks corresponding to the glucosides had disappeared after the reaction and were replaced by peaks with m/z consistent with the corresponding glucuronic acid conjugates. The MS/MS spectra of the reaction products were investigated and the observed fragment ion pattern corroborated the suggested structural change. A comparison in terms of retention times and product ion spectra between the glucuronides formed by the new method and those produced by liver microsomes indicated that the conjugates from the two different sources were identical, thus demonstrating the human relevance of the presented technique. Furthermore, the glucuronides formed by the presented method were readily hydrolyzed by β-glucuronidase which further gave evidence as to the fact that they were of β configuration. The investigated method was easy to perform, required a low input of work and had a low cost.

  • 185.
    Rydevik, Axel
    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.
    Thevis, Mario
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Turning drug glucosides into glucuronides - making fungal incubations work together with chemical derivatization2013Conference paper (Other academic)
  • 186.
    Rydevik, Axel
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hansson, Annelie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hellqvist, Anna
    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 novel trapping system for the detection of reactive drug metabolites using the fungus Cunninghamella elegans and high resolution mass spectrometry2015In: Drug Testing and Analysis, ISSN 1942-7603, E-ISSN 1942-7611, Vol. 7, no 7, p. 626-633Article in journal (Refereed)
    Abstract [en]

    A new model is presented that can be used to screen for bioactivation of drugs. The evaluation of toxicity is an important step in the development of new drugs. One way to detect possible toxic metabolites is to use trapping agents such as glutathione. Often human liver microsomes are used as a metabolic model in initial studies. However, there is a need for alternatives that are easy to handle, cheap, and can produce large amounts of metabolites. In the presented study, paracetamol, mefenamic acid, and diclofenac, all known to form reactive metabolites in humans, were incubated with the fungus Cunninghamella elegans and the metabolites formed were characterized with ultra high performance liquid chromatography coupled to a quadrupole time of flight mass spectrometer. Interestingly, glutathione conjugates formed by the fungus were observed for all three drugs and their retention times and MS/MS spectra matched those obtained in a comparative experiment with human liver microsomes. These findings clearly demonstrated that the fungus is a suitable trapping model for toxic biotransformation products. Cysteine conjugates of all three test drugs were also observed with high signal intensities in the fungal incubates, giving the model a further indicator of drug bioactivation. To our knowledge, this is the first demonstration of the use of a fungal model for the formation and trapping of reactive drug metabolites. The investigated model is cheap, easy to handle, it does not involve experimental animals and it can be scaled up to produce large amounts of metabolites. Copyright (c) 2014 John Wiley & Sons, Ltd.

  • 187.
    Rydevik, Axel
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hansson, Annelie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hellqvist, Anna
    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 novel trapping system for the detection of reactive drug metabolites using the fungus Cunninghamella elegans and high resolution mass spectrometryManuscript (preprint) (Other academic)
  • 188.
    Rydevik, Axel
    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.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    A mass spectrometric study of bupivacaine metabolites found in fungi and horse2012Conference paper (Other academic)
  • 189.
    Rydevik, Axel
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hellqvist, Anna
    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.
    Evaluation of the fungus Cunninghamella elegans as a model for the formation of reactive metabolites using glutathione trapping and UHPLCC/HRMS2013Conference paper (Other academic)
  • 190.
    Rydevik, Axel
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Lagojda, Andreas
    Bayer CropScience AG.
    Thevis, Mario
    Institute of Biochemistry and Center for Preventive Doping Research, German Sport University, Cologne.
    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.
    Isolation and characterization of a beta-glucuronide of hydroxylated SARM S1 produced using a combination of biotransformation and chemical oxidation2014In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 98, p. 36-39Article in journal (Refereed)
    Abstract [en]

    In this study, using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, it has been confirmed that biotransformation with the fungus Cunninghamella elegans combined with chemical oxidation with the free radical tetramethylpiperidinyl-1-oxy (TEMPO) can produce drug glucuronides of beta-configuration. Glucuronic acid conjugates are a common type of metabolites formed by the human body. The detection of such conjugates in doping control and other kinds of forensic analysis would be beneficial owing to a decrease in analysis time as hydrolysis can be omitted. However the commercial availability of reference standards for drug glucuronides is poor. The selective androgen receptor modulator (SARM) SARM Si was incubated with the fungus C elegans. The sample was treated with the free radical TEMPO oxidizing agent and was thereafter purified by SPE. A glucuronic acid conjugate was isolated using a fraction collector connected to an ultra high performance liquid chromatographic (UHPLC) system. The isolated compound was characterized by NMR spectroscopy and mass spectrometry and its structure was confirmed as a glucuronic acid beta-conjugate of hydroxylated SARM Si bearing the glucuronide moiety on carbon C-10.

