<p>Clopidogrel is an antiplatelet substance that prevents blood coagulation in the arteries. It is an inactive pro drug that becomes activated after first-pass metabolism by the liver. The active metabolite of clopidogrel is 2-oxoclopidogrel, which is unstable therefore pharmacokinetic data is obtained by measuring the inactive metabolite clopidogrel acid in plasma. Clopidogrel is taken orally in tablet form. The aim of this project was to develop a LC-MS/MS method for quantification of clopidogrel and its metabolite in plasma.</p><p> </p><p>The method has been developed by optimizing the sample preparation. Different extraction procedures and extraction columns were tested, for example, by changing the extraction column from a C8 silica sorbent to Oasis HLB (a polymer sorbent). Different internal standards were evaluated as a result of discovering the signal suppression of the previous internal standard clopidogrel acid.  Flupentixol was found to be the best candidate.</p>

The main research of this thesis includes three sections. The purpose of first part is to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of 8 macrolide antibiotics and lincosamides inside fish tissue, including erythromycin (ERM), oleandomycin (OLD), kitasamycin (KIT), tylosin (TYL), josamycin (JOS), spiramycin (SPM), tilmicosin (TIL), and lincomycin (LIN). Homogenized samples are first extracted with acetonitrile, dehydrated with sodium sulphate anhydrous, and then condensed. After the residue was redissolved in methanol and the extracts were partitioned with n-hexane to remove lipids, the sample is filterced and detected by LC/MS-MS using chromatography columns of Agilent HC-C18 (5£gm, 150 mm ¡Ñ4.6 mm). The mobile phase A was 5mM ammonium acetate containing 0.1% formic acid, while the mobile phase B was acetonitrile. The analysis of 8 macrolide antibiotics and lincosamides can be achieved within 10 minutes with electrospray ionization-tandem mass spectrometry in positive mode using multiple reaction monitoring (MRM) for simultaneous detection. The second part is to verify the method by regulation of European Union (EU) resolution scheme (2002/657/EC). In the case where the drug is set as allowed drug, the recovery rate under gradient addition according to MRL is between 93.64% to 106.67%, and the CV is between 0.27% to 7.17%. In the case where the drug is set as prohibited drug, the recovery rate under gradient addition according to MRPL is between 96.35%~104.88%, and the CV is between 6.77%~13.91%. As a result, the decision limit (CC£\) and the Detection capability (CC£]) of the 8 macrolide antibiotics and lincosamides is between 0.24 to 0.40£gg kg-1 and 0.33 to 0.49£gg kg-1. The last section is to evaluate the stability of drugs in fish body under domestic preservation and process methods on fish, including refrigeration at -20¢J and cold storage at 4 ¢J. The test is implemented by adding the drug into fish tissue according to MRL and detecting the antibiotics residue after regulated 40 days. Besides, the effect on activity of drug residue in fish body after boiling at 100 ¢J is compared. The results show that the residual amount of spiramycin, josamycin, tilmicosin, and lincomycin is below 35% while that of erythromycin, oleandomycin, kitasamycin, and tylosin will be below 20%. Therefore, the drugs including erythromycin, josamycin, tylosin, and lincomycin will stay stably in fish tissue if they are stored under -20 ¢J. However, it may affect human health if the fish contains such antibiotic residues is not boiled.

