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  • 1.
    Allard, Erik
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Åslund Tröger, Rikard
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Arvidsson, Björn
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Sjöberg, Per Johan Ragnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Quantitative aspects of analyzing small molecules - monitoring singly or doubly charged ions?: A case study of ximelagatran.2010In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 24, no 4, p. 429-435Article in journal (Refereed)
    Abstract [en]

    Precision, reproducibility and lower limit of quantitation (LLOQ) are important characteristics of a quantitative method. We have investigated these properties for Ximelagatran (Xi), which has a high tendency to form doubly charged ions in electrospray ionization (ESI), by studying the percentage of doubly charged species formed when varying the formic acid (FA) concentration, analyte concentration, amount of organic modifier and flow rate. It was found that the percentage of [Xi + 2H]2+ can be controlled to be more than 90% or less than 10% by varying the amount of FA present, and that the change between these values is dramatic. Furthermore, the percentage of [Xi + 2H]2+ formed decreases with increased analyte concentration and increased flow rate. No apparent relationship with the amount of organic modifier was found. The results have the implication that, by carefully controlling the selected parameters, the LLOQ, precision and reproducibility can be improved. We have compared the fragmentation of the singly and doubly charged species and concluded that the [Xi + 2H]2+ ion is more inclined to undergo fragmentation than [Xi + H]+. As a consequence, unusual instrumental settings had to be used for the experiments. The fragmentation patterns are to a great extent similar, but the doubly charged species is more inclined to generate low-mass product ions.

  • 2.
    Axelsson, Jan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Palmblad, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science. Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Håkansson, Kristina
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Håkansson, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Electron Capture Dissociation of Substance-P using a Commercially Available Fourier Transform Ion Cyclotron Resonance Mass Spectrometer1999In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 13, no 6, p. 474-477Article in journal (Refereed)
    Abstract [en]

    Electron capture dissociation of the peptide Substance P is reported for the first time, with an unmodified, commercially available Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. The fragmentation pattern is compared with that obtained with collisionally induced dissociation of the ions in the electrospray ion source, and note that electron capture dissociation gives a more easily interpreted spectrum, showing mainly C-fragments. With the exception of the proline residues, which require cleavage of two chemical bonds, we observe all C-fragmental we find the bias voltage of the electron gun not to be very critical.

  • 3.
    Axén, Jakob
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Malmström, David
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Axelsson, Bengt-Olof
    Petersson, Patrik
    Sjöberg, Per J. R.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Efforts to improve detection sensitivity for capillary electrophoresis coupled to atmospheric pressure photoionization mass spectrometry.2010In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 24, no 9, p. 1260-1264Article in journal (Refereed)
    Abstract [en]

    Electrospray ionization performs best with volatile buffers. However, generally the best separation performance for capillary electrophoresis (CE) is achieved with non-volatile buffers. Hyphenation of CE with mass spectrometry (MS) utilizing atmospheric pressure photoionization (APPI) enables use of a wider range of separation buffers without compromising detection sensitivity. As APPI is considered to be mass flow sensitive, the use of a larger inner diameter separation capillary (75 microm) allows larger volumes to be injected, without decreased separation performance, thus providing improved sensitivity (approx. a factor of 10), compared to the use of a 25 microm capillary. However, nebulizing gas flow and position of capillary tip in the sprayer have to be carefully optimized to prevent excessive band broadening. Further improvement in sensitivity (approx. a factor of 2) was obtained by decreasing the distance between the sprayer and ionization region, indicating that a specially designed CE/APPI-MS interface for low flow rates will be favourable.

  • 4.
    Bengtsson, Jörgen
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Division of Pharmacokinetics and Drug Therapy.
    Jansson, Britt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Division of Pharmacokinetics and Drug Therapy.
    Hammarlund-Udenaes, Margareta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Division of Pharmacokinetics and Drug Therapy.
    On-line desalting and determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in microdialysis and plasma samples using column switching and liquid chromatography/tandem mass spectrometry2005In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 19, no 15, p. 2116-2122Article in journal (Refereed)
    Abstract [en]

    A sensitive and reproducible method for the determination of morphine and the metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) was developed. The method was validated for perfusion fluid used in microdialysis as well as for sheep and human plasma. A C18 guard column was used to desalt the samples before analytical separation on a ZIC HILIC (hydrophilic interaction chromatography) column and detection with tandem mass spectrometry (MS/MS). The mobile phases were 0.05% trifluoroacetic acid (TFA) for desalting and acetonitrile/5 mM ammonium acetate (70:30) for separation. Microdialysis samples (5 microL) were directly injected onto the system. The lower limits of quantification (LLOQ) for morphine, M3G and M6G were 0.50, 0.22 and 0.55 ng/mL, respectively, and the method was linear from LLOQ to 200 ng/mL. For plasma, a volume of 100 microL was precipitated with acetonitrile containing internal standards (deuterated morphine and metabolites). The supernatant was evaporated and reconstituted in 0.05% TFA before the desalting process. The LLOQs for sheep plasma were 2.0 and 3.1 ng/mL and the ranges were 2.0-2000 and 3.1-3100 ng/mL for morphine and M3G, respectively. For human plasma, the LLOQs were 0.78, 1.49 and 0.53 ng/mL and the ranges were 0.78-500, 1.49-1000 and 0.53-500 ng/mL for morphine, M3G and M6G, respectively.

  • 5.
    Bergquist, Jonas
    et al.
    Institute of Clinical Neuroscience, Department of Psychiatry and Neurochemistry, Göteborg University, Sahlgrenska University Hospital/Mölndal.
    Silberring, J
    Faculty of Chemistry and the Regional Laboratory, Jagiellonian University, Poland.
    Identification of catecholamines in the immune system by electrospray ionization mass spectrometry.1998In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 12, no 11, p. 683-8Article in journal (Refereed)
    Abstract [en]

    The first evidence that catecholamines might be present in the immune system was provided by capillary electrophoresis combined with electrochemical detection. Here, we present the first structural characterization of the endogenous catecholamines isolated from human peripheral blood mononuclear cells. Dopamine, L-DOPA and norepinephrine were detected and were identified with electrospray ionization mass spectrometry by determination of the protonated molecular species of each catecholamine and their major fragments generated in the electrospray source with a nozzle-skimmer voltage method. This technique, in conjunction with accurate mass measurement, allowed us to identify in an unfractionated sample the content of catecholamines in extracted cells in a quantitative manner, with structure-specific methodology. The data unambiguously confirm our previous tentative findings, and also strengthen the importance of the regulatory function of catecholamines in the immune system and the existence of an autocrine loop, where lymphocytes may down-regulate their own activity.

