Studies On The Determination Of 5-hydroxyindoleacetic Acid In Human Urine And Gefitinib In Human Plasma

5-Hydroxyindoleacetic acid(5-HIAA),a monoamine neurotransmitter,is an inactive acidic metabolic end product of 5-hydroxytryptamine(5-HT)that is eventually excreted in urine.5-HIAA is an important neurotransmitter metabolite in the human body.Increasing or decreasing of 5-HIAA indirectly reflects certain pathological changes in the body.In patients with metastatic carcinoid urine 5-HIAA levels were significantly higher.Clinically,the over-secretion of 5-HIAA is the most specific biochemical indicator of carcinoid tumors.Detection of 24-hour urinary 5-HIAA levels can be used for the monitoring of carcinoid syndrome and evaluation of treatment efficacy.Carcinoid tumors belong to neuroendocrine neoplasms(NENs),a group of tumors that originate in stem cells and have neuroendocrine markers that produce bioactive amine or polypeptide kinases.Among them,gastroenteropancreatic neuroendocrine neoplasms(GEP-NENs)are the most common.In the treatment of gastrointestinal pancreatic neuroendocrine tumors drug research,some new drugs such as:gefitinib and thalidomide have played a certain effect.The subject of human urine 5-HIAA and human plasma gefitinib detection methods were studied.This thesis is divided into five chapters,the main contents include:Chapter ?:Introduced the significance of the research,the characteristics and diagnosis of carcinoid carcinogenesis,5-HIAA anabolism and summarized the 5-HIAA routine analysis methods,carcinoid and molecular targeted drug therapy are reviewed.Chapter ?:Established the method of ultra-high performance liquid chromatography-mass spectrometry for the determination of 5-HIAA in human urine,and validated the method.The method used a Waters HSS T3 analytical column(100×2.1 mm,1.8?m).ESI was applied and operated in negative ion mode.The artificial human urine UR-N-CONTROL LEVEL 2 NP was used for calibration curve preparation,artificial urine and natural urine were used for quality control samples preparation.5-hydroxyindoleacetic acid-d5(5-HIAA-d5)was used as internal standards,urine samples were diluted with acetonitrile.The supernatants were detected by UPLC-MS/MS.The linear calibration curve was observed in the concentration range of 0.0500 to 50.0?g/mL with r~2?0.9896.The lower limit of quantification was 0.0500?g/mL.The accuracy(intra-assay and inter-assay bias)for 5-HIAA was between-10.7%~4.8%with precision(CV)?8.4%.The mean recoveries were from 100.3%to 102.3%.The method is proved to be highly selective,sensitive and rapid.Artificial urine and natural urine are no different in quantitative analysis and suitable for determination of 5-HIAA in human urine.Chapter ?:Determination of 5-HIAA concentration in 24-hour urine in patients with advanced non-pancreatic neuroendocrine Tumors.The established method for the determination of 5-HIAA in human urine had been used for analyze the samples from a phase III clinical study of new generation of antitumor drugs treatment in patients with advanced non-pancreatic neuroendocrine tumors.So far,the baseline for total 5-HIAA in24 hours urine samples from 40 patients has been analyzed,the total amount of 5-HIAA ranged from 0.12 to 37.8 mg,the values for 7 patients were greater than 10 mg,the values for 15 patients were less than 2 mg.Chapter ?:Established the method of ultra-high performance liquid chromatography-mass spectrometry detection of gefitinib in human plasma and validated the method.The method used a Waters BEH C18 analytical column(100×2.1 mm,1.7?m).ESI was applied and operated in positive ion mode.Gefitinib-d3 was used as internal standards,plasma samples were protein precipitated with acetonitrile.The supernatants were detected by UPLC-MS/MS.The calibration curve range is from 5.00to 3000 ng/mL,the relationship between concentration and peak area ratio can be expressed as:peak area=A*concentration~2+B*concentration+C,with r~2?0.9962.The lower limit of quantification was 5.00 ng/mL.The accuracy(intra-assay and inter-assay bias)for gefitinib was between-3.8%~4.0%with precision(CV)?11.2%.The mean recoveries were from 98.9%to 101.8%.Gefitinib stock solution and plasma had good stability.The method is proved has good precision and accuracy and had used for determination of gefitinib in human plasma samples for clinical study.Chapter ?:Summarizes and prospects the full text.