Browsing by Subject "nestekromatografia"

Diseases of the posterior eye segment, such as age-related macular degeneration (AMD), diabetic retinopathy, diabetic macular edema and glaucoma are the leading cause of blindness worldwide. Current therapy to treat these vision-threatening diseases relies on intravitreal injections to maintain a desired therapeutic drug concentration in the back of the eye. Frequent intravitreal injections are uncomfortable with poor patient compliance and causes major burden to the healthcare systems as well as to the patients. Small molecule drugs have shorter half-life in the vitreous and are eliminated rapidly. This requires frequent intravitreal dosing intervals that are not feasible in the clinical settings. Also, intravitreally injected small molecule drugs are often poorly and non-specifically distributed to the ocular tissues causing adverse effects. To address these issues, controlled and sustained drug delivery systems in the form of drug conjugates are desirable. Conjugating small molecule drugs with enzymatically cleavable peptide linkers increases the residence time in the vitreous. The peptide linker gets cleaved by vitreal enzyme and the released drug reaches the target in retina and choroid. Aim of this thesis was to screen a library of 25 peptide linkers for cleavage in the presence of porcine vitreal enzymes. The peptide linkers were chemically synthesized and the in vitro stability of the peptide linkers were studied in freshly isolated porcine vitreous. Ten time point samples were collected over a period of 45 days and the peptide cleavage in porcine vitreous was assessed by LC-MS method. A TQ-S liquid chromatography-mass spectrometer was used to study the linker cleavage. LC-MS method development for the peptide library was carried out using IntelliStart wizard function. Out of the 25 peptide linker in the library, stability of eight linkers were not included in the LC-MS analysis as a mass method could not be developed. Out of 17 peptide linkers studied, 14 were categorized as fast cleaving linkers (>90% of the linker cleaved in porcine vitreous after 5 h). Three linker peptides; P4, P5 and P25 were categorized as slow cleaving linkers. Conjugating slow cleaving peptide linkers to small molecule drugs will increase the half-life and enhance the duration of drug action upon intravitreal injection. In this study, linkers that are hydrolyzed by specific enzymes present in vitreous or ocular tissues are exploited to investigate their potential for delivering small molecule drugs.

Tutkimuksen tarkoituksena oli selvittää hevosen nivelnesteen hyaluronihapon ja proteoglyknaanin konsentraatioiden vaihtelua eri artriittitiloissa korkean erotuskyvyn nestekromatografialla (HPLC). Hyaluronihappopitoisuuden muutokset heijastavat nivelen patofysiologista tilaa. Proteoglykaanipitoisuuksien nousun avulla voidaan mahdollisesti diagnosoida rustovauriot jo ennen muiden näkyvien muutosten havaitsemista. Nykyään ainoa menetelmä rustovaurion diagnosointiin on artroskopia. Nivelnesteet analysoitiin pakastuksen jälkeen HPLC-laitteella. Analysointi oli helppoa eikä muita edeltäviä näytteiden käsittelyjä tarvittu kuin sentrifugointi ja laimennus. Tämän puolesta laite sopisi mielestäni erittäin hyvin myös rutiinidiagnostiikkaan. Hyaluronihappopitoisuudet muodostivat samankaltaisen jakauman sekä kontrolli- että potilasnäytteissä. Tämän tutkimuksen mukaan hyaluronihappopitoisuuksien perusteella ei pystytä erottamaan akuutteja eikä kroonisia artriitteja kontrollinivelistä. Proteoglyknanipitoisuuksia ei määritetty sopivan standardiaineen puutteen takia. Tyydyttiin vain toteamaan pystytäänkö ko. laitteella detektoimaan pitoisuuden vaihtelut eri artriittitiloissa. Kaikissa infektiivisissä artriiteissa (5 kpl) todettiin proteoglykaania mutta myös muutamissa muissa artriiteissa sekä jopa kontrollinivelissä. Molempien yhdisteiden konsentraatiovaihteluiden arvostelua vaikeuttaa suuret yksilökohtaiset erot jopa terveiden nivelten välillä. Jatkuvaa perustutkimusta ko. yhdisteiden parissa tarvitaan, jotta menetelmästä saadaan kliinikkojakin hyödyntävä diagnoosimenetelmä.

