Study on the analytical application of matrix assisted laser desorption ionization mass spectrometry imaging technique for visualization of polyphenols

Study on the analytical application of matrix-assisted laser desorption/ionization mass spectrometry-imaging technique for visualization of polyphenols Nguyen Huu Nghi Kyushu University 2018 List of contents Chapter I Introduction Chapter II 11 Enhanced matrix-assisted laser desorption/ionization mass spectrometry detection of polyphenols 11 Introduction 11 Materials and methods 14 2.1 Materials 14 2.2 Sample and matrix preparations 14 2.3 MALDI-MS analyses 15 2.4 Statistical Analyses 15 Results and discussion 16 3.1 Screening of matrix reagents for negative MALDI-MS detection of monomeric and condensed catechins 16 3.2 Effect of concentration of nifedipine on negative MALDI-MS detection of monomeric and condensed catechins 23 3.3 Photobase reaction of nifedipine as matrix in MALDI 27 3.4 Proton-abstractive reaction of nifedipine in flavonol skeleton 32 3.5 Potential of nifedipine as matrix reagent for polyphenol detection 34 i Summary 39 Chapter III 40 Application of matrix-assisted laser desorption/ionization mass spectrometryimaging technique for intestinal absorption of polyphenols 40 Introduction 40 Materials and methods 42 2.1 Materials 42 2.2 Intestinal transport experiments using rat jejunum membrane in the Ussing Chamber system 42 2.3 LC-TOF-MS analysis 44 2.4 Preparation of intestinal membrane section and matrix reagent 45 2.5 MALDI-MS imaging analysis 46 Results and discussion 46 3.1 Optimization of MALDI-MS imaging for visualization of monomeric and condensed catechins in rat jejunum membrane 46 3.2 In situ visualization of monomeric and condensed catechins in rat jejunum membrane by MALDI-MS imaging 48 3.3 Absorption route(s) of monomeric and condensed catechins in rat jejunum membrane 52 3.4 Efflux route(s) of monomeric and condensed catechins in rat jejunum membrane 56 ii 3.5 Visualized detection of metabolites of monomeric and condensed catechins during intestinal absorption 60 Summary 68 Chapter IV 71 Conclusion 71 References 77 Acknowledgement 88 iii Abbreviations  1,5-DAN, 1,5-diaminonaphthalene  9-AA, 9-aminoacridine desorption/ionization  ABC, ATP-binding cassette spectrometry  ADME, absorption, distribution,  MCT, monocarboxylic transporter metabolism, and excretion  MeOH, methanol AMPK, adenosine monophosphate  MRP2, multidrug resistance protein    ANOVA, analysis of variance  BCRP, breast cancer resistance  Nd:YAG, neodymium-doped yttrium aluminum garnet  protein α-cyano-4- CHCA, mass activated-protein kinase  MALDI-MS, matrix-assisted laser OATP, organic anion transporting polypeptides hydroxycinnamic acid  PA, proton affinity  DHB, 2,5-dihydroxybenzoic acid  PepT1, peptide transporter  DMAN,  P-gp, P-glycoprotein amino)naphthalene  S/N, signal-to-noise ratio  DMSO, dimethyl sulfoxide  SA, sinapinic acid  EC, epicatechin  SD rat, Sprague-Dawley rat  ECG, epicatechin-3-O-gallate  SD, standard deviation  EGC, epigallocatechin  TF, theaflavin  EGCG,  TF3’G, theaflavin-3’-O-gallate  TF-33’diG, 1,8-bis(dimethyl- epigallocatechin-3-O- gallate theaflavin-3-3’-di-O-  ESI, electrospray ionization  FA, formic acid  TF3G, theaflavin-3-O-gallate  IAA, trans-3-indoleacrylic acid  THAP,  ITO, indium-tin oxide  KBR, Krebs-Bicarbonate Ringer’s  TJ, tight-junction  LC, liquid chromatography  TOF, time-of-flight  m/z, mass-to-charge ratio  UV, ultraviolet gallate trihydroxyacetophenone iv 2’,4’,6’- Chapter I Introduction A popular beverage of tea, derived from the leaves of the Camellia sinensis plant, has been consumed worldwide, and to date, it is considered that the tea intake would be of health-benefit owing to dietary flavonoids (polyphenols) In green or non-fermented tea, major components are monomeric catechins, e.g., epicatechin (EC), epicatechin-3-O-gallate (ECG), epigallocatechin (EGC), and epigallocatechin-3-O-gallate (EGCG) On the other hands, by fermentation of tea leaves to produce black