1. |
- Elhamili, Anisa
(författare)
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Development of Capillary Electrophoresis Methods Coupled to Mass Spectrometry for Biomedical and Pharmaceutical Analysis
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The analysis of large intact proteins and complex biological samples containing drug molecules is a common complicated task for many scientists. However, due to the importance of these molecules, there is a growing interest in pharmaceutical and medicinal research to develop rapid, highly sensitive and efficient analytical techniques. The advantages of capillary electrophoresis (CE) in combination with mass spectrometry (MS) provide a powerful analytical tool. However, further improvement and development of these techniques are required to extend their utility and to meet the challenges of selected analytes. Thus, the scope of this thesis deals with the development of novel analytical methods to achieve efficient and high performance analysis of peptides, intact proteins, digests of complex samples and basic pharmaceutical drug compounds in biological matrices. Implementation of CE for routine analysis of proteins and complex samples is constrained by the partial adsorption to the capillary wall. Consequently, the use of surface modified capillaries is required to control the surface properties and prevent analyte adsorption. In this thesis, analyte adsorption was successfully prevented using tailored covalent cationic (M7C4I) and electrostatic cationic (PVPy-Me) coatings. Rapid and efficient separations of peptides, proteins and digests of complex samples such as cerebrospinal fluids were obtained with these coatings. The M7C4I coating showed a distinct ability to handle large intact proteins with a molecular size of over 0.5 MDa. The highest peak efficiencies and surprisingly high peak stacking effects were obtained by adding salts to the protein samples. The effect of salt additives on peak efficiencies of intact proteins was further demonstrated and compared using different surface modified capillaries. Additionally, rapid CE-ESI-MS quantification of pharmaceutical drug molecules in human plasma was performed after a SCX-SPE sample preparation method using the M7C4I coating. In conclusion, the results presented in this thesis show the strong potential of CE in combination with MS using electrospray ionization (ESI) for the analysis of peptides and large intact proteins and the applicability for clinical monitoring of the levels of pharmaceutical drug molecules in human plasma with high sensitivity and efficiency.
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2. |
- Kollander, Barbro
(författare)
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Inductively Coupled Plasma Atomic Emission Spectrometry : Exploring the Limits of Different Sample Preparation Strategies
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes two different sample preparation strategies for inductively coupled plasma atomic emission spectrometry (ICP-AES), and their ability regarding multi element quantification in complex samples. Sensitivity, repeatability, reproducibility and accuracy were investigated. The aim was to increase the over all efficiency, the speed of analysis, and/or the sensitivity of the analytical method. The intention was to measure analytes with concentrations ranging from ng/g to mg/g simultaneously. The aim was additionally to study chemical and physical processes occurring during the sample preparation, the sample transport to the plasma, and the atomization therein. In the first sample preparation strategy, a hydrophilic highly cross-linked iminodiacetate-agarose adsorbent, IDA-Novarose, was used for preconcentration of metal ions, and matrix elimination in natural water samples. The sorbent was synthesized with different binding capacities. The effect of the capacity on preconcentration, matrix elimination, and uptake capability at high flow rates was studied. For a high capacity IDA-Novarose (≥ 45 µmole/ml) quantitative uptake was seen even at high flow rates (100 ml/min) for Cu2+ with a high affinity to the adsorbent, and for Cd2+ with a moderate affinity. For lower capacities the uptake of Cd2+ was affected by the sample matrix and the flow rate. A method based on the determination of the conditional stability constant of the metal sorbent complex was suggested for the prediction of the sorbent capacity needed to obtain quantitative recovery and optimal matrix elimination. The sorbent was used in a flow system with online buffering for the analysis of a certified riverine water (SLRS-3), tap water and lake water. With few exceptions the results obtained by ICP-AES after preconcentration agreed well with the certified concentrations and results obtained by ICP-MS. The other sample preparation strategy discussed is a method for non digested biological samples from different animal organs for the multi element analysis by ICP-AES. This “mix and measure method” consists of a simple homogenization of the sample with a mixing rod in a small amount of neutral media, followed by dilution and direct measurement with ICP-AES. The total time of analysis is only a few minutes. The ability of this fast method to accurately quantify some elements of toxic, environmental, and/or physiological concern with the lowest possible sample dilution and the highest possible plasma load was evaluated. In 10 % liver slurry Cd, Co, and Sr, at concentration levels around 0.05 µg/g were quantified simultaneously with P and K around 2000 µg/g and with several other elements in between (Al, Ca, Cu, Fe, Mg, Mn, Pb, and Zn). The relative standard deviation of repeated measurements of samples was around 5 - 6 % for regardless of the concentration of the element. The method was also used for fast screening of the elemental distribution in mice organs (brain, heart, kidney, liver, lung and spleen).
