| 1. |
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| 2. |
- Ahlgren, Joakim, 1972-
(författare)
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Organic Phosphorus Compounds in Aquatic Sediments : Analysis, Abundance and Effects
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Phosphorus (P) is often the limiting nutrient in lacustrine and brackish eco-systems, and enhanced input of P into an aquatic system might therefore negatively impact the environment. Because modern waste water manage-ment have reduced external P input to surface waters, internal P loading from the sediment has become one of the main P sources to aquatic ecosys-tems, in which relatively unknown organic P compounds seem to be more active in P recycling than previously thought. This thesis focus is on improving analysis methods for organic P com-pounds in lacustrine and brackish sediments, as well as determining which of these compounds might be degraded, mobilized and subsequently recycled to the water column and on what temporal scale this occur. In both lacustrine and brackish environments, the most labile P compound was pyrophosphate, followed by different phosphate diesters. Phosphate monoesters were the least labile organic P compounds and degraded the slowest with sediment depth. In regulated lakes, it was shown that pyrophosphate and polyphos-phate compound groups were most related to lake trophic status, thus indi-cating their involvement in P cycling. This thesis also indicates faster P turn-over in sediment from the brackish environment compared to sediment from the lacustrine environment. A comparison of organic P extraction procedures showed that pre-extraction with EDTA, and NaOH as main extractant, was most efficient for total P extraction. Using buffered sodium dithionite (BD) as a pre-extractant and NaOH as main extractant was most efficient for extracting the presuma-bly most labile organic P compound groups, pyrophosphate and polyphos-phate. Furthermore, it was determined that organic P compounds associated with humic substances were more recalcitrant than other P compounds, that the BD step used in traditional P fractionation might extract phosphate monoesters, and that NMR is a statistically valid method for quantification of organic P compounds in sediment extracts.
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| 3. |
- Ahlgren, Joakim, et al.
(författare)
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Sediment Depth Attenuation of Biogenic Phosphorus Compounds Measured by 31P NMR
- 2005
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Ingår i: Environmental Science and Technology. - Univ Uppsala, Dept Analyt Chem, SE-75124 Uppsala, Sweden. Univ Uppsala, Dept Ecol & Evolut, Uppsala, Sweden. Univ Uppsala, Dept Organ Chem, SE-75124 Uppsala, Sweden. : American Chemical Society (ACS). - 0013-936X .- 1520-5851. ; 39:3, s. 867-872
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Tidskriftsartikel (refereegranskat)abstract
- Being a major cause of eutrophication and subsequent loss of water quality, the turnover of phosphorus (P) in lake sediments is in need of deeper understanding. A major part of the flux of P to eutrophic lake sediments is organically bound or of biogenic origin. This P is incorporated in a poorly described mixture of autochthonous and allochthonous sediment and forms the primary storage of P available for recycling to the water column, thus regulating lake trophic status. To identify and quantify biogenic sediment P and assess its lability, we analyzed sediment cores from Lake Erken, Sweden, using traditional P fractionation, and in parallel, NaOH extracts were analyzed using 31P NMR. The surface sediments contain orthophosphates (ortho-P) and pyrophosphates (pyro-P), as well as phosphate mono- and diesters. The first group of compounds to disappear with increased sediment depth is pyrophosphate, followed by a steady decline of the different ester compounds. Estimated half-life times of these compound groups are about 10 yr for pyrophosphate and 2 decades for mono- and diesters. Probably, these compounds will be mineralized to ortho-P and is thus potentially available for recycling to the water column, supporting further growth of phytoplankton. In conclusion, 31P NMR is a useful tool to asses the bioavailability of certain P compound groups, and the combination with traditional fractionation techniques makes quantification possible.
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| 4. |
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| 5. |
- Amirkhani, Ardeshir, 1965-
(författare)
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Development of Techniques and Methods for the Quantitative Analysis of Endogenous Substances by Microcolumn Liquid Chromatography Coupled to Mass Spectrometry
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Liquid chromatography-mass spectrometry (LC-MS) is a powerful technique for the analysis of endogenous compounds. The introduction of electrospray ionization (ESI) as an interface between LC and MS has contributed strongly to a trend towards miniaturization of LC, due to the possibility to perform ESI at low flow rates. In this thesis, several aspects regarding the design of miniaturized LC systems and electrospray emitters were investigated. In addition miniaturized LC-ESI-MS have been used for the qualitative and quantitative analysis of endogenous polar compounds, peptides and protein digests.The performance of miniaturized LC-MS was compared using different electrospray emitter configurations. The results indicated that the efficiency of the LC system is rather independent of the configuration of the emitter.The lifetime of gold-coated fused silica electrospray emitters based on vapor deposited adhesion layers of titanium were investigated. The long lifetime of the emitter facilitates the use in LC-MS experiments, exemplified LC-MS by analysis of neuropeptides.The ESI voltage is shown to interfere with liquid chromatographic separations performed in packed porous graphitic carbon capillary column. This interference is ascribed to the presence of an electric field over the conductive column in absence of a ground point between the column and the ESI emitter.The solid supported enhanced microdialysis for analysis of neuropeptides were compared with conventional microdialysis. The difference between the two methodologies were evaluated by LC-MS analysis of the microdialysates. The solid supported method gave in general higher relative recoveries.Finally, a method of standard addition was developed to determine total level of tryptophan and two of its metabolites in human plasma by capillary LC-ESI tandem mass spectrometry. The method was applied in a clinical study of multiple scleroses patients treated with cytokines (IFN Beta 1a, 1b). The results show that the intervention effects the tryptophan metabolism.
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| 6. |
- Amirkhani, Ardeshir, et al.