tea, oxidation and polymerization reactions occur in leaves to form oligomeric catechins, such as theasinensins and theaflavins (TFs) including theaflavin (TF), theaflavin-3-Ogallate (TF3G), theaflavin-3’-O-gallate (TF3’G), and theaflavin-3-3’-di-Ogallate (TF-33’diG) [1] To date, extensive studies have been performed on health-benefits of tea polyphenols, and showed their potential in preventing cardiovascular diseases [2], diabetes [3], and cancers [4] Irrespective to the evidences on their preventive effects, it must be essential to know absorption, distribution, metabolism, and excretion (ADME) behavior, since the understanding of ADME is indispensable for elucidating the bioactive mechanism(s) and effective dosage of polyphenols in our body In general, polyphenols are thought to be absorbed into the circulation system, following distribution at organs, and/or excretion into urine and fecal via metabolism [1] Among catechins, EC and EGC have been reported to be highly bioavailable, compared to gallate catechins such as ECG and EGCG [5] In human study, EC, EGC, ECG, and EGCG were detected in plasma to be 174, 145, 50.6, and 20.1 pmol/mL, respectively, after the consumption of tea catechins (EC, 36.54 mg; EGC, 15.48 mg; ECG, 31.14 mg; EGCG, 16.74 mg) [6] Another human study also revealed the absorption of not only catechins, but also their conjugates in plasma at >50 ng/mL [7] They also clarified that ECG and EGCG were absorbed in their intact form, while EC and EGC were susceptible to metabolism to produce conjugated forms [7] Another research group reported high stability of EGCG during absorption process in human [8] In cell-line experiments using Caco-2 cell monolayers, non-gallate catechin, EC, was found to show lower cellular accumulation than gallate ECG, due to high efflux back of EC to apical side [9] After 50-µmol/L, 60-min, Caco-2 transport experiments of EC, ECG, and EGCG, only gallate catechins (ECG and EGCG) were predominantly accumulated in cells at 3037 ± 311 and 2335 ± 446 pmol/mg protein, respectively [10] There were few researches on absorption of black tea TFs In human study, even at high dose intake of 700 mg TFs, plasma and urine levels of TFs were as low as and ng/mL, respectively [11] In urine, TFs were not detected after consumption of 1000 mg of TF extract [12] Non-absorbable property of TFs was also confirmed by Caco-2 cell transport study, in which TF3’G was not detected in basolateral side after 60-min transport [13] Irrespective to poor absorption or low bioavailability of TFs, it was reported that they have potential in the regulation of intestinal absorption route(s); in turn, TFs may exert physiological function at the small intestine [14] However, the absorption behavior of TFs still remains unclear whether they could be incorporated into intestinal membrane or not Once being absorbed into the circulation system or organs, polyphenols undergo phase glucuronidation II [15][16] metabolism, namely, methylation, sulfation, and Phase II enzymes catalyzing the methylation, sulfation, and glucuronidation are catechol-O-methyltransferase, sulfotransferase, and uridine diphosphate-glucuronosyltransferase, respectively [17] These metabolic enzymes were found not only in the intestine, but also in the liver and the kidneys [18][19][20] It has been reported that higher absorbable catechins such as EC and EGC were more susceptible to such metabolic reactions, compared to gallate catechins (ECG and EGCG) [7] For EC absorption, a predominant sulfate conjugate of EC were effluxed from the enterocytes back to the intestinal perfusate, while glucuronide conjugate was absorbed into blood, bile and urine [21] When 500 mL of green tea was given to 10 volunteers, only intact ECG and EGCG were found in human plasma, whereas glucuronide, methyl-glucuronide, and methyl-sulfate conjugates of EC and EGC were detected studies of EGCG in mice [15] or ECG in Wistar rats [22] [5] In absorption , their sulfate and glucuronide conjugates were found in