Cytochrome P450 (CYP) -enzymes are one of the most important enzymes in the metabolism of xenobiotics. Because many xenobiotics are metabolized with each other by the same CYP-enzymes, it is possible that metabolic interactions will take place. These interactions can be the inhibition or induction of the metabolism of another xenobiotic. The interaction can be harmful e.g. when it causes an accumulation of a toxic metabolite or when it inhibits the metabolism of an active drug substance. The aim of this study was to develop a quantitative method for determining metabolic interactions between drugs and environmental chemicals in human liver microsome (HLM) incubations. HLMs contain high concentrations of CYP-enzymes, enabling the use of CYP-model reactions for observing interactions. The model reactions chosen for this study were O-deethylation of phenacetin (CYP1A2), 7-hydroxylation of coumarin (CYP2A6), 4'-hydroxylation of diclofenac (CYP2C9), 1'-hydroxylation of bufuralol (CYP2D6) and 6β-hydroxylation of testosterone (CYP3A4). Michaelis-Menten constants (Km) and maximal enzymatic activities (Vmax) were determined for each model reaction. The suitability of the model reactions for inhibition studies was assessed with specific inhibitors. The quantitative method was developed for an ultra-high performance liquid chormatograph (UPLC) and for a quadrupole time of flight mass spectrometer (QTOF). Samples were ionized with electrospray ionization (ESI) using positive mode. Device parameters were the same for all the metabolites. The analytical method validation was partly performed according to ICH (International Conference on Harmonisation) guidelines. A sufficient linearity (R2>0,99) and specificity was achieved for the quantitative method. The achieved limits of quantitation (LOQ) were low enough (1-120 nM) for quantitation of the small concentrations of the metabolites formed in the inhibition assays. The measurement reproducibility and the reproducibility and accuracy of the method did not fulfill the acceptance criteria for all the metabolites. Improvement of the results should be tried by e.g. exploring different device parameters. 1'-hydroxydiclofenac was found likely to degrade in the matrix solution because of the acidic conditions, making the reliability of the results poor for this metabolite. The Km value obtained for coumarin differed markedly from literature values, which can be due to a too long incubation time. Therefore, incubation conditions should be optimized for this model reaction in coming studies. The Km values obtained for the model reactions of CYP1A2, CYP2D6 and CYP3A4 were similar to those found in literature. Also the IC50 values were quite well within the range of values reported in literature for the inhibitors of the above mentioned model reactions. The effects of four different polymers, F68, F127, Tetronic 1307 and polyvinyl alcohol (PVA) on the enzyme activities were also studied, at a concentration of 1 mg/ml. In principal, at this concentration the polymers did not cause significant changes in the enzyme activities, although inhibition of the CYP2C9 could have been significant. However, the reliability of CYP2C9 model reaction was found to be poor with the used method. In the future this developed method should be further validated, and the incubation conditions for the model reaction of CYP2A6 should be optimized. After this, the IC50 values for the polymers could be studied to get more reliable information about their potential CYP-inhibition properties.

Cyanobacteria produce several secondary metabolites which can be assorted into larger groups like peptides, polyketides and alkaloids based on their structure and synthesis pathways. Though the importance of these compounds to the cyanobacteria is under discussion, some compounds are detected to express bioactivity towards the cells of other organisms. The most known of these compounds are probably toxins like microcystins and saxitoxins which pose a health risk to humans and animals. However, some compounds, when modified, might have a positive affect towards human health. These compounds could for example inhibit the growth of pathogens in human body or harm cancer cells. Therefore, cyanobacteria have risen among the most promising organisms when it comes to finding new drug leads. Unfortunately only a few cyanobacteria strains seem to produce compounds with high medical potential and these compounds of interest are among the hundreds of others metabolites produced by the strain. Therefore, efficient screening and purification methods are needed. This study aimed to isolate antileukemic fractions from cyanobacteria strains, which had expressed cytotoxic activity in previous studies. The compounds of interest would be identified and purified out of these fractions. Fractions were made using solid phase extraction and high performance liquid chromatography. The antileukemic activity of the fractions were tested using human leukemia patient-derived cell line. Using described methods we found apparently cytotoxic compounds resembling carotenoids from one of the strains. We also performed screening based on plate diffusion. The tests were done in order to determine whether the compounds of cyanobacteria strains would be able to cause hemolysis or prevent growth of different microbes. One of the strains tested seemed to express α-hemolytic activity.