  • 6.
    Boström, Emma
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Division of Pharmacokinetics and Drug Therapy.
    Jansson, Britt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Division of Pharmacokinetics and Drug Therapy.
    Hammarlund-Udenaes, Margareta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Division of Pharmacokinetics and Drug Therapy.
    Simonsson, Ulrika S. H.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Division of Pharmacokinetics and Drug Therapy.
    The Use of Liquid Chromatography/Mass Spectrometry for Quantitative Analysis of Oxycodone, Oxymorphone and Noroxycodone in Ringer Solution, Rat Plasma and Rat Brain Tissue2004In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 18, no 21, p. 2565-2576Article in journal (Refereed)
    Abstract [en]

    Sensitive and reproducible methods for the determination of oxycodone, oxymorphone and noroxycodone in Ringer solution, rat plasma and rat brain tissue by liquid chromatography/mass spectrometry are described. Deuterated analogs of the substances were used as internal standards. Samples in Ringer solution were analyzed by direct injection of 10 microL Ringer solution diluted by an equal volume of water. The limit of quantification was 0.5 ng/mL and the method was linear in the range of 0.5-150 ng/mL for all substances. To analyze oxycodone and oxymorphone in rat plasma, 50 microL of plasma were precipitated with acetonitrile, and the supernatant was directly injected onto the column. To analyze oxycodone, oxymorphone and noroxycodone in rat plasma, 100 microL of rat plasma were subjected to a C18 solid-phase extraction (SPE) procedure, before reconstituting in mobile phase and injection onto the column. For both methods the limit of quantification in rat plasma was 0.5 ng/mL and the methods were linear in the range of 0.5-250 ng/mL for all substances. To analyze the content of oxycodone, oxymorphone and noroxycodone in rat brain tissue, 100 microL of the brain homogenate supernatant were subjected to a C18 SPE procedure. The limit of quantification of oxycodone was 20 ng/g brain, and for oxymorphone and noroxycodone 4 ng/g brain, and the method was linear in the range of 20-1000 ng/g brain for oxycodone and 4-1000 ng/g brain for oxymorphone and noroxycodone. All methods utilized a mobile phase of 5 mM ammonium acetate in 45% acetonitrile, and a SB-CN column was used for separation. The total run time of all methods was 9 min. The intra-day precision and accuracy were <11.3% and <+/-14.9%, respectively, and the inter-day precision and accuracy were <14.9% and <+/-6.5%, respectively, for all the concentrations and matrices described.

  • 7. Conrotto, Paolo
    et al.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Lys Tag: an easy and robust chemical modification for improved de novo sequencing with a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer2008In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 22, no 12, p. 1823-33Article in journal (Refereed)
    Abstract [en]

    Mass spectrometry using a matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) instrument is a widespread technique for various types of proteomic analysis. Along with an expanding interest in proteomics, there is a strong requirement for the identification of proteins with high confidence from biological samples. Peptide modification by a wide variety of post-translational modifications (PTMs), the existence of different protein isoforms and the presence of uncharacterized genomes of many species, make protein identification through peptide mass fingerprinting (PMF) often unachievable. Peptide de novo sequencing has been proven to be a useful approach to overcome these variables, and efficient derivatization processes are important tools to achieve this goal. In the present work we describe the methodology and experimental applications of a fast, efficient and cheap lysine derivatization. This chemical modification improves the signals from lysine-terminated peptides and can be efficiently used as a lysine-blocking agent in combination with other derivatization techniques. Most importantly, upon peptide fragmentation it generates a neat series of predominantly y-ions, allowing the determination of unambiguous amino acid sequences. Moreover, this chemical compound was used with target-eluted samples, enabling a second, alternative analysis of the same sample in the MALDI mass spectrometer.

  • 8. Goloborodko, Anton A.
    et al.
    Mayerhofer, Corina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Biological Mass Spectrometry.
    Zubarev, Alexander R.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Biological Mass Spectrometry.
    Tarasova, Irina A.
    Gorshkov, Alexander V.
    Zubarev, Roman A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Biological Mass Spectrometry.
    Gorshkov, Mikhail V.
    Empirical approach to false discovery rate estimation in shotgun proteomics2010In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 24, no 4, p. 454-462Article in journal (Refereed)
    Abstract [en]

    Estimation of false discovery rate (FDR) for identified peptides is an important step in large-scale proteomic studies. We introduced an empirical approach to the problem that is based on the FDR-like functions of sets of peptide spectral matches (PSMs). These functions have close values for equal-sized sets with the same FDR and depend monotonically on the FDR of a set. We have found three of them, based on three complementary sources of data: chromatography, mass spectrometry, and sequences of identified peptides. Using a calibration on a set of putative correct PSMs these functions were converted into the FDR scale. The approach was tested on a set of similar to 2800 PSMs obtained from rat kidney tissue. The estimates based on all three data sources were rather consistent with each other as well as with one made using the target-decoy strategy.

  • 9.
    Goodwin, Richard JA
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Iverson, Suzanne L
    AstraZeneca R&D.
    Andrén, Per E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    The significance of ambient-temperature on pharmaceutical and endogenous compound abundance and distribution in tissues sections when analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging2012In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 26, no 5, p. 494-498Article in journal (Refereed)
    Abstract [en]

    RATIONALE: Mass spectrometry imaging has proven to be a complementary assay to the traditional labeled-compound studies employed in drug research and development. However, there has been limited examination of the technical limitations of the technique with respect to small molecule stability in samples.

    METHODS: Raclopride dosed rat brain tissue sections (single dose i.v. 2 mg/kg) were allowed to warm to room temperature for 0 to 5 min prior to either a solvent-based wet matrix-assisted laser desorption/ionization (MALDI) matrix or a solvent-free dry MALDI matrix being applied. Subsequent MS imaging analysis was at a spatial resolution of 200 mm, performed using a MALDI TOF/TOF (Ultraflex II, Bruker Daltonics).

    RESULTS: MALDI-MS has been used to monitor the time-dependent appearance and loss of small molecule abundance in tissue sections brought rapidly to room temperature for short periods of time. The abundances of a range of markers were seen to vary across the time course, both increasing and decreasing. The intensity of some markers changed significantly within 1 min. Importantly, the abundance of raclopride was seen to decrease over the 5-min time period examined.

    CONCLUSIONS: The results strongly indicate that considerable care is required to allow comparison of both pharmaceutical and endogenous compounds between MALDI-MSI experiments and also has implications for the standard practice of thaw-mounting multiple tissue sections onto MALDI-MS targets during MSI experiments.