blood, liver, and kidney, suggesting that overall absorption study is still required for further understanding of polyphenol bioavailability The low bioavailability of polyphenols is in part due to their pumping out (or efflux) to the apical compartment and/or metabolic degradation In vitro studies suggested that the routes involved in efflux of polyphenols are ATPbinding cassette (ABC) transporters such as multidrug resistance protein (MRP2) and P-glycoprotein (P-gp), which are located in the apical side [23] In Caco-2 cell transport experiments of monomeric catechin (EC), inhibition of MRP2 route by MK-571, an inhibitor of MRP2, significantly reduced the effluxes of EC and its sulfate conjugates to the apical compartment [24] In MRP2 transfected and P-gp transfected cells, it was demonstrated that the cellular accumulation of ECG was significantly increased by both MRP2 and P-gp efflux inhibitors, suggesting the involvement of ECG in both ABC transporters [10] In order to get inside into the absorption and metabolism behaviors of tea polyphenols, some analytical evaluations have been reported In in vivo evaluation, transport routes of polyphenols may not be fully explored [25][26] Thus, to elucidate intestinal absorption and metabolism of polyphenols, cell-based in vitro model, commonly Caco-2 cell, has been widely used Caco-2 cells, which are derived from human colon carcinoma, resemble the enterocytes and express transport systems as in small intestine [27] By using Caco-2 transport system in combination with transporter inhibitors, investigations on transport routes of polyphenols have been widely performed [10][28] Irrespective to easy set of cellline experiments, Caco-2 cell model remains some disadvantages such as different protease expression from actual intestinal membranes An alternative strategy for absorption study has been proposed by using ex vivo Ussing Chamber system, which is mounted with animal intestinal membranes [29][30] Miyake et al [29] evaluated intestinal absorption of drugs with different levels of membrane permeability using rat and human intestine mounted onto the Ussing Chamber system [29] The ex vivo system is considered to be a good tool for investigating transport mechanism as in vivo intestinal absorption events, and is used for transport of drugs [29][30], and peptides [31] It should be noted that analytical assays to monitor target analytes must be needed for absorption study, even though appropriate absorption systems are available To date, liquid chromatography-mass spectrometry (LC-MS) in electrospray ionization (ESI) mode is commonly used for absorption study of polyphenols [32], since LC-MS system could detect not only target polyphenols, but also metabolites simultaneously or one-in-run assay Irrespective to its high sensitivity and throughput characteristics, LC-based method remains some drawbacks; it requires tedious pre-treatments such as preparation and extraction steps, and could not obtain the localization of analytes in biological tissues [33] On the other hand, matrix-assisted laser desorption/ionization MS (MALDI-MS), Chapter III: Application of matrix-assisted laser desorption/ionization mass spectrometry-imaging technique for intestinal absorption of polyphenols Application of MALDI-MS imaging technique has been well established and applied in pharmacological science owing to its simultaneous visualization of targets and its metabolites in target organs The advantage of MS imaging is that it could provide the spatial localization of compounds distributed in tissue, which overcomes limitation of LC-based methods In Chapter III, we aim to challenge practical application of MALDI-MS imaging technique for studying intestinal absorption of polyphenols ECG, an absorbable polyphenol and TF3’G, a non-absorbable polyphenol, were targeted in this Chapter III These polyphenols were used for intestinal transport experiments (each at 50 µmol/L for 60 min) using SD rat jejunum membrane mounted onto an Ussing Chamber