  • 191.
    Rydevik, Axel
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Thevis, Mario
    Krug, Oliver
    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.
    The fungus Cunninghamella elegans can produce human and equine metabolites of selective androgen receptor modulators (SARMs)2013In: Xenobiotica, ISSN 0049-8254, E-ISSN 1366-5928, Vol. 43, no 5, p. 409-420Article in journal (Refereed)
    Abstract [en]

    1. Selective androgen receptor modulators (SARMs) are a group of substances that have potential to be used as doping agents in sports. Being a relatively new group not available on the open market means that no reference materials are commercially available for the main metabolites. In the presented study, the in vitro metabolism of SARMs by the fungus Cunninghamella elegans has been investigated with the purpose of finding out if it can produce relevant human and equine metabolites.

    2. Three different SARMs, S1, S4 and S24, were incubated for 5 days with C. elegans. The samples were analysed both with and without sample pretreatment using ultra performance liquid chromatography coupled to high resolution mass spectrometry.

    3. All the important phase I and some phase II metabolites from human and horse were formed by the fungus. They were formed through reactions such as hydroxylation, deacetylation, O-dephenylation, nitro-reduction, acetylation and sulfonation.

    4. The study showed that the fungus produced relevant metabolites of the SARMs and thus can be used to mimic mammalian metabolism. Furthermore, it has the potential to be used for future production of reference material.

  • 192.
    Salomonsson, Matilda
    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.
    Structural evaluation of glucuronides of morphine and formoterol using chemical derivatization with 1,2-dimethylimidazole-4-sulfonyl chloride and LC-MSn2008Conference paper (Other academic)
  • 193.
    Salomonsson, Matilda L.
    et al.
    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.
    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.
    Quantification of dimethylsulfoxide (DMSO) in equine plasma and urine using HILIC-MS/MS2017In: Drug Testing and Analysis, ISSN 1942-7603, E-ISSN 1942-7611, Vol. 9, no 6, p. 935-941Article in journal (Refereed)
    Abstract [en]

    This paper describes quantitative methods for the determination of dimethylsulfoxide (DMSO) in equine plasma and urine based on simple precipitation and dilution followed by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry (HILIC-MS/MS). DMSO is a polar solvent with analgesic and anti-inflammatory properties. Its pharmacological features make it prohibited in horse racing. However, since DMSO is naturally present in the horses' environment, international threshold values have been implemented for plasma and urine (1 and 15 mu g/mL, respectively). Previously presented quantitative methods for the determination of DMSO are based on gas chromatography, thus demanding a tedious extraction step to transfer the analyte from the aqueous bodily fluid to an injectable organic solvent. The column used in the presented method was an Acquity BEH HILIC and the mobile phase was a mixture of ammonium acetate buffer and acetonitrile delivered as a gradient. Hexadeuterated DMSO (H-2(6)-DMSO) was used as the internal standard. Validation was performed in the range of the international thresholds concerning selectivity, carry-over, linearity, precision, accuracy, stability and inter-individual matrix variation. The results fulfilled the predefined criteria and the methods were considered fit for purpose. Successful applications on real equine doping control samples were carried out with determined DMSO concentrations exceeding the international thresholds.