Hou-Chin stream, Love river, and Chien-Chen river, the three main rivers in Kaohsiung city, flow through the populous residential and industrial areas. A large portion of sewage from domestic and industrial sources are discharged into these rivers, then the Love river and Chien-Chen river pour into the harbor area. In order to understand the pollution of alkylphenol polyethoxylates in these areas, water and sediment samples in Hou-Chin stream, Love river, Chien-Chen river and harbor area in Kaohsiung city were collected and the contents of alkylphenol and corresponding polyethoxylates were analyzed in this study. LC/MS/MS was used as the analytical instrument which is relatively time-saving in comparison with other instruments. It is also more convenient due to the facts that no derivation or colorization are needed in sample pretreatment. The detection limit can reach to 0.03 ng/ml and recovery can be around 83.6~91.6%. It can analyze alkylphenols combinded with long ethoxylate chain with improved sensitivity and selectivity. In the four sampling areas, the concentration of NPs in water were between 7.4~241.8ng/ml, and OPs were between 0.66~64.2ng/ml. The most contaminated water samples were found at Chih-Ping Bridge on the mainstream of Love river and Pau-Chu-Kou Dam Station and Min-Tsu Bridge on the tributary of Love river where the concentrations of NPs were greater than 200ng/ml, OPs were greater than 30ng/ml. We found that the main pollution sources were from Lung-Hsin Bridge, Tzu-Yu Bridge, Lung -Hua Bridge, and Pau-Chu-Kou Dam Station. The pollution sources of the Chien-Chen river were mainly from Chung-An Bridge and Chen-Chuan Bridge. Concentration of NPs in upper sediments were between 633.1~2113.8ng/g, OPs were between 50.3~287.9ng/g. The highest concentration of NPs was at Ho-Ti Bridge on the mainstream of Love river, and the lowest concentration of NPs was at Chung-An Bridge on Chien-Chen river. The highest concentration of OPs was at Chen-Chuan Bridge in Chien-Chen river, and the lowest concentration of OPs was Min-Tsu Bridge on the tributary of Love river. The concentration of NPs in deeper sediments were between 523.9~1919.5ng/g, OPs were between 39.9~322.0ng/g. The highest concentration of NPs was at Chung-Hua Bridge on the tributary of Lover river, and the lowest concentration of NPs was at Chung-An Bridge on Chien-Chen river. The highest concentration of OPs was at Chi-Chin Fishing Port, and the lowest concentration of OPs was at Min-Tsu Bridge on the tributary of Love river. The salinity of water samples and the total organic carbon in sediment sample will influence the distribution coefficient of alkylphenol polyethoxylates with different length of ethoxylate chains, their distribution coefficients were between 0.48~2.67. In comparison with foreign studies, the concentrations of alkylphenol polyethoxylates of water and sediments amples in this study were between the highest and lowest values reported. However, the observed concentrations of alkylphenols in these study areas were higher then other rivers in Taiwan. These values were higher than the Probable No Effect Concentrations ( PNEC) of NP risk assessed by European Union. It can be concluded that the pollution of alkylphenol polyethoxylates of water and sediment is getting more serious in Hou-Chin stream, Love river, Chien-Chen river and harbor area in Kaohsiung city.

The plant peroxidases (EC1.11.17) exit as a large family of isozymes. These isozymes have more than 50% amino acid sequence differences. The function of Wasaba japonica peroxides plays the role as IAA oxidases. The kinetics result shows Wasabia japonica peroxidases displayed affinity (Km = 17.1 £gM) for IAA. The kinetics results in Wasabia japonica peroxidases display affinity (Km = 80.6 £gM) for syringaldazine. LC/MS/MS technique described the data that has proven to be a method for identification and characterization of proteins. The soluble proteins extracted form Wasabia japonica was purified by gel filtration chromatography and two-dimensional gel electrophoresis (2-DE). LC-MS/MS analyses of 2-DE gel spots and identify proteins structure based on the protein fragmentation characteristics. The Mascot Search Results showed that Wasabia japonica peroxidase has a significant similarity (10%) with Arabidopsis thaliana peroxidase.