(författare)
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Interferon-beta affects the tryptophan metabolism in multiple sclerosis patients
- 2005
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Ingår i: European Journal of Neurology. - : Wiley. - 1351-5101 .- 1468-1331. ; 12:8, s. 625-631
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Tidskriftsartikel (refereegranskat)abstract
- Tryptophan and its metabolites are of great interest in understanding the pathogenesis of multiple sclerosis (MS). The total levels of tryptophan and its metabolites, kynurenine and kynurenic acid were determined in plasma by capillary liquid chromatography electrospray ionisation tandem mass spectrometry. This is the first report of the plasma levels of these analytes in healthy controls and relapsing-remitting MS patients receiving long-term and acute interferon-beta (IFN-beta) treatment. Twenty-four hours post-administration increased kynurenine levels (first IFN MS versus healthy, P = 0.042) and kynurenine/tryptophan ratio (K/T; first IFN MS versus healthy, P =0.027; first IFN MS versus long-term IFN MS, P = 0.036) were found. The long-term IFN MS group had higher K/T ratios at 4 and 12 h post-administration (P = 0.015 and 0.009, respectively). The increase of K/T ratio in the first IFN MS group indicate an induction of the enzyme indolamine-2,3-dioxygenase (IDO), as reported earlier in experimental allergic encephalomyelitis. As IDO is participating in both inflammatory and neurodegenerative processes, further knowledge of its involvement in the pathogenesis of MS is of great importance.
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| 7. |
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| 8. |
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| 9. |
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| 10. |
- Bazoti, Fotini N., et al.
(författare)
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Localization of the noncovalent binding site between amyloid-beta-peptide and oleuropein using electrospray ionization FT-ICR mass spectrometry.
- 2008
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Ingår i: Journal of the American Society for Mass Spectrometry. - : American Chemical Society (ACS). - 1044-0305 .- 1879-1123. ; 19:8, s. 1078-1085:19, s. 1078-1085
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Tidskriftsartikel (refereegranskat)abstract
- Abnormal accumulation and aggregation of amyloid-alpha-peptide (AM) eventually lead to the formation and cerebral deposition of amyloid plaques, the major pathological hallmark in Alzheimer's disease (AD). Oleuropein (OE), an Olea europaea L. derived polyphenol, exhibits a broad range of pharmacological properties, such as antioxidant, anti-inflammatory, and antiatherogenic, which could serve as combative mechanisms against several reported pathways involved in the pathophysiology of AD. The reported noncovalent interaction between AM and OE could imply a potential antiamyloidogenic role of the latter on the former via stabilization of its structure and prevention of the adaptation of a toxic beta-sheet conformation. The established P-sheet conformation of the AM hydrophobic carboxy-terminal region and the dependence of its toxicity and aggregational propensity on its secondary structure make the determination of the binding site between AM and OF highly important for assessing the role of the interaction. In this study, two different proteolytic digestion protocols, in conjunction with high-sensitivity electrospray ionization mass spectrometric analysis of the resulting peptide fragments, were used to determine the noncovalent binding site of OE on AM and revealed the critical regions for the interaction.
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| 11. |
- Bazoti, Fotini N., et al.
(författare)
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Noncovalent interaction between amyloid-b-peptide (1-40) and oleuropein studied by electrospray ionization mass spectrometry.
- 2006
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Ingår i: Journal of the American Society for Mass Spectrometry. - : American Chemical Society (ACS). - 1044-0305 .- 1879-1123. ; 17:4, s. 568-575
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Tidskriftsartikel (refereegranskat)abstract
- Beta amyloid peptide (A beta) is the major proteinaceous component of senile plaques formed in Alzheimer's disease (AD) brain. The aggregation of A beta is associated with neurodegeneration, loss of cognitive ability, and premature death. It has been suggested that oxidative stress and generation of free radical species have implications in the fibrillation of A beta and its subsequent neurotoxicity. For this reason, it is proposed that antioxidants may offer a protective or therapeutic alternative against amyloidosis. This study is the first report of the formation of the noncovalent complex between A beta or its oxidized form and the natural derived antioxidant oleuropein (OE) by electrospray ionization mass spectrometry (ESI MS). ESI MS allowed the real time monitoring of the complex formation between A beta, OE, and variants thereof. Several experimental conditions, such as elevated orifice potential, low pH values, presence of organic modifier, and ligand concentration were examined, to assess the specificity and the stability of the formed noncovalent complexes.
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| 12. |
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| 13. |
- Bazoti, Fotini N, et al.
(författare)
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Study of the non-covalent interaction between amyloid-beta-peptide and melatonin using electrospray ionization mass spectrometry
- 2005
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Ingår i: Journal of Mass Spectrometry. - : Wiley. - 1076-5174 .- 1096-9888. ; 40:2, s. 182-192:40, s. 182-192
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Tidskriftsartikel (refereegranskat)abstract
- Oxidative stress and unregulated immune response are believed to play a key role in the processes inherent to Alzheimer's disease (AD). The fact that free radicals can result in neurodegeneration suggests that actions against reactive oxygen species may be beneficial in treating and preventing AD. In the light of the suggested link between oxidative stress and AD, it is proposed that antioxidants and, even more, endogenous antioxidants may offer a therapeutic regime for protection against the risk of this disease. For this reason, the formation of non-covalent complexes between amyloid-beta-peptide (A beta) or its oxidized forms and melatonin was studied by quadrupole and Fourier transform ion cyclotron resonance electrospray ionization mass spectrometry. The stability of the non-covalent complex was examined under several experimental conditions, such as orifice voltage, pH, presence of organic modifier, concentration and time. Two different digestion protocols combined with mass spectrometric analysis of the resulting peptide fragments were employed in order to locate the binding site of melatonin in A beta.
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| 14. |
- Bergquist, Jonas, et al.