  • 194.
    Salomonsson, Matilda Lampinen
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Fredriksson, Elisabeth
    Alfjorden, Anders
    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.
    Seafood sold in Sweden contains BMAA: A study of free and total concentrations with UHPLC-MS/MS and dansyl chloride derivatization2015In: Toxicology reports, ISSN 1972-6325, E-ISSN 2214-7500, Vol. 2, p. 1473-1481Article in journal (Refereed)
    Abstract [en]

    β-N-Methylamino-l-alanine (BMAA) is a potential neurotoxin associated with the aquatic environment. Validated analytical methods for the quantification of both free and total concentrations of BMAA were used in an investigation of seafood purchased from different grocery stores in Uppsala, Sweden. The analysis was performed using ultra high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI–MS/MS) and detection of BMAA as a dansyl derivate. The determined concentrations of free BMAA (after a simple trichloroacetic acid extraction) in mussels and scallops were up to 0.46 μg g−1 wet homogenate. The total BMAA (after hydrochloric acid hydrolysis) levels were between 0.29 and 7.08 μg g−1 wet mussel homogenate. The highest concentration of total BMAA was found in imported cooked and canned mussels which contained about ten times the quantity of BMAA measured in domestic cooked and frozen mussels. In this study it was also concluded that BMAA could be detected in seafood origin from four different continents. The risks associated with human exposure to BMAA through food are unknown today. However, the results of this study show that imported seafood in Sweden contain BMAA, indicating that this area needs more investigation, including a risk assessment regarding the consumption of e.g., mussels, scallops and crab.

  • 195.
    Salomonsson, Matilda Lampinen
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hansson, Annelie
    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.
    Development and in-house validation of a method for quantification of BMAA in mussels using dansyl chloride derivatization and ultra performance liquid chromatography tandem mass spectrometry2013In: Analytical Methods, ISSN 1759-9660, E-ISSN 1759-9679, Vol. 5, no 18, p. 4865-4874Article in journal (Refereed)
    Abstract [en]

    A new approach for the detection and quantification of beta-N-methylamino-L-alanine (BMAA) in mussels using chemical derivatization with dansyl chloride and ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) is presented. The method, using dansyl chloride as the reagent, is simple, robust and cost efficient. Comparing the fragmentation patterns for derivatized BMAA and its isomer L-2,4-diaminobutyric acid derivatized a selective fragment for the derivatized BMAA was formed m/z 585 > m/z 71. To ensure an actual detection of BMAA, the ion ratio of the daughter ions m/z 71 and m/z 277 was calculated and compared with the calculated mean ion ratio. The method development resulted in a simplified sample preparation excluding solid phase extraction and, instead, performing filtration and dilution of the samples before the derivatization. Validation was performed and the limit of quantification was determined to be 0.15 mu g g(-1) wet mussel homogenate (33 fmol per injection) and the limit of detection was estimated to be 16 ng g(-1) (4 fmol per injection). The intra-and inter-day precisions were within the accepted criteria and the recovery was about 83%. The stability of BMAA in the stock solution was at least 3 months and the stability of derivatized extracts in vials for the quantification was good for 27 days. This is the first quantification method for BMAA in mussels with extensive validation data published and the method was also applied on real mussel samples collected on the west coast of Sweden. The concentrations of BMAA in the mussel samples were determined to be between 0.27 and 1.6 mu g g(-1) wet mussel homogenates.

  • 196. Sanderson Nydahl, Katarina
    et al.
    Pierson, Johan
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Nyberg, Fred
    Caprioli, Richard M.
    Andrén, Per E.
    In vivo processing of LVV-hemorphin-7 in rat brain and blood utilizing microdialysis combined with electrospray mass spectrometry2003In: Rapid Communications In Mass Spectrometry, Vol. 17, p. 838–844-Article in journal (Refereed)
  • 197. Sandqvist, A
    et al.
    Henrohn, D
    Schneede, J
    Egerod, H
    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.
    Wikstrom, B
    Comparison of vardenafil and adenosine for vasoreactivity testing in patients with pulmonary hypertension2012Conference paper (Other academic)
  • 198. Sandqvist, A. M.
    et al.
    Henrohn, Dan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Schneede, J.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Egerod, H. C.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Bondesson, Ulf G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Wikström, Björn G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Renal Medicine.
    High inter-individual variability of vardenafil pharmacokinetics in patients with pulmonary hypertension2013In: European Journal of Clinical Pharmacology, ISSN 0031-6970, E-ISSN 1432-1041, Vol. 69, no 2, p. 197-207Article in journal (Refereed)
    Abstract [en]