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3. |
- Andersson, Mikael, 1964-
(författare)
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Characterisation of Chromatography Media Aimed for Purification of Biomolecules
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Chromatography media (resins) are very important for and widely used by the biopharma industry in large scale production of biopharmaceuticals, e.g. monoclonal antibodies. Today there are several hundred biopharmaceuticals released globally on the healthcare market. This thesis discusses various strategies and methods for the characterisation of chemical and functional stability of chromatography media. In addition, various analytical techniques used in these areas were evaluated and applied. Further, more specific physical and chemical characterisation methods were evaluated and applied to explore different properties of various chromatography media.In Papers I-III, established methodologies for performing chemical and functional stability studies were used. Mainly agarose-based chromatography media were investigated. For fast screening of the chemical stability, the total organic carbon analysis technique was evaluated and applied. This technique that measures the carbon leakage from the chromatography media at different conditions, proved to be very suitable and robust. For detection and/or identification of leakage compounds responsible for or for part of the measured carbon leakage, different methods such as (high performance) liquid chromatography and gas chromatography mass spectrometry were used.In Papers IV-VII, different properties (i.e. functional performance, ligand content and surface chemistry) were evaluated for different agarose-based chromatography media. Standard chromatographic methods (ion exchange chromatography) and spectroscopic methods (e.g. Fourier transform infrared spectroscopy and time-of-flight secondary ion mass spectrometry) were evaluated and applied. Chemometric methods were used for efficient evaluation of data.Information of chemical, functional and leakage data of chromatography media are valuable and important for the biopharmaceutical companies to be able to fulfil the regulatory requirements of biopharmaceuticals. In addition, information of various chemical, functional and physical properties of chromatography media is likewise important during development and set up of new biopharmaceutical processes.
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4. |
- Edström, Lena, 1978-
(författare)
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Adsorption Studies with Liquid Chromatography : Experimental Preparations for Thorough Determination of Adsorption Data
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Analytical chemistry is a field with a vast variety of applications. A robust companion in the field is liquid chromatography, the method used in this thesis, which is an established workhorse and a versatile tool in many different disciplines. It can be used for identification and quantification of interesting compounds generally present in low concentrations, called analytical scale chromatography. It can also be used for isolation and purification of high value compounds, called preparative chromatography. The latter is usually conducted in large scale with high concentrations. With high concentrations it is also possible to determine something called adsorption isotherms.Determination of adsorption isotherms is a useful tool for quite a wide variety of reasons. It can be used for characterisation of chromatographic separation systems, and then gives information on the retention mechanism as well as provides the possibility to study column-column and batch-batch reproducibility. If a protein is immobilised on a solid support, adsorption isotherms can be used for pharmacological characterisation of drug-protein interactions. Moreover, they can be used for the study of unexpected chromatographic phenomena.If the adsorption isotherm is known it is also possible to simulate chromatograms, and subsequently optimise the separation process numerically. The gain of a numerically optimised separation process is higher purity or yield of valuable compounds such as pharmaceuticals or antioxidants, as well as reducing the solvent usage. Taken all together, it saves time, money and the environment.However, the process of the adsorption isotherm determination requires a number of careful experimental considerations and preparations, and these are the main focus of the thesis. Important steps along the way include the choice of separation system and of suitable analytes, preparation of mobile phases and sample solutions, calibration, determination of injection profiles and column void, and of course the adsorption isotherm determination method itself. It is also important to keep track of parameters such as temperature and pH. These issues are discussed in this thesis.At the end, a description of useful methods for processing of the raw adsorption isotherm data is presented, as well as a brief passage on methods for numerical optimisation.