(författare)
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Peptide Mapping of Proteins in Human Body Fluids using Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
- 2002
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Ingår i: Mass spectrometry reviews (Print). - : Wiley. - 0277-7037 .- 1098-2787. ; 21:1, s. 2-15
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Tidskriftsartikel (refereegranskat)abstract
- Human body fluids have been rediscovered in the postgenomic era as great sources of biological markers and perhaps particularly as sources of potential protein biomarkers of disease. Analytical tools that allow rapid screening, low sample consumption, and accurate protein identification are of great importance in studies of complex biological samples and clinical diagnosis. Mass spectrometry is today one of the most important analytical tools with applications in a wide variety of fields. One of the fastest growing applications is in proteomics, or the study of protein expression in an organism. Mass spectrometry has been used to find post-translational modifications and to identify key functions of proteins in the human body. In this study, we review the use of human body fluids as sources for clinical markers and present new data that show the ability of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) to identify, and characterize proteins in four human body fluids: plasma, cerebrospinal fluid (CSF), saliva, and urine. The body fluids were tryptically digested without any prior separation, purification, or selection, and the digest was introduced into a 9.4 T FTICR mass spectrometer by direct-infusion electrospray ionization (ESI). Even though these samples represent complex biological mixtures, the described method provides information that is comparable with traditional 2D-PAGE data. The sample consumption is extremely low, a few microliters, and the analysis time is only a few minutes. It is, however evident that the separation of proteins and/or peptides must be included in the methodology in order to detect low-abundance proteins and other proteins of biological relevance.
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| 15. |
- Bergström, Sara, 1977-
(författare)
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Integrated Micro-Analytical Tools for Life Science
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Advances in life science require knowledge of active molecules in complex biological systems. These molecules are often only present for a certain time and at limited concentrations. Integrated micro-analytical tools for sampling, separation and mass spectrometric (MS) detection would meet these requests and are therefore continuously gaining interest. An on-line coupling of analytical functions provides shorter analysis time and less manual sample handling. In this thesis, improved compatibility of microdialysis sampling and multidimensional separations coupled to MS detection are developed and discussed. Microdialysis was used in vitro for determination of the non-protein bound fraction of the drug ropivacaine. The sampling unit was coupled on-line to capillary column liquid chromatography (LC) followed by ultraviolet or MS detection. For MS detection, the system was extended with a desalting step and an addition of internal standard. A method for MS screening of microdialysates, collected in vivo, was also developed. The method involved sampling and measurements of the chemical pattern of molecules that generally are ignored in clinical investigations. Chemometric tools were used to extract the relevant information and to compare samples from stimulated and control tissues. Complex samples often require separation in more than one dimension. On-line interfaces for sample transfer between LC and capillary electrophoresis (CE) were developed in soft poly(dimethylsiloxane) (PDMS). MS detection in the LC-CE system was optimised on frequent sampling of the CE peak or on high resolution in mass spectra using time-of-flight (TOF)MS or Fourier transform ion cyclotron resonance (FTICR)MS, respectively. Aspects on electrode positioning in the LC-CE interface led to development of an on-column CE electrode. A successful method for deactivation of the PDMS surface using a polyamine polymer was also developed. The systems were evaluated using peptides and proteins, molecules that are gaining increased attention in bioscience, and consequently also in chemical analysis.
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| 16. |
- Bergström, Sara K., et al.
(författare)
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A simplified multidimensional approach for analysis of complex biological samples : on-line LC-CE-MS
- 2006
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Ingår i: The Analyst. - : Royal Society of Chemistry (RSC). - 0003-2654 .- 1364-5528. ; 131:7, s. 791-798
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Tidskriftsartikel (refereegranskat)abstract
- Information on protein expression, disease biomarkers or surrogate markers and genetic disorders can nowadays be achieved from analysis of complex biological samples by liquid separation coupled to mass spectrometric (MS) detection. This paper describes fast multidimensional separation by on-line liquid chromatography (LC) and capillary electrophoresis (CE), followed by electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) MS detection. This detector provides ultrahigh resolution of the detected ions, mass accuracy at the ppm-level and high sensitivity. Most of the challenge of this system lies in the development of a new interface for the on-line coupling of LC to CE. The interface developed in poly(dimethylsiloxane) provides a RSD for injection repeatability of <3.5% and surface control for unspecific binding by deactivation with a cationic polymer, PolyE-323. We have evaluated the interface, as well as the overall system, with respect to robustness and deconvolution ability. Sequence coverage for bovine serum albumin (BSA) of 93% showed a high recovery of sample in the different transfer steps through the system. The detection limit for identification is 277 ng mL−1 (or 280 nM) on average for peptides. In the future, we expect LC-CE-MS to be a novel strategy for elucidating the chemistry of biological matrices.
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| 17. |
- Bergström, Sara K, et al.
(författare)
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Development of a Poly(dimethylsiloxane) Interface for On-Line Capillary Column Liquid Chromatography : Capillary Electrophoresis Coupled to Sheathless Electrospray Ionization Time-of-Flight Mass Spectrometry
- 2003
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 75:20, s. 5461-5467
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Tidskriftsartikel (refereegranskat)abstract
- An interface in elastomeric poly(dimethylsiloxane) (PDMS) for on-line orthogonal coupling of packed capillary liquid chromatography (LC) (i.d. = 0.2 mm) with capillary electrophoresis (CE) in combination with sheathless electrospray ionization (ESI) time-of-flight mass spectrometric (TOFMS) detection is presented. The new interface has a two-level design, which in combination with a continuous CE electrolyte flow through the interface provides integrity of the LC effluent and the CE separation until an injection is desired. The transparent and flexible PDMS material was found to have a number of advantages when combined with fused silica column technology, including ease to follow the process and ease to exchange columns. By combining conventional microscale systems of LC, CE, and ESI−MS, respectively, the time scales of the individual dimensions were harmonized for optimal peak capacity per unit time. The performance of the LC−CE−TOFMS system was evaluated using peptides as model substances. A S/N of about 330 was achieved for leucine-enkephaline from a 0.5 μL LC injection of 25 μg/mL peptide standard.