  • 10.
    Gupta, Anubha
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Division of Pharmacokinetics and Drug Therapy.
    Jansson, Britt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Division of Pharmacokinetics and Drug Therapy.
    Chatelain, Pierre
    Massingham, Roy
    Hammarlund-Udenaes, Margareta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Division of Pharmacokinetics and Drug Therapy.
    Quantitative determination of cetirizine enantiomers in guinea pig plasma, brain tissue and microdialysis samples using liquid chromatography/tandem mass spectrometry2005In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 19, no 12, p. 1749-1757Article in journal (Refereed)
    Abstract [en]

    Sensitive enantioselective liquid chromatographic assays using tandem mass spectrometric detection were developed and validated for the determination of S-cetirizine (S-CZE) and R-cetirizine (R-CZE) in guinea pig plasma, brain tissue, and microdialysis samples. Enantioselective separation was achieved on an alpha1-acid glycoprotein column within 14 min for all methods. A cetirizine analog, ucb 20028, was used as internal standard. Cetirizine and the internal standard were detected by multiple reaction monitoring using transitions m/z 389.1 --> 200.9 and 396.1 --> 276.1, respectively. The samples were prepared using protein precipitation with acetonitrile. For guinea pig plasma, the assay was linear over the range 0.25-5000 ng/mL for both S-CZE and R-CZE, with a lower limit of quantification (LLOQ) of 0.25 ng/mL. For the brain tissue and microdialysis samples, the assays were linear over the range 2.5-250 ng/g and 0.25-50 ng/mL, respectively, and the LLOQ values were 2.5 ng/g and 0.25 ng/mL, respectively. The intra- and inter-day precision values were < or =7.1% and < or =12.6%, respectively, and the intra- and inter-day accuracy varied by less than +/-8.0% and +/-6.0% of the nominal value, respectively, for both enantiomers in all the matrices investigated.

  • 11.
    Hansson, Annelie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Knych, Heather
    Univ Calif Davis, Sch Vet Med, KL Maddy Equine Analyt Chem Lab, Davis, CA 95616 USA.;Univ Calif Davis, Sch Vet Med, Dept Vet Mol Biosci, Davis, CA 95616 USA..
    Stanley, Scott
    Univ Calif Davis, Sch Vet Med, KL Maddy Equine Analyt Chem Lab, Davis, CA 95616 USA..
    Thevis, Mario
    German Sport Univ Cologne, Inst Biochem, Cologne, Germany.;German Sport Univ Cologne, Ctr Prevent Doping Res, Cologne, Germany..
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Natl Vet Inst SVA, Dept Chem Environm & Feed Hyg, SE-75651 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-75651 Uppsala, Sweden..
    Investigation of the selective androgen receptor modulators S1, S4 and S22 and their metabolites in equine plasma using high-resolution mass spectrometry2016In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 30, no 7, p. 833-842Article in journal (Refereed)
    Abstract [en]

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

  • 12.
    Hellman, Ulf
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Bhikhabhai, Rama
    Easy amino acid sequencing of sulfonated peptides using post-source decay on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer equipped with a variable voltage reflector2002In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 16, no 19, p. 1851-1859Article in journal (Refereed)
    Abstract [en]

    Tryptic peptides were labeled with sulfonic acid groups at the N-termini using an improved chemistry. The derivatization was performed in common aqueous buffers on peptides adsorbed onto a ZipTip trade mark C(18), thus allowing simultaneous desalting/concentration of the sample. When only Arg-terminating peptides were considered, the procedure from adsorption onto the ZipTip until analysis by MALDI-PSD took about 10 min and several samples could be worked on in parallel. The resulting improved post-source decay (PSD) fragmentation produced spectra containing only y-ions. PSD amino acid sequencing of underivatized and derivatized synthetic peptides was compared. From the sequence information obtained from derivatized peptides isolated by ion selection from tryptic in-gel digests, a protein was correctly identified which was difficult to analyze from an unclear peptide mass fingerprint analysis. The method was also applied to the identification and localization of phosphorylated Ser and Tyr residues in native and synthetic peptides.

  • 13.
    Jansson, Britt
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Elsherbiny, Doaa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Simonsson, Ulrika SH
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Enantiospecific separation and quantitation of mephenytoin and its metabolites nirvanol and 4'-hydroxymephenytoin in human plasma and urine by liquid chromatography/tandem mass spectrometry2006In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 20, no 3, p. 463-572Article in journal (Refereed)
    Abstract [en]

    A sensitive method using enantiospecific liquid chromatography/tandem mass spectrometry detection for the quantitation of S- and R-mephenytoin as well as its metabolites S- and R-nirvanol and S- and R-4'-hydroxymephenytoin in plasma and urine has been developed and validated. Plasma samples were prepared by protein precipitation with acetonitrile, while urine samples were diluted twice with the mobile phase before injection. The analytes were then separated on a chiral alpha(1)-acid glycoprotein (AGP) column and thereafter detected, using electrospray ionization tandem mass spectrometry. In plasma, the lower limit of quantification (LLOQ) was 1 ng/mL for S- and R-4'-hydroxymephenytoin and S-nirvanol and 3 ng/mL for R-nirvanol and S- and R-mephenytoin. In urine, the LLOQ was 3 ng/mL for all compounds. Resulting plasma and urine intra-day precision values (CV) were <12.4% and <6.4%, respectively, while plasma and urine accuracy values were 87.2-108.3% and 98.9-104.8% of the nominal values, respectively. The method was validated for plasma in the concentration ranges 1-500 ng/mL for S- and R-4'-hydroxymephenytoin, 1-1000 ng/mL for S-nirvanol, and 3-1500 ng/mL for R-nirvanol and S- and R-mephenytoin. The validated concentration range in urine was 3-5000 ng/mL for all compounds. By using this method, the metabolic activities of two human drug-metabolizing enzymes, cytochrome P450 (CYP) 2C19 and CYP2B6, were simultaneously characterized.

  • 14.
    Lampinen 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 the glucuronides of morphine and formoterol using chemical derivatization with 1,2-dimethylimidazole-4-sulfonyl chloride and liquid chromatography/ion trap mass spectrometry2008In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 22, no 17, p. 2685-2697Article in journal (Refereed)
    Abstract [en]

    For the first time chemical derivatization of isomeric drug glucuronides with 1,2-dimethylimidazole-4-sulfonyl chloride (DMISC) has been successfully applied as a tool for determining the site of conjugation. This provides a way to differentiate between glucuronide isomers containing aliphatic and phenolic hydroxyl groups. The analyses were performed with liquid chromatography/electrospray ion trap mass spectrometry (LC/ESI-MSn). DMISC has previously been shown to react selectively with phenols in estrogens, thus improving sensitivity in ESI-MS. The model compounds selected for this study were commercially available standards of formoterol, morphine, morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G). Formoterol glucuronides were produced with an enzymatic method in house. Both formoterol and morphine possess one phenolic and one aliphatic hydroxyl group where glucuronidation could take place. The product ion mass spectra of the native morphine glucuronides were indistinguishable due to the initial neutral loss of monodehydrated glucuronic acid (1.76u). However, a significant difference between the isomers was observed with DMISC derivatization, as only the form with a free phenol, M6G, gave a detectable reaction product. Formoterol formed two detectable glucuronide isomers in the enzymatic reaction. Their respective sites of conjugation could not be directly determined from the product ion spectra. Reaction with DMISC, however, gave a detectable product with only one of the isomers. Based on previous experience of the preferred DMISC reactions with phenols, and interpretation of the fragmentation pattern of the derivative, it was concluded that the reactive isomer had a free phenol, and was thus conjugated on the aliphatic chain.