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5. |
- Saleh, Aljona, 1983-
(författare)
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Analysis of drugs and proteins in biological matrices, using different types of sample preparations and mass spectrometry
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes bioanalysis of small and large molecules in biological matrices and includes screening of illegal drugs in urine with high resolution mass spectrometer, bioanalysis of MTH1 inhibitors in mice plasma and quantification of proteins in plasma and cell lysates.Screening of illegal drugs was based on high resolving power mass spectrometer (QTOF) and the results were compared to immunoassays. For the study, the nine most commonly abused drugs were selected for screening of a large number of authentic urine samples. Evaluation of the screening results showed that the QTOF generated a low rate of false positive and false negative results and can be used as an alternative or a complementary to immunoassays.In another study, a bioanalytical method for the two new anticancer targets TH588 and TH287 was developed and validated. The compounds inhibit the MTH1 protein that is required for cancer cell survival. To study the pharmacokinetic properties of the substances, a bioanalytical method was developed using a fast sample preparation followed by LC-MS/MS analysis. Validation showed that the method was linear, accurate and precise in the studied concentration range. Stability tests showed that the substances were stable in mice plasma and under different storage conditions. The study also addresses other important factors such as solubility, chromatography and fragmentation mechanisms.Two other studies focused on quantification of specific proteins in biological specimens; plasma and bacterial lysates. The matrices are rich in endogenous proteins making quantitative analysis of target proteins challenging. Therefore a new strategy is proposed that combines known procedures in a way it has not been used before. The methods are based on quantification with isotopically labelled peptides added after proteolytic digestion of the sample with immobilized thermolysin or pepsin. The sample digest was analysed on LC-MS/MS and LC/LC-MS/MS systems.
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6. |
- Sjödin, Marcus O.D. 1978-
(författare)
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Advances for Biomarker Discovery in Neuroproteomics using Mass Spectrometry : From Method Development to Clinical Application
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins offer a prominent group of compounds which may be ubiquitously affected in disease and used as biomarkers for early diagnosis, assessing treatment or drug development. Clinical proteomics aim to screen for protein biomarkers by a comprehensive analysis of all proteins expressed in a biological matrix during a certain pathology. Characterization of thousands of proteins in a complex biological matrix is from an analytical point of view a challenging task. Hence, sophisticated methods that are sensitive, specific and robust in a high-throughput manner are required. Mass spectrometry (MS) is able to perform this to a wide extent is.A prominent source for finding protein biomarkers related to neurological diseases is the central nervous system (CNS) due to close proximity of the pathogenesis. Neuroproteomic analysis of CNS tissue samples is thus likely to reveal novel biomarkers. Cerebrospinal fluid (CSF) bathes the entire CNS and offers a good balance between clinical implementation and usefulness. Both matrices put further requirements on the methodology due to a high dynamic range, low protein concentration and limited sample amount.The central objective of this thesis was to develop, assess and utilize analytical methods to be used in combination with MS to enable protein biomarker discovery in the CNS. The use of hexapeptide ligand libraries was exemplified on CSF from patients with traumatic brain injury and demonstrated the ability to compress the dynamic range to enable protein profiling in the order of mg/mL to pg/mL. Further, a method based on cloud-point extraction was developed for simultaneous enrichment and fractionation of hydrophobic/hydrophilic proteins in brain tissue. Comparison between label and label-free MS based strategies were carried out, mimicking the true conditions with a few differentially expressed proteins and a bulk of proteins occurring in unchanged ratio. Finally, a clinical application was carried out to explore the molecular mechanism underlying the analgesic effect of spinal cord stimulation (SCS) in patients with neuropathic pain. The CSF concentration of Lynx1 was found to increase upon SCS. Lynx1, acting as a specific modulator of the cholinergic system in the CNS, may act as a potential important molecular explanation of SCS-induced analgesia.
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