system During the transport experiment, intestinal membrane transporter inhibitors were used to elucidate the absorption route(s) of these polyphenols According to the finding of nifedipine as a novel matrix for negative MALDI-MS of polyphenols in Chapter II and phytic acid as a matrix additive for enhanced visualization of targets in biological tissue in our previous research [66] , nifedipine/phytic acid was selected to facilitate MALDI-MS visualization of these polyphenols in rat intestinal membrane Chapter III demonstrated that the nifedipine/phytic acid-aided MALDI-MS imaging technique provided adequate visualization of TF3’G and ECG in rat intestinal membrane Under the optimal nifedipine/phytic acid-aided MALDI-MS imaging, TF3’G was found to specifically distribute only in the apical region of the intestinal membrane, whereas ECG distributed in the whole membrane It suggested that the MALDI-MS imaging could be used as an in situ analytical tool for absorption study The visualized regions of TF3’G and ECG were reduced in phloretin and estrone-3-sulfate and expanded in cyclosporine A, 75 indicating that TF3’G and ECG could be incorporated into the intestinal membrane via MCT and OATP transport routes followed by efflux back to the apical compartment via ABC transporters These results suggested that the inhibitor-aided MALDI-MS imaging is a powerful and novel analytical tool for direct analysis of intestinal absorption route(s) of compounds Using the present MALDI-MS imaging for nontargeting visualization of TF3’G and ECG metabolites demonstrated that TF3’G was stable against phase II metabolism, whereas ECG was susceptible to phase II metabolism to form methylation, sulfation, and their combination conjugates in the intestinal membrane In conclusion, the present study demonstrated that a photobase generator, nifedipine, being able to form a nitrosophenyl pyridine under UV-irradiation, could act as a novel matrix reagent for negative MALDI-MS detection of polyphenols The nifedipine/phytic acid-aided MALDI-MS imaging, in combination with the inhibitoraided intestinal transport experiment using rat intestinal membrane mounted onto an Ussing Chamber system must be a novel and powerful analytical strategy for studying intestinal absorption and metabolism of polyphenols without any staining or labelling preparation, and tedious analytical extraction and separation steps 76 References [1] M G Sajilata, P R Bajaj, R S Singhal Tea polyphenols as nutraceuticals Compr Rev Food Sci Food Saf 2008, 7, 229 [2] V Stangl, M Lorenz, K Stangl The role of tea and tea flavonoids in cardiovascular health Mol Nutr Food Res 2006, 50, 218 [3] M C Sabu, K Smitha, R Kuttan Anti-diabetic activity of green tea polyphenols and their role in reducing oxidative stress in experimental diabetes J Ethnopharmacol 2002, 83, 109 [4] C S Yang, H Wang Cancer preventive activities of tea catechins Molecules 2016, 21, 1679 [5] A Stalmach, S Troufflard, M Serafini, A Crozier Absorption, metabolism and excretion of Choladi green tea flavan-3-ols by humans Mol Nutr Food Res 2009, 53, S44 [6] B A Warden, L S Smith, G R Beecher, D A Balentine, B A Clevidence Catechins are bioavailable in men and women drinking black tea throughout the day J Nutr 2001, 131, 1731 [7] H H S Chow, I A Hakim, D R Vining, J A Crowell, J Ranger-Moore, W M Chew, C A Celaya, S R Rodney, Y Hara, D S Alberts Effects of dosing condition on the oral bioavailability of green tea catechins after single-dose administration of polyphenon E in healthy individuals Clin Cancer Res 2005, 11, 4627 [8] H S Chow, Y Cai, D S Alberts, I Hakim, R Dorr, F Shahi, J A Crowell, C S Yang, Y Hara Phase I pharmacokinetic study of tea polyphenols following 77 single-dose administration of epigallocatechin gallate and polyphenon E Cancer Epidemiol Biomarkers Prev 2001, 10, 53 [9] M Kadowaki, N Sugihara, T Tagashira, K Terao, K Furuno Presence or absence of a gallate moiety on catechins affects their cellular transport J Pharm Pharmacol 2008, 60, 1189 [10] J B Vaidyanathan, T Walle Cellular uptake and efflux of the tea flavonoid (-)epicatechin-3-gallate in the human intestinal cell line Caco-2 J Pharmacol Exp Ther 2003, 307, 745 [11] T P J Mulder, C J van Platerink, P J Wijnand Schuyl, J M M van Amelsvoort Analysis of theaflavins in biological fluids using liquid chromatography–electrospray mass spectrometry J Chromatogr B 2001, 760, 271 [12] G Pereira-Caro, J M Moreno-Rojas, N Brindani, D Del Rio, M E J Lean, Y Hara, A Crozier Bioavailability of black tea theaflavins: absorption, metabolism, and colonic catabolism J Agric Food Chem 2017, 65, 5365 [13] J Takeda, H Y Park, Y Kunitake, K Yoshiura, T Matsui Theaflavins, dimeric catechins, inhibit peptide transport across Caco-2 cell monolayers via down-regulation of AMP-activated protein kinase-mediated peptide transporter PEPT1 Food Chem 2013, 138, 2140 [14] T Matsui Condensed catechins and their potential health-benefits Eur J Pharmacol 2015, 765, 495 [15] Y H Kim, Y Fujimura, T Hagihara, M Sasaki, D Yukihira, T Nagao, D Miura, S Yamaguchi, K Saito, H Tanaka, H Wariishi, K Yamada, H Tachibana In situ label-free imaging for visualizing the biotransformation of a 78 bioactive polyphenol Sci Rep 2013, 3, 2805 [16] M Margalef, Z Pons, F I Bravo, B Muguerza, A Arola-Arnal Tissue distribution of rat flavanol metabolites at different doses J Nutr Biochem 2015, 26, 987 [17] A Rodriguez-Mateos, D Vauzour, C G Krueger, D Shanmuganayagam, J Reed, L Calani, P Mena, D Del Rio, A Crozier Bioavailability, bioactivity and impact on health of dietary flavonoids and related compounds: an update Arch Toxicol 2014, 88, 1803 [18] H Lu, X Meng, C S Yang Enzymology of methylation of tea catechins and inhibition of catechol-O-methyltransferase by (-)-epigallocatechin gallate Drug Metab Dispos 2003, 31, 572 [19] J B Vaidyanathan, T Walle Glucuronidation and sulfation of the tea flavonoid (-)-epicatechin by the human and rat enzymes Drug Metab Dispos 2002, 30, 897 [20] W Teubner, W Meinl, S Florian, M Kretzschmar, H Glatt Identification and localization of soluble sulfotransferases in the human gastrointestinal tract Biochem J 2007, 404, 207 [21] L Actis-Goretta, A Lévèques, M Rein, A Teml, C Schäfer, U Hofmann, H Li, M Schwab, M Eichelbaum, G Williamson Intestinal absorption, metabolism, and excretion of (-)-epicatechin in healthy humans assessed by using an intestinal perfusion technique Am J Clin Nutr 2013, 98, 924 [22] T Kohri, M Suzuki, F Nanjo Identification of metabolites of (-)-epicatechin gallate and their metabolic fate in the rat J Agric Food Chem 2003, 51, 5561 [23] K M Giacomini, S.-M Huang, D J Tweedie, L Z Benet, K L R Brouwer, 79 X Chu, A Dahlin, R Evers, V Fischer, K M Hillgren, K A Hoffmaster, T Ishikawa, D Keppler, R B Kim, et al Membrane transporters in drug development Nat Rev Drug Discov 2010, 9, 215 [24] J Vaidyanathan, T Walle Transport and metabolism of the tea flavonoid (-)epicatechin by the human intestinal cell line Caco-2 Pharm Res 2001, 18, 1420 [25] P V Balimane, S Chong, R A Morrison Current methodologies used for evaluation of intestinal permeability and absorption J Pharmacol Toxicol Methods 2000, 44, 301 [26] J M Carbonell-Capella, M Buniowska, F J Barba, M J Esteve, A Frígola Analytical methods for determining bioavailability and bioaccessibility of bioactive compounds from fruits and vegetables: a review Compr Rev Food Sci Food Saf 2014, 13, 155 [27] P Artursson, K Palm, K Luthman Caco-2 monolayers in experimental and theoretical predictions of drug transport Adv Drug Deliv Rev 2001, 46, 27 [28] L Zhang, Y Zheng, M S S Chow, Z Zuo Investigation of intestinal absorption and disposition of green tea catechins by Caco-2 monolayer model Int J Pharm 2004, 287, [29] M Miyake, T Koga, S Kondo, N Yoda, C Emoto, T Mukai, H Toguchi Prediction of drug intestinal absorption in human using the Ussing chamber system: A comparison of intestinal tissues from animals and humans Eur J Pharm Sci 2017, 96, 373 [30] V Rozehnal, D Nakai, U Hoepner, T Fischer, E Kamiyama, M