| A novel analytical method based on integrated ionization approach of electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI) combining with rapid resolution liquid chromatography mass spectrometry/tandem mass spectrometry (RRLC-MS/MS) has been explored and established. Urinary metabonomic researches on lung cancer have been investigated by using integrated ionization method and multivariate data processing methods, and 121 endogenous metabolites have been found to have significant differences between lung cancer patients and healthy volunteers. The results indicate that this approach is appropriate for complex biological system and would overcome the low ionization efficiencies of endogenous metabolites due to diverse physicochemical properties as well as ion suppression in the maximal degree. It provides a new idea to metabonomic investigation by taking full advantage of every ionization method and achieving comprehensive analysis.Additionally, further metabonomic research based on RRLC-ESIMS in positive and negative ion modes and multivariate statistics have been employed on large amount healthy controls, newly diagnosed lung cancer patients, and lung cancer patients pre and post treatment. The biochemically significant metabolite biomarkers in lung cancer have been further verified. Meanwhile, the potential biomarkers related to clinical curative effects have been achieved by combining with clinical curative effects evaluation and the change trend of biochemically significant metabolite biomarkers at the state of health, pre and post treatment. These potential biomarkers related to clinical curative effects would offer important evidence to the clinical effect evaluation and prognosis prediction.1. Integrated ionization approach for LC-MS analysisMultiple ionization approach of ESI, APCI, and APPI were investigated to an integrated ionization approach combining with RRLC-MS/MS for global metabonomic analysis. Ionization capability and applicability were manifested by improved ionization efficiency and enlarged metabolite coverage in analysis on typical urinary metabolite standards and urine samples from healthy volunteers. The results demonstrate that the RRLC-MS method established is suitable for global and comprehensive metabonomic research on complex biological samples. Additionally, the methodology of integrated ionization approach for RRLC-MS analysis implemented above has been studied. The results indicate that this approach would improve the sensitivity of RRLC-MS analysis and have good precision. Therefore, an integrated ionization approach of ESI, APCI, and APPI combining with RRLC-MS is valuable for the analysis of a wide range of metabolites and obtains comprehensive metabolites for global and comprehensive metabonomic analysis.2. The integrated ionization approach of RRLC-MS-based urinary metabonomic study on lung cancerBased on integrated ionization approach of RRLC-MS/MS analysis in both positive and negative ion mode and multivariate statistics, the metabonomic research on lung cancer has been employed to investigate human urine samples to find biochemically significant metabolite biomarkers in lung cancer. The results indicate the advantages of integrating multiple ionizations in metabonomic studies to overcome the low ionization efficiencies of endogenous metabolites due to diverse physicochemical properties as well as ion suppression. This is a further indication that an integrated ionization approach for RRLC-MS/MS-based metabonomic studies can gain more comprehensive metabolites and avoid losing diverse metabolite information. Additionally this proposed approach further verifies identical biomarkers that have been obtained simultaneously using different ionization methods. By using this method,121 metabolites have been found to have significant differences between lung cancer patients and healthy volunteers. These potential biomarkers have close relationship to abnormal changed metabolites in lung cancer and they might become new specific biomarkers for lung cancer. Until now, we have identified 11 metabolites as potential biomarkers including amino acids, nucleosides, and a metabolite of indole as high resolution MS and MS/MS analyses, as well as fragmentation regularities of metabolites have been performed for the identification of the metabolites of interest.3. RRLC-MS-based urinary metabonomics study on large amount healthy controls and lung cancer patientsA metabonomics strategy based on rapid resolution liquid chromatography/tandem mass spectrometry (RRLC-MS/MS) and multivariate statistics has been implemented to validate biochemically significant metabolite biomarkers which have been found in small amount lung cancer samples. Until now,66 potential biomarkers have been found by RRLC-ESIMS in positive and negative ion modes in this metabonomic validation study, 21 of which are identical to the previously found potential biomarkers. Five potential biomarkers have been identified as phenylalanine, tyrosine, tryptophan, proline betaine and uric acid which are elevated in lung cancer patients'urine. The validated biochemically significant metabolite biomarkers would prove biochemical insights into changes in lung cancer and offer the evidence of biology, clinic diagnose and individual treatment.4. Comparison of urinary metabonomic study on lung cancer patients pre and post treatmentBased on the research and analysis mentioned above, RRLC-MS analysis in both positive and negative ion modes and multivariate statistics method have been employed to the metabonomic research on lung cancer patients pre and post treatment. Based on clinic efficacy, potential biomarkers related to clinical curative effects have been investigated based on the change trend of biochemically significant metabolite biomarkers at the state of health, before and after treatment in the metabonomic analysis. Until now,55 metabolites have been detected to have significant differences between lung cancer patients before and after treatment. Comparison and correlation analysis have been implemented between potential biomarkers before and after treatment and those found from healthy and lung cancer patients.14 metabolites have been found elevated in lung cancer after eliminating the effects of drug metabolites. Finally, we identified four metabolites as potential biomarkers including phenylalanine, tyrosine, tryptophan, and uric acid. The potential biomarkers found by this metabonomic investigation not only serve as potential biomarkers for diagnosis but also as those related to efficacy. They would provide an important basis to curative effect evaluation and prognosis judgement.
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