Nitrofurans have been widely used either in waterbath or feed additives for the prevention and treatment of aquatic products. The European Union was able to assign a maximum residue limit and prohibited nitrofurans used to animals in 1995, because of the potential carcinogenic effects of their residues on human health. This study is focusing on the analytical method of four kinds of commonly used nitrofurans and corresponding residual metabolites by LC-MS/MS. The detection limits of furazolidone, furaltadone, nitrofurazone and nitrofurntoin were 6.11, 3.63, 4.52 and 6.20 £gg kg-1,respectively. The detection limits of AOZ, AMOZ, SC and AH were 0.23, 0.30, 0.36, 0.53 £gg kg-1, respectively. The lightness is the main factor to cause the decomposition of nitrofurans. It is not significant for temperature to depredate nitrofurans. The adsorbtion of metabolites by the plastic tube was in the extraction procedure. Equipments in glass are suggested to be used for the sample pretreatment and plastic meterials are averted to be exercised. About the comparation of determination of AOZ by ELISA and LC-MS/MS. The result demonstrated that the ELISA method might overestimate the residual AOZ content at low concentrations. The detection limit and recovery of the known addition were 0.05 £gg kg-1 and 108% for the LC-MS/MS method and 0.31 £gg kg-1 and 305% for the ELISA method, respectively. The amounts of residual nitrofurans and metabolites in muscle, liver, gill and skin tissue of tilapia which were treated in different conditions were compared. The depletion data of bathing treatment group obtained showed similar be haviors of furazolidone, furaltadone, nitrofurazone, nitrofurantoin in tilapia which the residual time was less than 24 hr. The amounts of residual nitrofurans appeared the highest concentration in gill and the lowest concentration in muscle. Bonded residues of metabolites can be detected for at least 4 weeks after administration in muscle, skin, liver and gill. The concentrations of residual bonded metabolites were higher than non-bonded metabolites in gill and muscle besides liver during depletion periods. After bathing medication, there were more residual nitrofurans and corresponding metabolites in sea water tilapia than fresh water group, because sea water fish survives in high osmotic condition to reduce their urination. Nitrofurans and metabolites were deconstructed by enzyme in gills, livers, intestines and muscles. Then tissues of fish accumulated nitrofurans and metabolites soon after medication. The maturity of fish is one of facters to effect different residual concentration during depletion periods. Liver is the main tissue to deconstruct nitrofurans and metabolites for the bathing medication and intestine is the major tissue to decompose antibiotics for the feeding medicaton. In this research, we built a completed way to determine nitrofurans and corresponding metatbolites. Comparation of fish in different conditions and different medicative ways were in this investigation. These results could be helpful for aquacultures and government institutions.

Stress conditions such as heat shock, UV, alkylating agents, and H2O2 have been shown to result in the modification of a variety of protein targets via the production of reactive electrophiles. These modifications can directly impact protein function or can alter posttranslational modifications, thus leading to a disruption of cellular regulatory processes. Recent studies have shown that stress-induced protein modifications can modulate posttranslational modification by the small ubiquitin related modifier (SUMO) family of proteins. Unlike ubiquitination, which primarily targets proteins for proteasomal degradation, sumoylation exerts a variety of effects including protein stabilization, subcellular localization, and the alteration of protein-protein interactions and transcriptional activity. To investigate the effects of alkylation and oxidative stress on sumoylation, HEK293 cells were treated with iodoacetamide, hydroquinone, benzoquinone, Texas Red C5 bromoacetamide, hydrogen peroxide, and 4-hydroxynonenal (HNE), a highly reactive product of lipid peroxidation associated with oxidative stress. Western blot analysis revealed that the agents tested resulted in concentration-dependent changes in the patterns of SUMO-1 and SUMO-2/3 protein conjugation. Localization studies using western blot analysis and confocal immunofluorescence microscopy demonstrated that SUMO-1 protein conjugates were located primarily in the nucleus, whereas SUMO-2/3 protein conjugates were more equally distributed between the nucleus and the cytoplasm. SUMO-associated proteins were harvested from vehicle- and HNE-treated non-transfected HEK293 cells using agarose conjugated anti-SUMO-1 antibodies or from HA-SUMO-1- and HA-SUMO-3-expressing HEK293 cells using immunoaffinity chromatography. Multidimensional liquid chromatography-tandem mass spectrometry analyses resulted in the identification of 54 HA-SUMO-1-associated proteins and 37 HA-SUMO-3-associated proteins in vehicle-treated cells and 21 HA-SUMO-1- and HA-SUMO-3-associated proteins in HNE treated cells. Additionally, 27 SUMO-1-associated proteins were identified in the HNE-treated non-transfected cells. The functional classes of proteins targeted included RNA binding and processing proteins, metabolic enzymes, cytoskeletal regulators, and chaperone proteins. HNE treatment resulted in a near complete redistribution of both SUMO-1 and SUMO-3 to different targets. There was a 15% overlap in SUMO-1 and SUMO-3 associated proteins in vehicle-treated cells and a 10% overlap in HNE-treated cells indicating that SUMO proteins target distinct protein groups. These results indicate that protein modifying reactive electrophiles can regulate protein functions through the indirect alteration of endogenous posttranslational modifications.