  • 15.
    Misharin, Alexander S.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Zubarev, Roman
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Coaxial multi-electrode cell (' O-trap ') for high-sensitivity detection at a multiple frequency in Fourier transform ion cyclotron resonance mass spectrometry: main design and modeling results2006In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 20, no 21, p. 3223-3228Article in journal (Refereed)
    Abstract [en]

    Separation of the functions of ion excitation and detection between different cell compartments allows for implementation of excitation and detection techniques unattainable in a single compartment of the conventional ion cyclotron resonance (ICR) cell. In particular, multi-electrode detection at a multiple of the main cyclotron frequency can be utilized without the loss of sensitivity and other negative effects. The new O-trap designed exclusively for ion detection adds an additional, internal coaxial cylinder around which ions with excited cyclotron orbits rotate. Comparison of simulated performance characteristics of the new O-trap with those of the same-size conventional cylindrical cell shows that the O-trap can provide higher sensitivity and ion capacity. Multiplexing of the O-traps can further increase the analysis speed. Future efforts will be aimed at building and testing experimentally the coaxial O-trap, including optimization of the method of ion transfer between the compartments of the cell.

  • 16.
    Nilsson, S L
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Andersson, C
    Astra Zeneca R&D Lund.
    Sjöberg, P J R
    Gyros AB, Uppsala Science Park.
    Bylund, D
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Petersson, P
    Astra Zeneca R&D Lund.
    Jörntén-Karlsson, M
    Astra Zeneca R&D Lund.
    Markides, K E
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Phosphate buffers in capillary electrophoresis/mass spectrometry using atmospheric pressure photoionization and electrospray ionization.2003In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 17, no 20, p. 2267-72Article in journal (Refereed)
    Abstract [en]

    Capillary electrophoresis (CE) has been combined with atmospheric pressure photoionization (APPI) and electrospray ionization (ESI) for mass spectrometric (MS) detection. Separation conditions using potassium phosphate buffer and ammonium formate buffer have been compared for analysis of eleven pharmaceutical bases. The results showed improvements in separation efficiency and peak symmetry when phosphate buffer was used. The low flow in CE may enable utilization of these advances with MS detection. Compared with ESI, the APPI technique provided a cluster-free background. The enhanced signal-to-noise ratio in the total ion current (TIC) and the reduced spectral background indicated that the APPI process is less affected by non-volatile salts in the CE buffers. This results in a wider range of choice of CE buffers in CE/MS analysis when APPI is the ionization method.

  • 17.
    Nilsson, Stefan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Nyholm, Leif
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Markides, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    A simple and robust conductive graphite coating for sheathless electrospray emitters used in capillary electrophoresis/mass spectrometry2001In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 15, no 21, p. 1997-2000Article in journal (Refereed)
    Abstract [en]

    A graphite-polyimide mixture was used as a conductive coating for sheathless electrospray emitters. The coating procedure described is simple and inexpensive compared to previously described methods. An investigation of the stability of the conductive coating carried out by electrochemical methods revealed good performances during oxidative stress. In addition, no decrease in emitter performance was seen during continuous electrospray in the positive electrospray mode for two weeks. Fast capillary electrophoresis with attomole sensitivity demonstrated the excellent performance of the described sheathless interface when used in conjunction with an orthogonal time-of-flight mass spectrometer. The overall simplicity, stability and low cost of this type of sheathless emitter make the described approach highly suitable for any on-column coupling of low flow rate separation techniques to a mass spectrometer.

  • 18.
    Nilsson, Tomas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Martínez, Eriel
    Laboratori de Microbiolgia, Facultat de Farmacia, Universitat de Barcelona.
    Manresa, Angeles
    Laboratori de Microbiolgia, Facultat de Farmacia, Universitat de Barcelona.
    Oliw, Ernst H.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Liquid chromatography/tandem mass spectrometric analysis of 7,10-dihydroxyoctadecenoic acid, its isotopomers, and other 7,10-dihydroxy fatty acids formed by Pseudomonas aeruginosa 42A22010In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 24, no 6, p. 777-783Article in journal (Refereed)
    Abstract [en]

    Pseudomonas aeruginosa is an opportunistic pathogen, which oxidizes oleic acid to 7(S),10(S)-dihydroxy-8(E)-octadecenoic acid (7,10-(OH)(2)-18:1) of biological and industrial interest. Electrospray tandem mass spectrometric (MS/MS) analysis of hydroxylated fatty acids usually generates characteristic fragments containing the carboxylate anion and formed by alpha-cleavage at the oxidized carbon. These fragments indicate the positions of the hydroxyl group. In contrast, liquid chromatography (LC)/MS/MS analysis of 7,10-(OH)(2)-18:1 yielded a series of other ions with structural information. To study the fragmentation mechanism, we prepared (2)H- and (18)O-labeled isotopomers. We also performed MS(3) analysis of the major ions, and for comparison we generated the corresponding 7,10-dihydroxy metabolites of 16:1n-7, 18:2n-6, and 20:1n-11 with a protein extract of P. aeruginosa. The MS/MS spectra of 7,10-(OH)(2)-18:1 and its isotopomers, 7,10-(OH)(2)-16:1, and 7,10-(OH)(2)-20:1, contained a series of prominent fragments that all hold the omega end. The 8,9-double bond was not essential for this fragmentation, as 7,10-(OH)(2)-18:0, and its isotopomers, formed essentially the same fragments in the lower mass range. In contrast, 7,10-dihydroxy-8(E),12(Z)-octadecadienoic acid (7,10-(OH)(2)-18:2) fragmented by alpha-cleavage at the oxidized carbons with formation of carboxylate anions. Our results demonstrate that C(16)-C(20) fatty acids with a 7,10-dihydroxy-8(E) functionality undergo charge-driven fragmentation after charge migration to the omega-end, whereas the main ions of 7,10-(HO)(2)-18:2 retain charge at the carboxyl group.

