Work related musculoskeletal disorders are the dominating causes of reported ill-health in industrialized countries. These chronic pain conditions are one of the most costly public health problems in Europe and North America. When work related musculoskeletal disorders are considered to be of muscular origin and the trapezius muscle is affected, the common appellation is trapezius myalgia. Since little is known about the genesis or how it is maintained, it is of great importance to better understand the pathophysiology of trapezius myalgia; doing so will better enable recommendations for prevention, treatment and rehabilitation. Several hypotheses have been presented based on biochemical alterations in the muscle, suggesting increased signaling of inflammatory substances and altered metabolism. Previous research has not been able to present the comprehensive picture of the muscle in pain. Thus there is a demand for more comprehensive research regarding the biochemical milleu of the chronic trapezius muscle. Proteomic and metabolomic methods allow non-targeted simultaneous analyses of a large number of proteins and metabolites. The main emphasis in this thesis is on a proteomic method, two-dimensional differential gel electrophoresis (2D-DIGE). The method is validated to human skeletal muscle biopsy research with laboratory specific settings. In the baseline study, there were 14 metabolic, contractile, structural and regulatory proteins that differed significantly in abundance when trapezius and vastus lateralis muscles were compared. Using the validated 2D-DIGE method and the baseline study, a comparison between healthy and myalgic muscles was made. Biopsies from female cleaners with and without myalgia were compared to obtain results from women with the same type of work exposure. In the multivariate model, 28 identified unique proteins separated healthy and myalgic muscle and were grouped according to function: metabolic (n=10), contractile (n=9), regulatory (n=3), structural (n=4), and other (n=2). Finally, a second screening method, metabolomics, was introduced to analyze differences in metabolite content as a complement to and verification of the proteomic results. Gas chromatography-mass spectrometry (GC-MS) was performed on muscle interstitial fluid samples obtained with microdialysis, and differences in the abundance of extracellular metabolites were revealed.  The 2D-DIGE method is a reliable method to analyze human skeletal muscle. The outcomes of the proteomic analyses were dependant on the statistical approach. Systematic differences in protein and metabolite content were detected using a multivariate approach. Univariate analyses were used to analyze individual proteins for their significance. The significant proteins in the baseline study were predominately related to muscle fiber type which correlated with the differences in fiber type content between trapezius and vastus lateralis. The proteomic and metabolomics studies where myalgic and healthy muscles were compared provide us with new clues and new aspects regarding the pathophysiology of the myalgic muscle. Technically advanced methods employed in the thesis enabled an explorative screening of proteins of relevance for the pathophysiology of the myalgic muscle. The results of these analyses may contribute to the formulation of future hypothesis that need to be further evaluated.

<p>Electrospray ionization (ESI) mass spectrometry (MS) in combination with liquid chromatography (LC) is an excellent tool for the identification of drug metabolites. Utilizing this hyphenated technique in combination with proper sample pretreatment, the metabolic pathways of the analgesic drug ketobemidone were investigated in human urine and rat microdialysate from blood and brain. Two novel phase I metabolites (ketobemidone N-oxide and meta-hydroxymethoxyketobemidone) and three novel phase II metabolites (glucuronic acid conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone) were identified in human urine. Further, norketobemidone and ketobemidone N-oxide were identified in rat microdialysate from brain after regional distribution of ketobemidone in striatum. This indicates that the brain itself has the possibility to metabolize ketobemidone. </p><p>Synthetic ketobemidone metabolites were used for comparison of retention times and tandem MS spectra with the possible metabolites recovered from the biological samples. The conjugated metabolites were identified by accurate mass measurements and tandem MS spectra of the aglycones. The accuracy of the estimated masses was better than 2.1 ppm for two out of three conjugates in presence of internal standard.</p><p>On-line micro-SPE was successfully used for trapping and desalting of the microdialysates. The small SPE pre-column made it possible to inject approximately 100 times more sample on the analytical column compared to injection without pre-column. Selective trapping was demonstrated for the polar catechol amine metabolite, dihydroxyketobemidone, which forms covalent complexes with phenylboronic acid (PBA). A fluorinated silica type stationary phase was the only column out of several tested that was able to separate ketobemidone and all relevant phase I metabolites. </p><p>Liquid chromatography and mass spectrometry are independently valuable tools in the field of analytical pharmaceutical chemistry. The present study showed that the combination of LC-MS, with its excellent selectivity and sensitivity, offers an outstanding tool in the qualitative analysis of drugs and metabolites in biological fluids. </p>

Electrospray ionization (ESI) mass spectrometry (MS) in combination with liquid chromatography (LC) is an excellent tool for the identification of drug metabolites. Utilizing this hyphenated technique in combination with proper sample pretreatment, the metabolic pathways of the analgesic drug ketobemidone were investigated in human urine and rat microdialysate from blood and brain. Two novel phase I metabolites (ketobemidone N-oxide and meta-hydroxymethoxyketobemidone) and three novel phase II metabolites (glucuronic acid conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone) were identified in human urine. Further, norketobemidone and ketobemidone N-oxide were identified in rat microdialysate from brain after regional distribution of ketobemidone in striatum. This indicates that the brain itself has the possibility to metabolize ketobemidone. Synthetic ketobemidone metabolites were used for comparison of retention times and tandem MS spectra with the possible metabolites recovered from the biological samples. The conjugated metabolites were identified by accurate mass measurements and tandem MS spectra of the aglycones. The accuracy of the estimated masses was better than 2.1 ppm for two out of three conjugates in presence of internal standard. On-line micro-SPE was successfully used for trapping and desalting of the microdialysates. The small SPE pre-column made it possible to inject approximately 100 times more sample on the analytical column compared to injection without pre-column. Selective trapping was demonstrated for the polar catechol amine metabolite, dihydroxyketobemidone, which forms covalent complexes with phenylboronic acid (PBA). A fluorinated silica type stationary phase was the only column out of several tested that was able to separate ketobemidone and all relevant phase I metabolites. Liquid chromatography and mass spectrometry are independently valuable tools in the field of analytical pharmaceutical chemistry. The present study showed that the combination of LC-MS, with its excellent selectivity and sensitivity, offers an outstanding tool in the qualitative analysis of drugs and metabolites in biological fluids.