A highly sensitive and rapid method for the analysis of isradipine in human plasma using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed. The procedure involves a simple liquid-liquid extraction of isradipine and amlodipine (IS, internal standard) with methyl-t-butyl ether after alkaline treatment and separation by RP-HPLC. Detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 372.1 → m/z 312.2 and m/z 408.8 → m/z 237.9, for quantification of isradipine and IS, respectively. The standard calibration curves showed good linearity within the range of 10 to 5000 pg/mL (r2 ≥ 0.9998). The lower limit of quantitation (LLOQ) was 10 pg/mL. The retention times of isradipine (0.81 min) and IS (0.65 min) suggested the potential for high throughput of the proposed method. In addition, no significant metabolic compounds were found to interfere with the analysis. This method offered good precision and accuracy and was successfully applied for the pharmacokinetic and bioequivalence studies of 5 mg of sustained-release isradipine in 24 healthy Korean volunteers.

The detection and quantification of hormones are important to assess the reproductive and stress status of experimental models and for the diagnosis of diseases in human and veterinary clinics. The peptide secretoneurin (SN) has been proposed as a new sex hormone, but effective quantification methods are challenging. Traditional methods require the use of antibodies with either radioactive or non-radioactive tracers. There are difficulties with these methods in terms of sensitivity, specificity, and inter-laboratory repeatability. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can circumvent many of these challenges. Another source of variation is the extraction of lipophilic steroidal compounds, which is incompatible with the extraction of hydrophilic peptide hormones. I have developed efficient extraction and sensitive detection methods of SN with numerous other peptide and steroid hormones in the same tissue sample in mice and zebrafish. The extraction efficiency for both peptide and steroid analytes is over 85%. The standard deviation for extraction and LC-MS/MS analysis for each compound varies between 5-10%. The steroid hormones can be quantified in the low to medium fmol/µL range. We quantified peptide hormones in the high fmol/µL to low pmol/µL range. Mouse SN levels were measured and compared against the levels of GnRH 1, oxytocin, vasopressin, E2, and P4 in multiple tissues at 3 important periods through the estrous cycle. In addition, SN levels were found to be moderately related to GnRH 1 levels in the hypothalamus in the estrous cycle. This is important because it is GnRH 1 that stimulates the luteinizing hormone surge in the pituitary that regulates ovulation in all vertebrate species. I also determined that SNa and SNb were both within the 2-8 pmol/µL range in the brain or pituitary harvested from a single female zebrafish. This makes it feasible for the first time to study the correlation between the SNs and other peptides and steroid hormones by quantifying them simultaneously in very small tissue samples. Untargeted peptidomics determined that the SN peptides in zebrafish can be further processed into smaller discrete fragments. This implies not only active synthesis and selective peptide processing but suggests that the are unknown functions of the SN peptide fragments that await discovery. This cost-effective package was used for the detailed assessment of hypothalamic-pituitary-gonadal function in mice and zebrafish and may be adaptable to many other hormones across species.