  • 19.
    Onnerfjord, P
    et al.
    Department of Analytical Chemistry, Lund University.
    Ekström, S
    Department of Analytical Chemistry, Lund University.
    Bergquist, Jonas
    Institute of Clinical Neuroscience, Department of Psychiatry and Neurochemistry, Sahlgrenska University Hospital.
    Nilsson, J
    Department of Electrical Measurements, University of Lund.
    Laurell, T
    Department of Electrical Measurements, University of Lund.
    Marko-Varga, G
    Astra Draco AB, Bioanalytical Chemistry, Preclinical Research and Development.
    Homogeneous sample preparation for automated high throughput analysis with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.1999In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 13, no 5, p. 315-22Article in journal (Refereed)
    Abstract [en]

    This work presents a simple method for obtaining homogeneous sample surfaces in matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the automated analysis of peptides and proteins. The sample preparation method is based on applying the sample/matrix mixture onto a pre-deposited highly diluted matrix spot. The pre-deposited crystals act as seeds for the new sample containing crystals which become much smaller in size and more evenly distributed than with conventional methods. This 'seed-layer' method was developed, optimised and compared with the dried-droplet method using peptides and proteins in the 1000-20,000 Da range. The seed-layer method increases the surface homogeneity, spot to spot reproducibility and sample washability as compared with the commonly used dried-droplet method. This methodology is applicable to alpha-cyanohydroxycinnamic acid, sinapinic acid and ferulic acid, which all form homogeneous crystal surfaces. Within-spot variation and between-spot variation was investigated using statistics at a 95% confidence level (n = 36). The statistical values were generated from more than 5000 data points collected from 500 spectra. More than 90% of the sample locations results in high intensity spectra with relatively low standard deviations (RSDs). Typically obtained data showed an RSD of 19-35% within a sample spot as well as in-between spots for proteins, and an RSD of < or = 50% for peptides. Linear calibration curves were obtained within one order of magnitude using internal calibration with a point-RSD of 3% (n = 10). The sample homogeneity allows mass spectra (average of 16 laser shots) to be obtained on each individual sample within 15 sec, whereby a 100 spot target plate can be run in 25 min. High density target plates using the seed-layer method were prepared by spotting approximately 100 picoliter droplets onto the target, resulting in sample spots < or = 500 microns in diameter using a flow-through piezo-electric micro-dispenser. By using this automated sample preparation step lower standard deviations are obtained in comparison to manually prepared samples.

  • 20.
    Palmblad, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science.
    Tsybin, Youri O
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science.
    Ramström, Margareta
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Håkansson, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Liquid Chromatography and Electron Capture Dissociation in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry2002In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 16, no 10, p. 988-992Article in journal (Refereed)
    Abstract [en]

    Liquid separation methods in combination with electrospray mass spectrometry as well as the recently introduced fragmentation method electron capture dissociation (ECD) have become powerful tools in proteomics research. This paper presents the results of the first successful attempts to combine liquid chromatography (LC) and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) with ECD in the analysis of a mixture of standard peptides and of a bovine serum albumin tryptic digest. A novel electron injection system provided conditions for ECD sufficient to yield extensive sequence information for the most abundant peptides in the mixtures on the time-scale of the chromatographic separation. The results suggest that LC/ECD-FTICRMS can be employed in the characterization of peptides in enzymatic digests of proteins or protein mixtures and identify and localize posttranslational modifications.

  • 21.
    Palmblad, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science. Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Markides, Karin E
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Håkansson, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Analysis of Enzymatically Digested Proteins and Protein Mixtures using a 9.4 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer2000In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 14, no 12, p. 1029-1034Article in journal (Refereed)
    Abstract [en]

    A commercially available 9.4 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was applied in the analysis of tryptic digests of protein mixtures without any separation. First, the method was demonstrated on a mixture of tryptic digests of equine cytochrome c, equine myoglobin and bovine serum albumin. The same method was then applied to human plasma from a healthy blood donor. Computer programs were employed to simplify analysis of the complex spectra. The 2745 peaks in the human plasma electrospray ionization FTICR spectrum could be reduced to 1165 isotopic clusters and 669 unique masses. Out of these, 82 masses matched tryptic fragments of serum albumin with mass measurement errors less than 10 ppm, covering 93% of the sequence. Another 16 masses were assigned to tryptic fragments of transferrin, covering 41% of the sequence on the 10 ppm mass measurement error level (14 within 2 ppm). The mass measurement errors were approximately normal distributed with a standard deviation of 1.7 ppm. This demonstrates the feasibility of combining the ultra-high mass resolving power and accuracy of FTICR mass spectrometry with automated computer analysis for investigating complex biological matrices.

  • 22.
    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.

  • 23.
    Salehpour, Mehran
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Ion Physics.
    Ekblom, Jonas
    Sabetsky, Vladimir
    Håkansson, Karl
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Ion Physics.
    Possnert, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Ion Physics.
    Accelerator mass spectrometry offers new opportunities for microdosing of peptide and protein pharmaceuticals2010In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 24, no 10, p. 1481-1489Article in journal (Refereed)
    Abstract [en]

    Accelerator Mass Spectrometry (AMS) is an ultra-sensitive analytical method which has been instrumental in developing microdosing as a strategic tool in early drug development. Considerable data is available for AMS microdosing using typical pharmaceutical drugs with a molecular weight of a few hundred Daltons. The so-called biopharmaceuticals such as proteins offer interesting possibilities as drug candidates; however, experimental data for protein microdosing and AMS is scarce. The analysis of proteins in conjunction with early drug development and microdosing is overviewed and three case studies are presented on the topic. In the first case study AMS experimental data is presented, for the measured concentration of orally administered recombinant insulin in the blood stream of laboratory rabbits. Case study 2 concerns minimum sample size requirements. AMS samples normally require about 1 mg of carbon (10 mu L of blood) which makes AMS analysis unsuitable in some applications due to the limited availability of samples such as human biopsies or DNA from specific cells. Experimental results are presented where the sample size requirements have been reduced by about two orders of magnitude. The third case study concerns low concentration studies. It is generally accepted that protein pharmaceuticals may be potentially more hazardous than smaller molecules because of immunological reactions. Therefore, future first-in-man microdosing studies might require even lower exposure concentrations than is feasible today, in order to increase the safety margin. This issue is discussed based on the current available analytical capabilities.

  • 24.
    Salehpour, Mehran
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Forsgard, Niklas
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Possnert, Goran
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Accelerator mass spectrometry of small biological samples2008In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 22, no 23, p. 3928-3934Article in journal (Refereed)
    Abstract [en]

    Accelerator mass spectrometry (AMS) is an ultra-sensitive technique for isotopic ratio measurements. In the biomedical field, AMS can be used to measure femtomolar concentrations of labeled drugs in body fluids, with direct applications in early drug development such as Microdosing. Likewise, the regenerative properties of cells which are of fundamental significance in stem-cell research can be determined with an accuracy of a few years by AMS analysis of human DNA. However, AMS nominally requires about 1 mg of carbon per sample which is not always available when dealing with specific body substances such as localized, organ-specific DNA samples. Consequently, it is of analytical interest to develop methods for the routine analysis of small samples in the range of a few tens of mu g. We have used a 5 MV Pelletron tandem accelerator to study small biological samples using AMS. Different methods are presented and compared. A 12 C-carrier sample preparation method is described which is potentially more sensitive and less susceptible to contamination than the standard procedures.