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| 18. |
- Bergström, Sara K., et al.
(författare)
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On-line coupling of microdialysis to packed capillary column liquid chromatography-tandem mass spectrometry demonstrated by measurement of free concentrations of ropivacaine and metabolite from spiked plasma samples
- 2002
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Ingår i: Journal of chromatography. B. - 1570-0232 .- 1873-376X. ; 775:1, s. 79-87
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Tidskriftsartikel (refereegranskat)abstract
- An on-line coupling of microdialysis to a packed capillary column switching liquid chromatographic system (0.2 mm I.D.) and mass spectrometric detection was developed. The microdialysates were collected in the loop of the first of three valves, coupled in direct series. A deuterated internal standard was added on-line by the middle valve and the third valve operated both a pre-column, for desalting of the physiological buffer used in the sampling procedure, and a separation column. The on-line system was used to study free concentrations of ropivacaine and its metabolite (PPX) in human plasma samples. The analytes were detected by electrospray ionization in a tandem mass spectrometer operating in multiple reaction monitoring mode. The free fractions of ropivacaine (200 nM total concentration) and PPX (20 nM total concentration) in spiked plasma samples were 12±3 and 47±5% (±standard deviation for day-to-day variations, n=3), respectively.
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| 19. |
- Bergström, Sara K., et al.
(författare)
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Polyamine deactivation of integrated poly(dimethylsiloxane) structures investigated by radionuclide imaging and capillary electrophoresis experiments
- 2005
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 77:3, s. 938-942
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Tidskriftsartikel (refereegranskat)abstract
- The poly(dimethylsiloxane) (PDMS) material provides a number of advantageous features, such as flexibility, elasticity, and transparency, making it useful in integrated analytical systems. Hard fused-silica capillary structures and soft PDMS channels can easily be combined by a tight fit, which offers many alternatives for structure combinations. PDMS and fused silica are in different ways prone to adsorption of low levels of organic compounds. The need for modification of the inner wall surface of PDMS channels may often be necessary, and in this paper, we describe an easy and effective method using the amine-containing polymer PolyE-323 to deactivate both fused-silica and PDMS surfaces. The adsorption of selected peptides to untreated surfaces was compared to PolyE-323-modified surfaces, using both radionuclide imaging and capillary electrophoresis experiments. The polyamine modification displayed a substantially reduced adsorption of three hydrophobic test peptides compared to the native PDMS surface. Filling and storage of aqueous solution were also possible in PolyE-323-modified PDMS channels. In addition, hybrid microstructures of fused silica and PDMS could simultaneously be deactivated in one simple coating procedure.
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| 20. |
- Bergström, Sara K., et al.
(författare)
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Screening of microdialysates taken before and after induced liver damage; on-line solid phase extraction-electrospray ionization-mass spectrometry
- 2006
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1120:1-2, s. 21-26
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Tidskriftsartikel (refereegranskat)abstract
- A novel method is described to follow known and unknown compounds in biological processes using microdialysis sampling and mass spectrometric detection. By implementation of internal standard, desalting/enrichment for the sample work-up, and multivariate data analysis, this methodology is a basis for future applications in early diagnosis of diseases and organ damage, as a complement to the routinely used clinical methods for biological samples. The present study includes screening without specific target analytes, of samples collected by microdialysis from liver of anaesthetized rats before and after local damage to this organ. Sample series were classified by principal component analysis, and the stimulation was identified in the chemical patterns produced by the presented analytical tool.
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| 21. |
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| 22. |
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| 23. |
- Bylund, Dan, et al.
(författare)
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Chromatographic alignment by warping and dynamic programming as a pre-processing tool for PARAFAC modelling of liquid chromatography-mass spectrometry data
- 2002
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 961:2, s. 237-244
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Tidskriftsartikel (refereegranskat)abstract
- Solutes analysed with LC–MS are characterised by their retention times and mass spectra, and quantified by the intensities measured. This highly selective information can be extracted by multiway modelling. However, for full use and interpretability it is necessary that the assumptions made for the model are valid. For PARAFAC modelling, the assumption is a trilinear data structure. With LC–MS, several factors, e.g. non-linear detector response and ionisation suppression may introduce deviations from trilinearity. The single largest problem, however, is the retention time shifts not related to the true sample variations. In this paper, a time warping algorithm for alignment of LC–MS data in the chromatographic direction has been examined. Several refinements have been implemented and the features are demonstrated for both simulated and real data. With moderate time shifts present in the data, pre-processing with this algorithm yields approximately trilinear data for which reasonable models can be made.
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| 24. |
- Bylund, Dan, et al.
(författare)
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Peak purity assessment in liquid chromatography-mass spectrometry
- 2001
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 915:1/2, s. 43-52
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Tidskriftsartikel (refereegranskat)abstract
- Fixed-size moving window evolving factor analysis and base peak chromatograms have been used for peak purity detection in data generated with LC–MS. The two methods were evaluated with both real and simulated data and were found to be fast and complementary to each other. When a possibly impure peak is detected, it is suggested that further information can be obtained from local principal component analysis modelling and comparative mass chromatogram plots.
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| 25. |
- Bökman, C.F., et al.