    To evaluate the pharmacokinetic parameters of a single oral dose of vardenafil in patients with pulmonary hypertension (PH). Sixteen patients with PH received vardenafil in single oral doses (20, 10 or 5 mg), and repeated blood sampling for up to 9 h was performed. Vardenafil plasma concentration was determined using liquid chromatography tandem mass spectrometry. Pharmacokinetic parameters were calculated using model-independent analysis. The plasma vardenafil concentration increased rapidly and exhibited a median time to maximum plasma concentration (t(max)) of 1 h and a mean elimination half-life (t(1/2)) of 3.4 h. The geometric mean and standard deviation of (1) the peak plasma concentration (C-max) was 21.4 +/- 1.7 mu g/L, (2) the normalized C-max (C-max,C- norm) 79.1 +/- 1.6 g/L, (3) the area under the time-concentration curve (AUC) 71.5 +/- 1.6 mu g center dot h/L and (4) the normalized AUC (AUC(norm)) 261.6 A +/- 1.7 g center dot h/L. Patients co-medicated with bosentan reached t(max) later and had a 90% reduction of C-max, C-max,C- norm, AUC and AUC(norm). The pharmacokinetic profile of vardenafil overall revealed considerable inter-individual variability in patients with PH. Co-medication with bosentan resulted in a pharmacokinetic drug interaction, leading to significantly decreased plasma concentrations of vardenafil. Therapeutic drug monitoring for individual dose optimization may be warranted.

  • 199.
    Sandqvist, Anna
    et al.
    Umea Univ, Div Clin Pharmacol, Dept Pharmacol & Clin Neurosci, Umea, Sweden..
    Henrohn, Dan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Egeröd, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Natl Vet Inst SVA, Dept Chem, Uppsala, Sweden..
    Wernroth, Lisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, UCR-Uppsala Clinical Research Center.
    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, Uppsala, Sweden..
    Schneede, Jorn
    Umea Univ, Div Clin Pharmacol, Dept Pharmacol & Clin Neurosci, Umea, Sweden..
    Wikström, Gerhard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cardiology.
    Acute vasodilator response to vardenafil and clinical outcome in patients with pulmonary hypertension2015In: European Journal of Clinical Pharmacology, ISSN 0031-6970, E-ISSN 1432-1041, Vol. 71, no 10, p. 1165-1173Article in journal (Refereed)
    Abstract [en]

    Purpose Acute vasodilator testing is recommended in patients with pulmonary arterial hypertension to identify individuals who may benefit from long-term treatment with oral calcium channel blockers. The aim of this study was to investigate the use of vardenafil in acute vasoreactivity testing compared to adenosine. Methods A total of 20 patients eligible for right heart catheterisation were enrolled. Acute vasoreactivity testing was carried out with intravenous (iv) adenosine (n = 18) followed by oral vardenafil (n = 20). Haemodynamic responses were recorded at baseline and after 60 min (vardenafil). Responders were defined according to consensus guideline criteria. Results Both vardenafil and adenosine significantly decreased mean pulmonary arterial pressure (mPAP, p < 0.001 and p = 0.026, respectively) and pulmonary vascular resistance (p < 0.001 and p > 0.001, respectively), and significantly increased cardiac output (p = 0.001 and p = 0.005, respectively). Vardenafil reduced mPAP more than adenosine (p = 0.044), while adenosine resulted in higher responses of cardiac index (p = 0.009) and pulmonary arterial oxygen saturation (p = 0.042). Acute adverse reactions were common with adenosine, while no side effects were observed after a single oral dose vardenafil. Vardenafil identified five responders (out of 20), while adenosine identified three responders (out of 18). During a 7-year follow-up, vardenafil responders had significantly lower NT-proBNP levels compared to non-responders. Conclusions Vardenafil may be safely used for acute vasoreactivity testing in patients with PH. A single oral dose of vardenafil is better tolerated than iv adenosine and may identify additional responders who could benefit from long-term vasodilator treatment.

  • 200. Sjögren, E
    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.
    Lennernäs, H
    Verapamil Affects the Pharmacokinetics and the Hepatic Disposition of Fexofenadine in Pigs2012Conference paper (Other academic)
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