Analysis of drugs and proteins in biological matrices, using different types of sample preparations and mass spectrometry

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.