Browsing by Subject "virtsa"
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(2010)The most important part in bioanalysis is the sample cleanup process which is usually the most laborious and time consuming part of the analysis and very susceptible to errors. A functional bioanalysis has to be quick, easily automated, sensitive, selective and stable. It also needs to be suitable for high throughput analysis. Desorption atmospheric pressure photoionization (DAPPI) is a novel direct desorption/ionization technique for mass spectrometry that enables direct analysis of solids from surfaces or liquid samples from a suitable sample plate often without any sample preparation. The suitability of DAPPI-MS for biological samples was investigated by measuring the limits of detection for selected opioids and benzodiazepines and screening them from authentic urine samples. Limits of detection were measured for standard solutions and spiked urine. Opioids and benzodiazepines were analyzed from post mortem urine samples with an optimized DAPPI-MS method. Post mortem urine samples were analyzed with and without sample preparation. Sample preparation improved the sensitivity of the method remarkably. About 50 % of the analytes were detected without sample preparation and almost 100 % after sample cleanup. It is however difficult to estimate the suitability of DAPPI-MS as a screening method because not all analyte concentrations of the urine samples were known. Therefore we cannot be certain weither the results obtained without sample preparation are caused by the suppression of the urine matrix or if the concentrations of the analytes are below the limits of detection. The reliability of the method can further be improved by investigating the metabolites of the analytes and improving the system towards automation. On grounds of this research DAPPI-MS should be used cautiously as a screening method for urine samples without sample preparation and with only high enough analyte concentrations. DAPPI-MS shows promise as a screening method for opioids and benzodiazepines from urine when the sample cleanup is used before the analysis.
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(2012)Neurosteroids are steroids which are active in the central nervous system. They have many biological and physiological functions in human body. Fluctuations of the neurosteroid concentrations are related to many diseases such as depression, schizophrenia and epilepsy. Neurosteroid levels are measured to understand their role in brain function and human behavior. The aim of the work was to develop a gas chromatographic-atmospheric pressure fotoionization-tandem mass spectrometric (GC-APPI-MS/MS) method for analyzing 19 neurosteroids and their metabolites in urine. Neurosteroids are excreted in urine mainly as conjugates, so they have to be hydrolyzed before analysis. Sample purification is done by liquid-liquid extraction and the analytes are subsequently derivatized to enhance their volatility. Because widely used β-glucuronidase/arylsulfatase-enzyme from Helix pomatia oxidases 3β-hydroxy-5-ene and 3β-hydroxy-5α-reduced steroids, we decided to use β-glucuronidase from Escherichia coli and acid hydrolysis instead of H. pomatia. The quantification of the total neurosteroid concentration in urine was challenging because β-glucuronidase enzyme from E. coli did not hydrolyze glucuronides completely and acid hydrolysis deconjugated also glucuronides in addition to sulfate conjugates. In addition the internal standard d4-allopregnanolone was noticed to be impure and degrade during acid hydrolysis. The limits of detection were reasonably low for the method (2 pg/ml-1 ng/ml). The retention times of the analyte peaks were very repeatable (RSD 0,06-0,11%) and the repeatability of the method was acceptable for all compounds (RSD < 27%). Urine samples from two males and two females were analyzed with the preliminary validated method. We could determine estimated concentrations for dehydroepiandrosterone, dihydrotestosterone, androstenedione, testosterone, estrone, β-estradiol, estriol, 5α-tetrahydrodeoxocorticosterone, cortisone, corticosterone and hydrocortisone. The developed method did not meet all the aims of this work. The method needs further validation and more exact investigation about the effect of the selected hydrolysis method on intact steroids. Also the internal standard should be changed to some other compound, preferably a non-deuterated one.
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