In the early 19th century a ground-breaking discovery was made that linked a dietary deficiency of a fat-soluble vitamin with the childhood disease known as rickets. The vitamin was named vitamin D and extensive research regarding the physiological importance of this vitamin followed ever since. It is currently known that vitamin D plays an important role in maintaining the calcium and phosphate homeostasis in the human body. Less clear evidence states the medical importance of vitamin D in the prevention and cancer, autoimmune disease and diabetes. Current literature shows that vitamin D has five distinct forms, vitamin D1 to D5, of which vitamin D2 and D3 are the most studied forms. The term “vitamin D” is often wrongfully used to include the vitamin D mother molecule, the vitamin D status indicator (25(OH)D), the biologically active form (1,25(OH)2D) and biologically inactive form (24,25(OH)2D). The interest for measurement of these vitamin D analogues is a continuously growing field both on individual and epidemiological level. For decades laboratories have struggled to produce a robust method capable of quantifying these different vitamin D analogues and uncovered a new form of complexity regarding the analysis of these analogues. The identification of the C3-epimeric forms of vitamin D metabolites has forced laboratories to rethink their analytical methods and several concerns were raised regarding the overestimation of the true vitamin D status by current analytical methods. The quantification of the biologically active and inactive forms of vitamin D is reported to be difficult and to date very few LC-MS/MS methods reported in the literature are able to quantify various vitamin D analogues. However, to our knowledge none of these methods are able to include the precursor vitamin D, the 25-hydroxylated metabolites, the biologically active and inactive metabolites, C3-epimers and isobaric compounds in a single run. Therefore the aim of this study was to develop, optimise and validate a LC-MS/MS method for the quantification of twelve vitamin D analogues in a single run. This was done by optimising the underlying LC-MS/MS parameters to ensure optimal analytical sensitivity in positive ESI mode and sufficient chromatographic separation between analytes with similar chemical properties. Furthermore, the optimised method was validated to ensure the accuracy and precision of the method before implementation into a clinical environment. The vitamin D analogues included in this study were vitamin D2, vitamin D3, 25(OH)D2, 25(OH)D3, 1,25(OH)2D2, 1,25(OH)2D3, 24,25(OH)2D2, 24,25(OH)2D3, 3-epi-25(OH)D2, 3-epi- 25(OH)D3, 7(OH)4C3 and 1α(OH)D3. A double liquid-liquid extraction with hexane and ethyl acetate were found to be the most efficient at extracting the vitamin D analogues from a serum matrix after matrix modification with sodium hydroxide. Recoveries of > 95 % (CV <10 %) were achieved for all the analytes. It was noted that a precursor adduct other than the molecular mass ion for a specific vitamin D analogue can produce a more abundant MS1 signal and that the ESI source parameters vary between analytes with different chemical properties and should therefore be optimised individually for each analyte. Various columns were assessed and sufficient chromatographic separation between the relevant analytes was achieved with an Agilent Technologies Pentafluorophenyl column. Baseline separation was achieved between 25(OH)D3 and 3-epi-25(OH)D3 as well as 25(OH)D2 and 3-epi-25(OH)D2, which is a requirement for this method to be viable. The method was subjected to a series of validation steps to ensure the accuracy and precision of the method. These included the assessment of the analytical range, LOD, LOQ, inaccuracy, imprecision, stability, interference and recovery. It was found that the optimised method had good linearity (r > 0.995), acceptable repeatability (CV < 10 %) and within-lab precision (CV < 15%) and excellent method accuracy (systematic error < 6.60 %). Furthermore, all the analytes proved to be stable for 48 hours after sample preparation with no interferences found for co-eluting analytes. Finally, based on the sigma metric scale specifications, it was calculated that this method proved to be “world class” and very little QC is needed to ensure the quality of the data derived from this method. Based on the findings in this study, it was concluded that a novel LC-MS/MS method for the quantification of twelve vitamin D analogues in a single run was successfully developed. All the LC-MS/MS parameters were optimised to ensure optimal analytical sensitivity for each analyte and the method was validated based on a series of method validation steps required for implementation into a clinical laboratory. This validation proved this method to be ready for implementation into a clinical environment. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