  • 25.
    Salehpour, Mehran
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Forsgård, Niklas
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Possnert, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    FemtoMolar measurements using accelerator mass spectrometry2009In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 23, no 5, p. 557-563Article in journal (Refereed)
    Abstract [en]

    Accelerator mass spectrometry (AMS) is an ultra-sensitive analytical  method suitable for the detection of sub-nM concentrations of labeled   biological substances such as pharmaceutical drugs in body fluids. A  limiting factor in extending the concentration measurements to the   sub-pM range is the natural C-14 content in living tissues. This was  circumvented by separating the labeled drug from the tissue matrix,  using standard high-performance liquid chromatography (HPLC)   procedures. As the separated total drug amount is in the few fg range,  it is not possible to use a standard AMS sample preparation method,  where mg sizes are required. We have utilized a sensitive carbon carrier method where a C-14-deficient compound is added to the HPLC fractions and the composite sample is prepared and analyzed by AMS.   Using 50 mu L human blood plasma aliquots, we have demonstrated concentration measurements below 20 JFM, containing sub-amol amounts of   the labeled drug. The method has the immediate potential of operating in the sub-fM region.

  • 26.
    Salehpour, Mehran
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy.
    Håkansson, Karl
    Höglund, Urban
    Grahn-Westin, Annika
    Nilsson, Sten
    Márquez, Marcela
    Possnert, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy.
    Holmberg, Anders R.
    Application of accelerator mass spectrometry to macromolecules: preclinical pharmacokinetic studies on a polybisphosphonate2011In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 25, no 17, p. 2453-2458Article in journal (Refereed)
    Abstract [en]

    Data on the use of accelerator mass spectrometry (AMS) in conjunction with in vivo studies of macromolecular drugs are scarce. The present study shows the versatility of this technique when investigating the pharmacokinetics (PK) of a macromolecular drug candidate, a polybisphosphonate conjugate (ODX). The aforementioned is a polymer (molecular weight similar to 30 kDa) constituting a carbohydrate backbone with covalently linked ligands (aldendronate and aminoguanidine) and is intended for treatment of osteoporosis and the therapy of bone metastasis from prostate cancer. The conjugate is prepared through partial oxidation of the carbohydrate and sequential coupling of the ligands by reductive amination. (14)C was incorporated in the conjugate by means of coupling a commercially available (14)C-lysine in the conjugation sequence. Fifteen rats were injected intravenously with (14)C-labelled ODX (150 mu g, 14 Bq/rat) and blood samples were collected at 1, 2, 4, 6, and 24 h post-injection (3 rats/time point). Liver, spleen and kidney samples were collected at 4 and 24 h post-injection. Blood from each time point (triplicate) were collected for AMS measurement determining the isotopic ratio ((14)C/(12)C) and consequently the drug concentration in blood. ODX showed a transient presence in blood circulation; 93% of the total dose was cleared from the circulation within 1 h. The half-life after 1 h was estimated to be about 3 h; 0.7% of the administered (14)C dose of ODX remained in circulation after 24 h. The major (14)C accumulation was in the liver, the spleen and the kidneys indicating the probable route of metabolism and excretion. This study demonstrates the versatility of AMS for pharmacological in vivo studies of macromolecules. Labelling with (14)C is relatively simple, inexpensive and the method requires minimal radioactivity, eliminating the need for radioprotection precautions in contrast to methods using scintillation counting.

  • 27.
    Samgina, T. Yu.
    et al.
    Organic Chemistry Department, Moscow State University.
    Gorshkov, V. A.
    Organic Chemistry Department, Moscow State University.
    Artemenko, Konstantin A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Kovalev, S. V.
    N. N. Blokhin Russian Cancer Research Center, Russian Academy of Medicial Sciences.
    Ogourtsov, S. V.
    Biological Department, Moscow State University.
    Zubarev, Roman A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Lebedev, A. T.
    Organic Chemistry Department, Moscow State University.
    Novel natural peptides from Hyla arborea schelkownikowi skin secretion2010In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 24, no 12, p. 1749-1754Article in journal (Refereed)
    Abstract [en]

    Hyla arborea schelkownikowi is one of the leaf frog species inhabiting the southern territories of Russia and the former USSR. This frog species is a member of the Hylidae Rafinesque, 1815 batrachians family. The present study deals with the previously uninvestigated peptidome of the Hyla arborea schelkownikowi skin secretion. Nano-electrospray ionization Fourier transform mass spectrometry (nanoESI-FTMS) of the skin secretion, in the intact form and after acetylation, was selected as the general method of analysis. Electron-capture dissociation (ECD) and collision-induced dissociation (CID) fragmentation were both employed, while de novo sequencing was performed by manual interpretation of the MS data. The suppression of the cyclization of b-ions in the mass spectrometer by the acetylation reaction proved to be very efficient for the de novo sequencing of short peptides. Ten skin peptides were found and all of them, except for bradykinin, had not previously been reported. Six of the peptides belong to the tryptophyllins and related peptides, while three peptides are similar to the aureins.

  • 28. Samgina, T. Yu.
    et al.
    Gorshkov, V. A.
    Vorontsov, Ye. A.
    Artemenko, Konstantin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Zubarev, R. A.
    Lebedev, A. T.
    Mass spectrometric study of bradykinin-related peptides (BRPs) from the skin secretion of Russian ranid frogs2011In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 25, no 7, p. 933-940Article in journal (Refereed)
    Abstract [en]

    Amphibian skin secretion is known to contain biologically active peptides. Bradykinins and related peptides (BRPs) can be found in these animals, while frogs from the genus Rana are considered to be leaders in the levels and variety of these peptides. A reasonable rationalization of this fact is that bradykinins are efficient defense compounds against predators. Forty-four various BRPs have been identified in the skin secretions of five ranid frog species (R. ridibunda, R. lessonae, R. esculenta, R. temporaria, R. arvalis) from the Zvenigorod region (Moscow district, Russia). Some of these peptides are already known, but the novel ones constitute a significant portion. An interesting group of novel peptides was isolated from R. lessonae. These are bradykinin analogues bearing a tyrosine residue in the 5th or 8th position. [Arg(0), Trp(5), Leu(8)] bradykinin and [Thr(6), Leu(8)] bradykinin that had been isolated from fish and avian species, respectively, were also detected in the frog secretion, supporting the predator defense hypothesis. Furthermore, a novel group of BRPs named 'lessonakinins' was discovered in R. lessonae and R. esculenta. All of them include the [Arg(0), Trp(5), Leu(8)] bradykinin sequence and have some structural resemblance to the precursor of this peptide cloned by Chen and coworkers recently. However, the C-terminal part of the lessonakinins does not match the sequence predicted by Chen, demonstrating possible incompleteness of information obtained by cDNA cloning.