(författare)
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Relating chromatographic retention and electrophoretic mobility to the ion distribution within electrosprayed droplets
- 2006
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Ingår i: Journal of the American Society for Mass Spectrometry. - : American Chemical Society (ACS). - 1044-0305 .- 1879-1123. ; 17:3, s. 318-324
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Tidskriftsartikel (refereegranskat)abstract
- Ions that are observed in a mass spectrum obtained with electrospray mass spectrometry can be assumed to originate preferentially from ions that have a high distribution to the surface of the charged droplets. In this study, a relation between chromatographic retention and electrophoretic mobility to the ion distribution (derived from measured signal intensities in mass spectra and electrospray current) within electrosprayed droplets for a series of tetraalkylammonium ions, ranging from tetramethyl to tetrapentyl, is presented. Chromatographic retention in a reversed-phase system was taken as a measure of the analyte's surface activity, which was found to have a large influence on the ion distribution within electrosprayed droplets. In addition, different transport mechanisms such as electrophoretic migration and diffusion can influence the surface partitioning coefficient. The viscosity of the solvent system is affected by the methanol content and will influence both diffusion and ion mobility. However, as diffusion and ion mobility are proportional to each other, we have, in this study, chosen to focus on the ion mobility parameter. It was found that the influence of ion mobility relative to surface activity on the droplet surface partitioning of analyte ions decreases with increasing methanol content. This effect is most probably coupled to the decrease in droplet size caused by the decreased surface tension at increasing methanol content. The same observation was made upon increasing the ionic strength of the solvent system, which is also known to give rise to a decreased initial droplet size. The observed effect of ionic strength on the droplet surface partitioning of analyte ions could also be explained by the fact that at higher ionic strength, a larger number of ions are initially closer to the droplet surface and, thus, the contribution of ionic transport from the bulk liquid to the liquid/air surface interface (jet and droplet surface), attributable to migration or diffusion will decrease.
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| 26. |
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| 27. |
- Dahlin, Andreas, 1976-
(författare)
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Microscale Tools for Sample Preparation, Separation and Detection of Neuropeptides
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The analysis of low abundant biological molecules is often challenging due to their chemical properties, low concentration and limited sample volumes. Neuropeptides are one group of molecules that fits these criteria. Neuropeptides also play an important role in biological functions, which makes them extra interesting to analyze. A classic chemical analysis involves sampling, sample preparation, separation and detection. In this thesis, an enhanced solid supported microdialysis method was developed and used as a combined sampling- and preparation technique. In general, significantly increased extraction efficiency was obtained for all studied peptides. To be able to control the small sample volumes and to minimize the loss of neuropeptides because of unwanted adsorption onto surfaces, the subsequent analysis steps were miniaturized to a micro total analysis system (µ-TAS), which allowed sample pre-treatment, injection, separation, manipulation and detection. In order to incorporate these analysis functions to a microchip, a novel microfabrication protocol was developed. This method facilitated three-dimensional structures to be fabricated without the need of clean room facilities. The sample pre-treatment step was carried out by solid phase extraction from beads packed in the microchip. Femtomole levels of neuropeptides were detected from samples possessing the same properties as microdialysates. The developed injection system made it possible to conduct injections from a liquid chromatographic separation into a capillary electrophoresis channel, which facilitated for advanced multidimensional separations. An electrochemical sample manipulation system was also developed. In the last part, different electrospray emitter tip designs made directly from the edge of the microchip substrate were developed and evaluated. The emitters were proven to be comparable with conventional, capillary based emitters in stability, durability and dynamic flow range. Although additional developments remain, the analysis steps described in this thesis open a door to an integrated, on-line µ-TAS for neuropeptides analysis in complex biological samples.
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| 28. |
- Dahlin, Andreas P., et al.
(författare)
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Poly(dimethylsiloxane)-Based Microchip for Two-Dimensional Solid-Phase Extraction-Capillary Electrophoresis with an Integrated Electrospray Emitter Tip
- 2005
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 77:16, s. 5356-5363
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Tidskriftsartikel (refereegranskat)abstract
- A microchip in poly(dimethylsiloxane) (PDMS) for in-line solid-phase extraction-capillary electrophoresis-electrospray ionization-time-of-flight mass spectrometry (SPE-CE-ESI-TOF-MS) has been developed and evaluated. The chip was fabricated in a novel one-step procedure where mixed PDMS was cast over steel wires in a mold. The removed wires defined 50-um cylindrical channels. Fused-silica capillaries were inserted into the structure in a tight fit connection. The inner walls of the inserted fused-silica capillaries and the PDMS microchip channels were modified with a positively charged polymer, PolyE-323. The chip was fabricated in a two-level cross design. The channel at the lower level was packed with 5-um hyper-cross-linked polystyrene beads acting as a SPE medium used for desalting. The upper level channel acted as a CE channel and ended in an integrated emitter tip coated with conducting graphite powder to facilitate the electrical contact for sheathless ESI. An overpressure continuously provided fresh CE electrolyte independently of the flows in the different levels. Further studies were carried out in order to investigate the electrophoretic and flow rate properties of the chip. Finally, six-peptide mixtures, in different concentrations, dissolved in physiological salt solution was injected, desalted, separated, and sprayed into the mass spectrometer for analysis with a limit of detection in femtomole levels.
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| 29. |
- Danielsson, Rolf, et al.
(författare)
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Matched filtering with background suppression for improved quality of base peak chromatograms and mass spectra in liquid chromatography-mass spectrometry
- 2002
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Ingår i: Analytica Chimica Acta. - 0003-2670. ; 454:2, s. 167-184
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Tidskriftsartikel (refereegranskat)abstract
- A time domain filter that combines the properties of matched filtering and two-fold differentiation is presented. The filter coefficients are given by the second derivative of a Gaussian model peak, controlled by the setting of two parameters related to the chromatographic system. The fundamental characteristics of the filter were derived, and its applicability demonstrated for real liquid chromatography–mass spectrometry (LC–MS) data. The filter is primarily intended as a fast pre-processing step, for a mass chromatogram with 320 scans over 700 mass channels the computation time was 0.6 s on a standard PC. Base peak chromatograms with improved peak detection capability and mass spectra useful for compound identification were obtained with filtered data. The most significant effect of the described filter is background reduction due to the differentiation, which in combination with the matched filter can be performed with maintained or even improved signal-to-noise ratio.
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| 30. |
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| 31. |
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| 32. |
- Galanakis, Petros A., et al.