Takahashi, S Yasuda, J Mueller Human small intestinal and colonic tissue mounted in the Ussing chamber as a tool for characterizing the intestinal absorption of drugs 80 Eur J Pharm Sci 2012, 46, 367 [31] M Tanaka, S M Hong, S Akiyama, Q Q Hu, T Matsui Visualized absorption of anti-atherosclerotic dipeptide, Trp-His, in Sprague-Dawley rats by LC-MS and MALDI-MS imaging analyses Mol Nutr Food Res 2015, 59, 1541 [32] R K Bonta Application of HPLC and ESI-MS techniques in the analysis of phenolic acids and flavonoids from green leafy vegetables (GLVs) J Pharm Anal 2017, 7, 349 [33] A R Buchberger, K DeLaney, J Johnson, L Li Mass spectrometry imaging: a review of emerging advancements and future insights Anal Chem 2018, 90, 240 [34] M C Menet, S Sang, C S Yang, C T Ho, R T Rosen Analysis of theaflavins and thearubigins from black tea extract by MALDI-TOF mass spectrometry J Agric Food Chem 2004, 52, 2455 [35] M Monagas, J E Quintanilla-López, C Gómez-Cordovés, B Bartolomé, R Lebrón-Aguilar MALDI-TOF MS analysis of plant proanthocyanidins J Pharm Biomed Anal 2010, 51, 358 [36] M W Duncan, D Nedelkov, R Walsh, S J Hattan Applications of MALDI mass spectrometry in clinical chemistry Clin Chem 2016, 62, 134 [37] M Dilillo, R Ait-Belkacem, C Esteve, D Pellegrini, S Nicolardi, M Costa, E Vannini, E L De Graaf, M Caleo, L A McDonnell Ultra-high mass resolution MALDI imaging mass spectrometry of proteins and metabolites in a mouse model of Glioblastoma Sci Rep 2017, 7, 603 [38] T Goto, N Terada, T Inoue, K Nakayama, Y Okada, T Yoshikawa, Y Miyazaki, M Uegaki, S Sumiyoshi, T Kobayashi, T Kamba, K Yoshimura, O 81 Ogawa The expression profile of phosphatidylinositol in high spatial resolution imaging mass spectrometry as a potential biomarker for prostate cancer PLoS One 2014, 9, e90242 [39] B Prideaux, L E Via, M D Zimmerman, S Eum, J Sarathy, P O’Brien, C Chen, F Kaya, D M Weiner, P Y Chen, T Song, M Lee, T S Shim, J S Cho, et al The association between sterilizing activity and drug distribution into tuberculosis lesions Nat Med 2015, 21, 1223 [40] A Nilsson, A Peric, M Strimfors, R J A Goodwin, M A Hayes, P E Andrén, C Hilgendorf Mass spectrometry imaging proves differential absorption profiles of well-characterised permeability markers along the crypt-villus axis Sci Rep 2017, 7, 6352 [41] J Chen, Y Hsieh, I Knemeyer, L Crossman, W A Korfmacher Visualization of first-pass drug metabolism of terfenadine by MALDI-imaging mass spectrometry Drug Metab Lett 2008, 2, [42] S Sang, J D Lambert, C T Ho, C S Yang The chemistry and biotransformation of tea constituents Pharmacol Res 2011, 64, 87 [43] W Pongsuwan, T Bamba, K Harada, T Yonetani, A Kobayashi, E Fukusaki High-throughput technique for comprehensive analysis of Japanese green tea quality assessment using ultra-performance liquid chromatography with time-offlight mass spectrometry (UPLC/TOF MS) J Agric Food Chem 2008, 56, 10705 [44] M Nishidate, K Yamamoto, C Masuda, H Aikawa, M Hayashi, T Kawanishi, A Hamada MALDI mass spectrometry imaging of erlotinib administered in combination with bevacizumab in xenograft mice bearing B901L, EGFR- 82 mutated NSCLC cells Sci Rep 2017, 7, 16763 [45] R Shroff, L Rulísek, J Doubsky, A Svatos Acid-base-driven matrix-assisted mass spectrometry for targeted metabolomics Proc Natl Acad Sci U S A 2009, 106, 10092 [46] T C Baker, J Han, C H Borchers Recent advancements in matrix-assisted laser desorption/ionization mass spectrometry imaging Curr Opin Biotechnol 2017, 43, 62 [47] R L Vermillion-Salsbury, D M Hercules 9-Aminoacridine as a matrix for negative mode matrix-assisted laser desorption/ionization Rapid Commun Mass Spectrom 2002, 16, 1575 [48] R Shroff, A Svatoš Proton sponge: a novel and versatile MALDI matrix for the analysis of metabolites using mass spectrometry Anal Chem 2009, 81, 7954 [49] M Ohnishi-Kameyama, A Yanagida, T Kanda, T Nagata Identification of catechin oligomers from apple (Malus pumila cv Fuji) in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and fast-atom