  • 29. Samgina, Tatiana Yu.
    et al.
    Artemenko, Konstantin A.
    Gorshkov, Vladimir A.
    Ogourtsov, Sergey V.
    Zubarev, Roman A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Lebedev, Albert T.
    De novo sequencing of peptides secreted by the skin glands of the Caucasian Green Frog Rana ridibunda2008In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 22, no 22, p. 3517-3525Article in journal (Refereed)
    Abstract [en]

    Amphibian skin glands are known to secrete various types of bioactive peptides. The array of these peptides is specific for every frog species. The present research deals with the identification of peptides isolated from the skin secretion of the Marsh frog R. ridibunda inhabiting the Kolkhida Canyon of the Caucasian region. The research is based on comprehensive high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) analysis of intact and chemically modified peptides. In particular, an oxidation procedure was applied directly to the crude skin secretion to open S-S loops whereas N-terminal acetylation was additionally carried out for one individual peptide. Sequences were determined by manual interpretation of electron capture dissociation (ECD) and collisionally induced dissociation (CID) tandem mass spectra. A total of 29 peptides were identified in the skin secretion of the Caucasian Marsh frog. The peptide profile is represented with disulfide-containing peptides belonging to the brevinin, esculentin and ranatuerin families, neuropeptides of the bradykinin and bombesin families. Two identified peptides belonging to the ranatuerins are the first peptides of this family discovered in the skin secretions of European frogs. Ten of the identified peptides coincide with those reported earlier for the European Edible frog. Another ten are identical to those found in R. ridubunda from the Moscow region. This fact verifies the described method as being art efficient analytical tool to compare intra- and interspecific variabilities.

  • 30.
    Samgina, Tatiana Yu.
    et al.
    Moscow MV Lomonosov State Univ, Dept Chem, Moscow, Russia..
    Tolpina, Miriam D.
    Moscow MV Lomonosov State Univ, Dept Chem, Moscow, Russia..
    Trebse, Polonca
    Univ Ljubljana, Ljubljana, Slovenia..
    Torkar, Gregor
    Univ Ljubljana, Ljubljana, Slovenia..
    Artemenko, Konstantin A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Lebedev, Albert T.
    Moscow MV Lomonosov State Univ, Dept Chem, Moscow, Russia..
    LTQ Orbitrap Velos in routine de novo sequencing of non-tryptic skin peptides from the frog Rana latastei with traditional and reliable manual spectra interpretation2016In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 30, no 2, p. 265-276Article in journal (Refereed)
    Abstract [en]

    RATIONALE: Mass spectrometry has shown itself to be the most efficient tool for the sequencing of peptides. However, de novo sequencing of novel natural peptides is significantly more challenging in comparison with the same procedure applied for the tryptic peptides. To reach the goal in this case it is essential to select the most efficient methods of triggering fragmentation and combine all the possible complementary techniques. METHODS: Collision-induced dissociation (CID), high-energy collision dissociation (HCD), and electron-transfer dissociation (ETD) tandem mass spectra recorded with a LTQ Orbitrap Velos instrument were used for the elucidation of the sequence of the natural non-tryptic peptides from the skin secretion of Rana latastei. Manual interpretation of the spectra was applied. RESULTS: The combined approach using CID, HCD, and ETD tandem mass spectra of the multiprotonated peptides in various charge states, as well as of their proteolytic fragments, allowed the sequences of seven novel peptides from the skin secretion of Rana latastei to be established. CONCLUSIONS: Manual mass spectrometry sequencing of natural non-tryptic peptides from the skin secretion of Rana latastei provided the opportunity to work successfully with these species and demonstrated once again its advantage over automatic approaches.

  • 31. Samgina, Tatyana Yu
    et al.
    Vorontsov, Egor A
    Gorshkov, Vladimir A
    Artemenko, Konstantin A
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Zubarev, Roman A
    Lebedev, Albert T
    Mass spectrometric de novo sequencing of natural non-tryptic peptides: comparing peculiarities of collision-induced dissociation (CID) and high energy collision dissociation (HCD)2014In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 28, no 23, p. 2595-2604Article in journal (Refereed)
    Abstract [en]

    RATIONALE: Mass spectrometry has shown itself as the most efficient tool for the sequencing of peptides. However, de novo sequencing of novel natural peptides is significantly more challenging in comparison with the same procedure applied for the tryptic peptides. To reach the goal in this case it is essential to select the most useful methods of triggering fragmentation and combine complementary techniques.

    METHODS: Comparison of low-energy collision-induced dissociation (CID) and higher energy collision-induced dissociation (HCD) modes for sequencing of the natural non-tryptic peptides with disulfide bonds and/or several proline residues in the backbone was achieved using an LTQ FT Ultra Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a 7 T magnet and an LTQ Orbitrap Velos ETD (Thermo Fisher Scientific, Bremen, Germany) instrument. Peptide fractions were obtained by high-performance liquid chromatography (HPLC) separation of frog skin secretion samples from ten species of Rana temporaria, caught in the Kolomna district of Moscow region (Russia).

    RESULTS: HCD makes the b/y series longer and more pronounced, thus increasing sequence coverage. Fragment ions due to cleavages at the C-termini of proline residues make the sequencing more reliable and may be used to detect missed cleavages in the case of tryptic peptides. Another HCD peculiarity involves formation of pronounced inner fragment ions (secondary yn bm ion series formed from the abundant primary y-ions). Differences in de novo sequencing of natural non-tryptic peptides with CID and HCD, involving thorough manual expert interpretation of spectra and two automatic sequencing algorithms, are discussed.

    CONCLUSIONS: Although HCD provides better results, a combination of CID and HCD data may notably increase reliability of de novo sequencing. Several pairs of b2 /a2 -ions may be formed in HCD, complicating the spectra. Automatic de novo sequencing with the available programs remains less efficient than the manual one, independently of the collision energy.

  • 32.
    Tevell Åberg, Annica
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Löfgren, Helena
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Structural elucidation of N-oxidized clemastine metabolites by liquid chromatography/tandem mass spectrometry and the use of Cunninghamella elegans to facilitate drug metabolite identification2010In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 24, no 10, p. 1447-1456Article in journal (Refereed)
    Abstract [en]

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

  • 33.
    Thevis, M.
    et al.
    German Sport Univ Cologne, Ctr Prevent Doping Res, Inst Biochem, Sportpk Mungersdorf 6, D-50933 Cologne, Germany.;European Monitoring Ctr Emerging Doping Agents Eu, Cologne, Germany..
    Machnik, M.
    German Sport Univ Cologne, Ctr Prevent Doping Res, Inst Biochem, Sportpk Mungersdorf 6, D-50933 Cologne, Germany..
    Schenk, I.
    German Sport Univ Cologne, Ctr Prevent Doping Res, Inst Biochem, Sportpk Mungersdorf 6, D-50933 Cologne, Germany..
    Krug, O.
    German Sport Univ Cologne, Ctr Prevent Doping Res, Inst Biochem, Sportpk Mungersdorf 6, D-50933 Cologne, Germany.;European Monitoring Ctr Emerging Doping Agents Eu, Cologne, Germany..
    Piper, T.
    German Sport Univ Cologne, Ctr Prevent Doping Res, Inst Biochem, Sportpk Mungersdorf 6, D-50933 Cologne, Germany..
    Schaenzer, W.
    German Sport Univ Cologne, Ctr Prevent Doping Res, Inst Biochem, Sportpk Mungersdorf 6, D-50933 Cologne, Germany..
    Duee, M.
    Deutsch Reiterliche Vereinigung eV FN, D-48231 Warendorf, Germany..
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Natl Vet Inst SVA, Dept Chem Environm & Feed Hyg, SE-75189 Uppsala, Sweden..
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Natl Vet Inst SVA, Dept Chem Environm & Feed Hyg, SE-75189 Uppsala, Sweden..
    Nickel in equine sports drug testing - pilot study results on urinary nickel concentrations2016In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 30, no 7, p. 982-984Article in journal (Refereed)
    Abstract [en]