(författare)
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Study of the Interaction Between the Amyloid Beta Peptide (1-40) and Antioxidant Compounds by Nuclear Magnetic Resonance Spectroscopy
- 2011
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Ingår i: Biopolymers. - : Wiley. - 0006-3525 .- 1097-0282. ; 96:3, s. 316-327
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid beta peptide (Aβ) aggregation leads to the senile plaque formation, a process that is strongly influenced by oxidative stress and is considered as the molecular basis of various neurodegenerative diseases, such as Alzheimer's Disease (AD). Endogenous antioxidants or dietary derived compounds may down-regulate this process. In this study, the interaction of two antioxidants, oleuropein (OE) and melatonin (M) with Aβ, is monitored through NMR spectroscopy and Mass Spectrometry. The concerted application of these two analytical techniques provides new experimental evidence and residue-specific insights into the interacting Aβ peptide amino acids that are implicated in this process. Both antioxidant compounds interact in a similar way with the peptide and cause chemical shift variations. The most pronounced resonance changes have been observed for the (1)H-(15)N signals of N-terminal region and Leu(17)-Phe(20) residues, as monitored by NMR titration studies.
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| 33. |
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| 34. |
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| 35. |
- Isaac, Giorgis, et al.
(författare)
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Analysis of phosphatidylcholine and sphingomyelin molecular species from brain extracts using capillary liquid chromatography electrospray ionization mass spectrometry
- 2003
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Ingår i: Journal of Neuroscience Methods. - 0165-0270 .- 1872-678X. ; 128:1-2, s. 111-119
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Tidskriftsartikel (refereegranskat)abstract
- One feature of complex lipids is that many subtypes of these molecules exist as a diverse mixture in a biological sample. Qualitative and quantitative analysis of these closely related molecules require sensitive and specific analytical methods to detect intact phospholipids (PL) and sphingomyelin (SM) species and to differentiate between them. Conventional analytical methods require laborious procedures including separation by column, argentation thin-layer chromatography or liquid chromatography (LC) after pre- or post-column derivatization. In the present work, a method based on reversed phase capillary LC coupled on-line to electrospray ionization mass spectrometry (LC/ESI/MS) has been developed to gather tools for lipidomic studies, i.e. the profiling of complex mixtures of lipids in small amounts of various cells and tissues. The LC/MS system used consisted of an LC pump in an isocratic elution, a reversed phase capillary column and a single quadrupole mass spectrometer operated in the positive ion mode. A successful separation of phosphatidylcholine (PC) and SM molecular species was obtained with a minimum detectable quantity (MDQ) in the low fmol range injected on column. The method was applied to human brain extracts. Furthermore, the extraction efficiencies of the traditional Folch method and pressurized fluid extraction (PFE) were compared using the human brain. It was found that the intensity of the PC and SM molecular species extracted by PFE is two times that of Folch.
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| 36. |
- Isaac, Giorgis, 1974-
(författare)
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Development of Enhanced Analytical Methodology for Lipid Analysis from Sampling to Detection : A Targeted Lipidomics Approach
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis covers a wide range of analytical method development for lipid analysis in complex biological samples; from sample preparation using pressurized fluid extraction (PFE) and separation with reversed phase capillary liquid chromatography (RP-LC) to detection by electrospray ionization mass spectrometry (ESI/MS) and tandem MS.The requirements for fast, reliable and selective extraction methods with minimal usage of solvents have accelerated the development of new extraction techniques. PFE is one of the new automated, fast and efficient liquid extraction techniques which use elevated temperature and pressure with standard liquid solvents. In this thesis the reliability and efficiency of the PFE technique was investigated for the extraction of total lipid content from cod, herring muscle and human brain tissue as well as for pesticides from fatty foodstuffs. Improved or comparable efficiencies were achieved with reduced time and solvent consumption as compared to traditional methods. A RP-LC coupled online to ESI/MS for the analysis of phosphatidylcholine (PC) and sphingomyelin (SM) molecular species was developed and used for the analysis of brain lipids from eight groups of mice treated with vehicle and various neuroleptics. The effect of postnatal iron administration in lipid composition and behavior was investigated. Whether or not these effects could be altered by subchronic administration of the neuroleptics (clozapine and haloperidol) were examined. The results support the hypothesis that an association between psychiatric disorders, behavior abnormalities and lipid membrane constitution in the brain exists.Finally, a tandem MS precursor ion scan was used to analyze the developmental profile of brain sulfatide accumulation in arylsulfatase A (ASA) deficient (ASA -/-) as compared to wild type control (ASA +/+) mice. The ASA -/- mice were developed as a model of the monogenic disease metachromatic leukodystrophy with an established deficiency of the lysosomal enzyme ASA. The results showed that an alteration in the composition of sulfatide molecular species was observed between the ASA -/- and ASA +/+ mice.This thesis shows that modern analytical methods can provide new insights in the extraction and analysis of lipids from complex biological samples.
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| 37. |
- Isaac, Giorgis, et al.
(författare)
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Total Lipid Extraction of Homogenized and Intact Lean Fish Muscles Using Pressurized Fluid Extraction and Batch Extraction Technique
- 2005
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Ingår i: Journal of Agricultural and Food Chemistry. - : American Chemical Society (ACS). - 0021-8561 .- 1520-5118. ; 53:14, s. 5506-5512
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Tidskriftsartikel (refereegranskat)abstract
- The reliability and efficiency of pressurized fluid extraction (PFE) technique for the extraction of total lipid content from cod and the effect of sample treatment on the extraction efficiency have been evaluated. The results were compared with two liquid-liquid extraction methods, traditional and modified methods according to Jensen. Optimum conditions were found to be with 2-propanol/n-hexane (65:35, v/v) as a first and n-hexane/diethyl ether (90:10, v/v) as a second solvent, 115 oC, and 10 min of static time. PFE extracts were cleaned up using the same procedure as in the methods according to Jensen. When total lipid yields obtained from homogenized cod muscle using PFE were compared yields obtained with original and modified Jensen methods, PFE gave significantly higher yields, ~10% higher (t test, P < 0.05). Infrared and NMR spectroscopy suggested that the additional material that inflates the gravimetric results is rather homogeneus and is primarily consists of phospholipid with headgroups of inositidic and/or glycosidic nature. The comparative study demonstrated that PFE is an alternative suitable technique to extract total lipid content from homogenized cod (lean fish) and herring (fat fish) muscle showing a precision comparable to that obtained with the traditional and modified Jensen methods. Despite the necessary cleanup step, PFE showed important advantages in the solvent consumption was cut by ~50% and automated extraction was possible.