In the early 19th century a ground-breaking discovery was made that linked a dietary deficiency of a fat-soluble vitamin with the childhood disease known as rickets. The vitamin was named vitamin D and extensive research regarding the physiological importance of this vitamin followed ever since. It is currently known that vitamin D plays an important role in maintaining the calcium and phosphate homeostasis in the human body. Less clear evidence states the medical importance of vitamin D in the prevention and cancer, autoimmune disease and diabetes. Current literature shows that vitamin D has five distinct forms, vitamin D1 to D5, of which vitamin D2 and D3 are the most studied forms. The term “vitamin D” is often wrongfully used to include the vitamin D mother molecule, the vitamin D status indicator (25(OH)D), the biologically active form (1,25(OH)2D) and biologically inactive form (24,25(OH)2D). The interest for measurement of these vitamin D analogues is a continuously growing field both on individual and epidemiological level. For decades laboratories have struggled to produce a robust method capable of quantifying these different vitamin D analogues and uncovered a new form of complexity regarding the analysis of these analogues. The identification of the C3-epimeric forms of vitamin D metabolites has forced laboratories to rethink their analytical methods and several concerns were raised regarding the overestimation of the true vitamin D status by current analytical methods. The quantification of the biologically active and inactive forms of vitamin D is reported to be difficult and to date very few LC-MS/MS methods reported in the literature are able to quantify various vitamin D analogues. However, to our knowledge none of these methods are able to include the precursor vitamin D, the 25-hydroxylated metabolites, the biologically active and inactive metabolites, C3-epimers and isobaric compounds in a single run. Therefore the aim of this study was to develop, optimise and validate a LC-MS/MS method for the quantification of twelve vitamin D analogues in a single run. This was done by optimising the underlying LC-MS/MS parameters to ensure optimal analytical sensitivity in positive ESI mode and sufficient chromatographic separation between analytes with similar chemical properties. Furthermore, the optimised method was validated to ensure the accuracy and precision of the method before implementation into a clinical environment. The vitamin D analogues included in this study were vitamin D2, vitamin D3, 25(OH)D2, 25(OH)D3, 1,25(OH)2D2, 1,25(OH)2D3, 24,25(OH)2D2, 24,25(OH)2D3, 3-epi-25(OH)D2, 3-epi- 25(OH)D3, 7(OH)4C3 and 1α(OH)D3. A double liquid-liquid extraction with hexane and ethyl acetate were found to be the most efficient at extracting the vitamin D analogues from a serum matrix after matrix modification with sodium hydroxide. Recoveries of > 95 % (CV <10 %) were achieved for all the analytes. It was noted that a precursor adduct other than the molecular mass ion for a specific vitamin D analogue can produce a more abundant MS1 signal and that the ESI source parameters vary between analytes with different chemical properties and should therefore be optimised individually for each analyte. Various columns were assessed and sufficient chromatographic separation between the relevant analytes was achieved with an Agilent Technologies Pentafluorophenyl column. Baseline separation was achieved between 25(OH)D3 and 3-epi-25(OH)D3 as well as 25(OH)D2 and 3-epi-25(OH)D2, which is a requirement for this method to be viable. The method was subjected to a series of validation steps to ensure the accuracy and precision of the method. These included the assessment of the analytical range, LOD, LOQ, inaccuracy, imprecision, stability, interference and recovery. It was found that the optimised method had good linearity (r > 0.995), acceptable repeatability (CV < 10 %) and within-lab precision (CV < 15%) and excellent method accuracy (systematic error < 6.60 %). Furthermore, all the analytes proved to be stable for 48 hours after sample preparation with no interferences found for co-eluting analytes. Finally, based on the sigma metric scale specifications, it was calculated that this method proved to be “world class” and very little QC is needed to ensure the quality of the data derived from this method. Based on the findings in this study, it was concluded that a novel LC-MS/MS method for the quantification of twelve vitamin D analogues in a single run was successfully developed. All the LC-MS/MS parameters were optimised to ensure optimal analytical sensitivity for each analyte and the method was validated based on a series of method validation steps required for implementation into a clinical laboratory. This validation proved this method to be ready for implementation into a clinical environment. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015