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

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

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

  • 35.
    Tsybin, Youri O.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Witt, Matthias
    Baykut, Gökhan
    Håkansson, Per
    Electron Capture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry in the Electron Energy Range 0-50 eV2004In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 18, no 14, p. 1607-1613Article in journal (Refereed)
    Abstract [en]

    Electron capture dissociation (ECD) of polypeptide cations was obtained with pencil and hollow electron beams for both sidekick and gas-assisted dynamic ion trapping (GADT) using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) with an electrostatic ion transfer line. Increasing the number of trapped ions by multiple ICR trap loads using GADT improved the ECD sensitivity in comparison with sidekick ion trapping and ECD efficiency in comparison with single ion trap load by GADT. Furthermore, enhanced sensitivity made it possible to observe ECD in a wide range of electron energies (0-50 eV). The degree, rate and fragmentation characteristics of ECD FTICR-MS were investigated as functions of electron energy, electron irradiation time, electron flux and ion trapping parameters for this broad energy range. The results obtained show that the rate of ECD is higher for more energetic (>1 eV) electrons. Long electron irradiation time with energetic electrons reduces average fragment ion mass and decreases efficiency of formation of c- and z-type ions. The obtained dependencies suggest that the average fragment ion mass and the ECD efficiency are functions of the total fluence of the electron beam (electron energy multiplied by irradiation time). The measured electron energy distributions in low-energy ECD and hot ECD regimes are about 1 eV at full width half maximum in employed experimental configurations.

  • 36.
    Zubarev, Roman A.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Radiation Sciences.
    Abeywarna, U
    Håkansson, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Radiation Sciences.
    Demirev, Plamen A.
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Radiation Sciences.
    Sundqvist, Bo
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Radiation Sciences.
    Kinetic energies of secondary ions in MeV and keV particle-induced desorption1996In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 10, no 15, p. 1966-1974Article in journal (Refereed)
    Abstract [en]

    Kinetic energy distributions of secondary ions produced by MeV or keV primary-ion bombardment of molecularsolids were measured in a reflectron time-of-flight mass spectrometer. It was found that the energy distributionsof many ions exhibit tails extending towards energies lower than the accelerating potential. The degree of tailingdepends upon the nature of the desorbed ion and the conditions of the target surface. Some light ions (H' andC' ), ions from inorganic (alkali halide) samples, radical molecular ions (Cg) and pre-formed molecuiar ions (e.g.complexes of valinomycin with alkali metal ions) exhibit almost no tailing. For positive adduct-type molecularions, such as MH', more extensive tailing was observed for MeV compared with keV primary ions. The originof the energy tailing is attributed to gas-phase formation of a fraction of the secondary ions. For molecular ionsof peptides, the characteristic time of formation of that fraction of ions (*lo% of the whole molecular-ionpopulation) was estimated to be of the order of 10 ns, while the characteristic distance from the target surfacewas of the order of 10 pm.

  • 37.
    Zubarev, Roman A.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Abeywarna, UK
    Demirev, Plamen A.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Eriksson, J
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Papaleo, R
    Håkansson, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Sundqvist, Bo U. R.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Delayed, gas-phase ion formation in plasma desorption mass spectrometry1997In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 11, no 9, p. 963-972Article in journal (Refereed)
    Abstract [en]

    The formation mechanisms of secondary ions from organic targets under MeV ion bombardment were studied with a high-resolution time-of-flight (TOF) mass spectrometer. Promptly formed ions Hn+, Cn+ and CnH+ were used for calibrating the TOF scale. Theoretical flight times of other ions were calculated according to the calibration curve and compared to experimentally determined values. The TOF values of non-specific low mass fragments formed via rearrangement or breaking of several bonds and/or abstraction of several atoms, agree well with the theoretical values. On the other hand, target-specific organic ions, including molecular ions of peptides, have longer TOF values than predicted by the calibration curve. Time delays of a few hundred picoseconds were found for low-mass specific fragments, and a few nanoseconds for peptide molecular ions. For protonated species and non-covalent clusters, the delays are larger than for pre-formed and radical molecular ions. Metals contained in organic samples, as contamination, also give delayed ions. For inorganic targets of LiBF4, significant delays were found for the clusters (LiF)nLi+ with n >3. A strong correlation was observed between the delay of an ion and the tailing of its kinetic energy distribution. The conclusion was made that the majority of target-specific ions are formed in the gas phase, at a distance from the target surface of the order of 1 μm.

  • 38.
    Zubarev, Roman A.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Radiation Sciences.
    Håkansson, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Radiation Sciences.
    Sundqvist, Bo
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Radiation Sciences.
    Accurate monoisotopic mass measurements of peptides: Possibilities and limitations of high resolution time-of-flight particle desorption mass spectrometry1996In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 10, no 11, p. 1386-1392Article in journal (Refereed)
    Abstract [en]

    The possibilities and limitations of time-of-flight particle-desorption mass spectrometry are analysed from the standpoint of accuracy of monoisotopic mass determination. For an instrument with mass reflector, typical mass accuracy was found to be ca. 20 ppm (standard deviation). The main limitation was found to be the mechanism of secondary ion production itself. A fraction of biological ions are formed in the gas phase and therefore exhibit both time delay and energy deficit. This leads to a limited resolving power and non-symmetric ion peaks. A peak shape model, taking into account gas-phase ion formation, is built and analysed, and a simple correction procedure proposed. The application of the correction has increased the mass accuracy to 12 ppm (standard deviation). It is shown that further progress cannot be achieved by a correction procedure without instrumental improvements.

  • 39.
    Zuberovic, Aida
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Ullsten, Sara
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    E. Markides, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Capillary electrophoresis off-line matrix-assisted laser desorption/ionisation mass spectrometry of intact and digested proteins using cationic-coated capillaries2004In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 18, no 23, p. 2946-2952Article in journal (Refereed)
    Abstract [en]

    Capillary electrophoresis (CE) was coupled off-line with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the analysis of proteins and peptides. CE fractions were collected directly on a matrix-coated MALDI target, using a sheath-flow interface. Protein adsorption during CE separations was prevented by coating the capillaries with the physically adsorbed, cationic polymer PolyE-323. The CE/MALDI-MS system was used for the analysis of model proteins and peptides at physiological pH as well as analysis of proteins in tear fluid. Moreover, tryptic on-target digestion of the collected protein fractions, with subsequent MALDI-MS and MS/MS peptide analysis, was demonstrated.

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