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| 38. |
- Jansson, Christer, et al.
(författare)
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A new multi-residue method for analysis of pesticide residues in fruit and vegetables using liquid chromatography with tandem mass spectrometric detection
- 2004
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1023:1, s. 93-104
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Tidskriftsartikel (refereegranskat)abstract
- A new multi-residue method for determination of pesticide residues in a wide variety of fruit and vegetables, using the National Food Administration (NFA) ethyl acetate extraction and determination by means of LC-MS/MS, is presented. The method includes pesticides normally detected by LC-UV or LC-fluorescence such as benzimidazoles, carbamates, N-methylcarbamates and organophosphorus compounds with an oxidisable sulphide group as well. After extraction with ethyl acetate, the extract is concentrated and an aliquot of the extract is evaporated to dryness and redissolved in methanol before injection on LC-MS/MS. The method has been validated for 57 different pesticides and metabolites. Representative species from different commodity groups were chosen as matrices in order to study the influence from different matrices on recoveries. The fortification levels studied were 0.01-0.5 mg kg(-1). Matrix effects were tested for all matrices by means of standard addition to blank extracts. The matrix effect, expressed as signal in solvent compared to signal in matrix, was in general found to be small. The obtained recoveries are, with a few exceptions, in the range 70-100%. The proposed method is quick and straightforward and no additional clean-up steps are needed. The method can be used for the analysis of all 57 pesticides in one single determination step at 0.01 mg kg(-1).
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| 39. |
- Johannesson, Nina, 1975-
(författare)
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Column Development in Capillary Electrophoresis and Electrochromatography for Bioanalytical Applications
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Analysis of biological samples can be a difficult task. This thesis covers a broad aspect of the analytical areas of capillary electrophoresis (CE) and capillary electrochromatography (CEC) in combination with mass spectrometry (MS) that are of great importance for achieving fast, accurate and sensitive bioanalyses. A significantly time reduced and automated system for sample cleanup was developed to greatly simplify the pretreatment process of biological samples with a complex matrix. Desalting and preconcentration of species in urine was conducted and the limit of detection for the antidepressant escitalopram was lowered 10 times. This extraction devise was also successfully incorporated in a chip based platform for the possibility to be a part of multidimensional separation systems. The reduced risk of sample loss leads to improved detection limits, which are usually one the most challenging parts when working with bioanalyses. In the area of separation, a monomer surface with tailored hydrophobicity was developed to achieve rapid, high efficient separations of complex mixtures. Within five minutes a tryptic digest of a protein could be separated and then identified by a Mascot search. The applications addressed have been focused on medical conditions which are of highest interest for both physicians and patients. A high throughput analysis of the kynurenine metabolites with CE-MS offers a new method to rapidly examine samples from patients with neurological disorders. A screening study of possible biomarkers for the two different types of appendicitis, gangraenous and phlegmonous was conducted. Indicative patterns were found for both pre and post surgery of the two types of inflammation as well as between them. The divergences were traced back to the MS peaks obtained in the CE- and CEC-MS setups as possible biomarkers for the two forms of appendicitis. A preliminary study of polycystic ovary syndrome also offered some valuable results for future biomarker identification.
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| 40. |
- Johannesson, Nina, et al.
(författare)
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Monomer surface modifications for rapid peptide analysis by capillary electrphoresis and capillary electrochromatography coupled to electrospray ionization mass spectrometry
- 2004
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 25:6, s. 809-816
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Tidskriftsartikel (refereegranskat)abstract
- In this study, positively charged alkylaminosilyl monomers were used to modify the inner surface of fused silica capillaries, which subsequently were employed in capillary electrophoresis (CE) and capillary electrochromatography (CEC). The obtained surfaces yield a reversed electroosmotic flow (EOF) and have varying carbon chain lengths, that interact with the analytes and give chromatographic retention. The coating procedure is very simple and fast. The performance of the modified capillaries was evaluated regarding pH influence on EOF and chromatographic interactions. The experiments were conducted with UV and mass spectrometry (MS) and applied to the separation of various neuropeptides. The derivatized surfaces showed a linear (R2 ∼0.99) pH dependence with isoelectric points (pI) at 8.6–8.8. Rapid separations of peptide standards and a protein digest with efficiencies as high as 5×105 plates/m were performed.
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| 41. |
- Johannesson, Nina, et al.
(författare)
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On-Line Biological Sample Cleanup for Electrospray Mass Spectrometry Using Sol-Gel Columns
- 2006
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Ingår i: Journal of chromatography. B. - : Elsevier BV. - 1570-0232 .- 1873-376X. ; 842:1, s. 70-74
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Tidskriftsartikel (refereegranskat)abstract
- Using a slight overpressure, a urine sample is loaded onto a monolithic photopolymerized sol-gel column that has been derivatized with hydrophobic carbon chains and then the complex urine matrix is washed with aqueous solution. A buffer containing organic solvent is used to elute the adsorbed peptides by an applied voltage and the sample is then introduced into a mass spectrometer by sheath flow electrospray. The importance of desalting this type of sample is demonstrated by an experiment that shows that the signal intensity of a test solution with neurotensin, sprayed directly into the mass spectrometer, decreased from 4.5 x 10(4) Cps to no detectible signal when just 10% urine is added to the sample solution. We suggest that this procedure may find general application for desalting biological samples prior to mass spectrometric analysis.