Indoor gardening is continuously increasing among consumers. Consumers and greenhouse entrepreneurs are looking for ways to optimize growing conditions for their plants to produce high quality and good yield. So far, the effects of LED lights on the plant biomass production and on the composition of volatile aroma compounds have been investigated. However, the effects of different lights on the taste and odor of homegrown herbs is yet to be discovered. The aim of this study was to investigate how different light conditions would affect the composition of compounds that are mainly responsible for the specific flavor of selected model plants. The main goal was to investigate what type of volatile compounds could be obtained in plants grown under different light conditions. The second goal was to investigate how non-volatile saliva soluble compounds could be modified due to the different growing conditions. Coriander and dill were selected as model plants. Coriander is globally utilized herb while dill is one of the most used herbs in the Nordic countries. Due to their strong flavor, both of these herbs divide consumer opinions and therefore investigation of their flavor modification is important. Coriander and dill were grown in domestic smart gardens manufactured by Plantui Oy. Used light conditions included control, green and blue light. Control light composed of a combination of blue, green and red light. Light source was LED lights. The herbs were grown at +22 °C and at humidity of 56.5 %. Used nutrients were ready-made mixtures by Plantui Oy. The composition of artificial saliva was optimized with commercial coriander and dill for the investigation of flavor compounds. Coriander and dill samples were extracted with the developed artificial saliva after which the volatile compounds were analyzed by a combination of gas chromatography and mass spectrometry (GC-MS) and non-volatile compounds by a combination of liquid chromatography and mass spectrometry (LC-MS). Principal component analysis (PCA) was utilized to investigate the composition of volatile compounds while partial least squares-discriminant analysis (PLS-DA) was used to investigate the differences in the non-volatile compounds. Used light conditions altered the chemical composition of herb leaves. Also, light conditions had a visible effect on plant growth. For example, herbs grown in blue light germinated weakly and produced less biomass. The profile of volatile compounds in corianders grown under green and blue light differed from those grown under control light. For dill, the profiles differed only for the samples grown under blue light. Majority of the volatile compounds were components of the essential oils of herbs and compounds that enhance stress tolerance. When looking at saliva soluble non-volatile compounds, coriander grown under blue light was different from the one grown under control light while for dill a difference was observed both under blue and green light. Based on the results, special light recipes can be developed to modify the flavor of coriander and dill. Further research is still needed, especially on the effects of light conditions in plant cell signaling and thereby on the morphological changes in plants and as a consequence on their flavor compounds.

The literature review deals with dyes in particular azo compounds and their chemistry, which are banned or permitted for the use in foodstuff. The literature review also deals with the analytical methods used for the determination of dyes in foodstuff. The main focus is on liquid chromatography and mass spectrometry. The aim of the experimental work was to develop and validate an UHPLC-MS/MS meth-od for the Finnish Customs laboratory for the simultaneous determination of multiple dyes in spices. The method included 37 different dyes that are either banned or permit-ted in foodstuff. The LC-MS/MS method development started by determining the pre-cursor ion as well as tree most abundant product ions for individual dyes. The next step was to optimize the liquid chromatographic method. This was carried out by running a standard mixture that included all of the dyes. The method for the extraction of dyes with acetonitrile in spices was developed so that it would be fast, easy and the maximum number of dyes would be extracted. Finally, the method was validated. The mass spectrometric method was developed for 37 dyes. Retention times were found for 35 of these dyes. The final runtime was 33 minutes. The extraction with acetonitrile was proven to work for 29 dyes. For the rest of the dyes, the extraction could be carried out with a different method. Only two of the colorants could not be extracted with the tested methods with the applied concentrations. For most of the dyes, the LOQs were 0,05-0,5 mg/kg. The method was repeatable concerning most of the dyes.