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| 42. |
- Koivisto, Pernilla, et al.
(författare)
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Determination of the free concentration of ropivacaine in plasma by packed capillary liquid chromatography : A comparison of ultrafiltration and microdialysis as sample preparation methods
- 2001
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Ingår i: Journal of Microcolumn Separations. - : Wiley. - 1040-7685 .- 1520-667X. ; 13:5, s. 197-201
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Tidskriftsartikel (refereegranskat)abstract
- In this study, ultrafiltration and microdialysis have been compared as sample preparation methods to separate the free fraction of ropivacaine from the protein bound in 150 μL plasma. A liquid chromatographic system with packed capillary columns (0.2 mm internal diameter) was used to enhance sensitivity when analyzing the small sample volumes obtained after the ultrafiltration and the microdialysis. The microdialysis was performed with probes of our own construction, and to save analysis time, the microdialysis sampling was coupled on-line to the liquid chromatographic system. The reduction of back pressure in the microdialysis outlet tube and in the injector was found to be essential. The free fraction obtained with each method was equivalent: both gave a free fraction of 6%.
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| 43. |
- Lavén, Martin, et al.
(författare)
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Analysis of microsomal metabolic stability using high-flow-rate extraction coupled to capillary liquid chromatography-mass spectrometry
- 2004
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Ingår i: Journal of Chromatography B. ; :806, s. 119-126
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Tidskriftsartikel (refereegranskat)abstract
- A method described for on-line high-speed extraction of microsomal samples and analysis by capillary liquid chromatography - mass spectrometry (LC-MS) for the determination of metabolic stability in connection with the development of positron emission tomography (PET) tracers. the method allowed direct injections of large sample volumes at a fast extraction rate, providing a gain in both sensitivity and sample preparation time. The calibration curve of the test compound flumazenil (Ro 15-1788) was linear in the concentration range of 1-150 nM, with a correlation coefficient exceeding 0.999. The accuracy of the method ranged from 98 to 101%. A high precision was obtained, with mean intra-assay and inter-assay relative standard deviations of at most 1.4 and 1.5%, respectively, for quality control (QC) samples. The extraction efficiency was determined to be 99.4%, the total recovery 96% and the carryover to <0.23%. Extractions were performed in a concentration interval of 30-300 nM without any sign of column overload. The method was successfully used for determining the microsomal metabolic stability of flumazenil. As a result, the described analysis system is currently used for metabolic screening of PET tracer candidates in our laboratory.
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| 44. |
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| 45. |
- Lavén, Martin, et al.
(författare)
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Determination of flumazenil in human plasma by liquid chromatography - electrospray ionisation tandem mass spectrometry
- 2004
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Ingår i: Journal of Chromatography B. ; :808, s. 221-227
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Tidskriftsartikel (refereegranskat)abstract
- A liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) method was developed to determine unlabelled flumazenil (Ro 15-1788) in human plasma in (11 C)flumazenil positron emission tomagraphy (PET) studies.N-Methyl tri-deuterated flumazenil was used as an internal standard. The analyte and internal standard were extracted from plasma samples using solid-phase extraction, with a recovery of 78%. This was determined through the convenience of radioactivity measurements of 11 C-labelled flumazenil. The evaporated and reconstituted eluate was analysed by LC-ESI-MS/MS. The calibration curve was linear over the tested concentration range of 0.05-0.5 nM (15-150 pg/ml) with a correlation coefficient, R 2, of 0.998 +- 0.001. A high precision was achieved, with mean intra-assay and inter-assay relative standard deviations of at most 6 and 7%, respectively. The accuracy of the method ranged from 95 to 104%. As a proof of concept, the validated method was applied in the determination of flumazenil in plasma from two healthy volunteers participating in a PET study with three repeated investigations. A bolus-infusion protocol was used to achieve a constant concentration level of flumazenil. The average plasma concentrations ranged from 0.11 and 0.19 nM and all measurements were within the calibration standard range. The flumazenil concentrations were relatively constant within each scan and the average intra-scan precision was 15%.
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| 46. |
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| 47. |
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| 48. |
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| 49. |
- Lavén, Martin, et al.
(författare)
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Imaging of peptide adsorption to microfluidic channels in a plastic compact disc using a positron emitting radionuclide
- 2005
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Ingår i: Lab Chip. ; :5, s. 756-763
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Tidskriftsartikel (refereegranskat)abstract
- A method for studying peptide-surface interactions within microfluidic channels by radionuclide imaging is described. With the high surface area-to-volume ratio of channels in miniaturised devices, combined with low amunts of analyte, non-specific peptide adsorption is a critical issue. The objective of the study was therefore to develop a method capable of direct detection of adsorbed peptide within microfluidic channels. A micro-device consisting of channels moulded in a plastic compact disc was chosen for the study, together with two selected peptides of different lengths and isolelectric point (pI) values. A bifunctional chelator, DOTA, was attached to the peptide by conjugation and labelled with the short-lived positron emitting radionuclide 68Ga. Quantitative images of radiotracer distribution within the microfluidic channels were obtained using a PhosphorImager system. The power of the method was demonstrated by the ability to clearly measure changes in adsorption when varying a number of parameters that typically affect peptide adsorption. These included surface modifications, analyte concentration, pH, and ionic strength. Additionally, two quantification methods were developed and compared. Radionuclide imaging also permitted visualisation of adsorption and release processes in microchannel chromatographic columns. The results suggest that radionuclide imaging is a suitable tool not only for the study of peptide adsorption to the microchannels presented in this study but also as a versatile tool to measure peptide-surface interactions in a wide variety of miniaturised structures and devices.
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| 50. |
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