bombardment mass spectrometry Rapid Commun Mass Spectrom 1997, 11, 31 [50] W Maafi, M Maafi Modelling nifedipine photodegradation, photostability and actinometric properties Int J Pharm 2013, 456, 153 [51] T Tanaka, S Watarumi, Y Matsuo, M Kamei, I Kouno Production of theasinensins A and D, epigallocatechin gallate dimers of black tea, by oxidation-reduction dismutation of dehydrotheasinensin A Tetrahedron 2003, 59, 7939 [52] Y Yang, M Chien Characterization of grape procyanidins using highperformance liquid chromatography/mass spectrometry and matrix-assisted laser 83 desorption/ionization time-of-flight mass spectrometry J Agric Food Chem 2000, 48, 3990 [53] K Suyama, M Shirai Photobase generators: Recent progress and application trend in polymer systems Prog Polym Sci 2009, 34, 194 [54] S Onoue, N Igarashi, Y Yamauchi, N Murase, Y Zhou, T Kojima, S Yamada, Y Tsuda In vitro phototoxicity of dihydropyridine derivatives: A photochemical and photobiological study Eur J Pharm Sci 2008, 33, 262 [55] S Zhang, J Liu, Y Chen, S Xiong, G Wang, J Chen, G Yang A novel strategy for MALDI-TOF MS analysis of small molecules J Am Soc Mass Spectrom 2010, 21, 154 [56] J M Koomen, W K Russell, J M Hettick, D H Russell Improvement of resolution, mass accuracy, and reproducibility in reflected mode DE-MALDITOF analysis of DNA using fast evaporation-overlayer sample preparations Anal Chem 2000, 72, 3860 [57] H Görner Nitro group photoreduction of 4-(2-nitrophenyl)- and 4-(3nitrophenyl)-1,4-dihydropyridines Chem Phys 2010, 373, 153 [58] S P Mirza, N P Raju, M Vairamani Estimation of the proton affinity values of fifteen matrix-assisted laser desorption/ionization matrices under electrospray ionization conditions using the kinetic method J Am Soc Mass Spectrom 2004, 15, 431 [59] A Moser, K Range, D M York Accurate proton affinity and gas-phase basicity values for molecules important in biocatalysis J Phys Chem B 2010, 114, 13911 [60] E P Hunter, S G Lias Evaluate gas phase basicities and proton affinity of 84 molecules: an update J Phys Chem Ref Data 1998, 27, 413 [61] A Fülöp, M B Porada, C Marsching, H Blott, B Meyer, S Tambe, R Sandhoff, H D Junker, C Hopf 4-Phenyl-α-cyanocinnamic acid amide: screening for a negative ion matrix for MALDI-MS imaging of multiple lipid classes Anal Chem 2013, 85, 9156 [62] B A Graf, C Ameho, G G Dolnikowski, P E Milbury, C.-Y Chen, J B Blumberg Rat gastrointestinal tissue metabolite quercetin J Nutr 2006, 136, 39 [63] M N Clifford, J J van der Hooft, A Crozier Human studies on the absorption, distribution, metabolism, and excretion of tea polyphenols Am J Clin Nutr 2013, 98, 1619S [64] M Zhu, Y Chen, R C Li Oral absorption and bioavailability of tea catechins Planta Med 2000, 66, 444 [65] H Y Park, Y Kunitake, N Hirasaki, M Tanaka, T Matsui Theaflavins enhance intestinal barrier of Caco-2 Cell monolayers through the expression of AMP-activated protein kinase-mediated Occludin, Claudin-1, and ZO-1 Biosci Biotechnol Biochem 2015, 79, 130 [66] S.-M Hong, M Tanaka, S Yoshii, Y Mine, T Matsui Enhanced visualization of small peptides absorbed in rat small intestine by Phytic-acid-aided matrixassisted laser desorption/ ionization-imaging mass spectrometry Anal Chem 2013, 85, 10033 [67] B Li, Y Terazono, N Hirasaki, Y Tatemichi, E Kinoshita, A Obata, T Matsui Inhibition of glucose transport by tomatoside A, a tomato seed steroidal saponin, through the suppression of GLUT2 expression in Caco-2 cells J Agric Food 85 Chem 2018, 66, 1428 [68] C Shen, R Chen, Z Qian, X Meng, T Hu, Y Li, Z Chen, C Huang, C Hu, J Li Intestinal absorption mechanisms of MTBH, a novel hesperetin derivative, in Caco-2 cells, and potential involvement of monocarboxylate transporter and multidrug resistance protein Eur J Pharm Sci 2015, 78, 214 [69] A Kondo, K Narumi, J Ogura, A Sasaki, K Yabe, T Kobayashi, A Furugen, M Kobayashi, K Iseki Organic anion-transporting polypeptide (OATP) 2B1 contributes to the cellular uptake of theaflavin Drug Metab Pharmacokinet 2017, 32, 145 [70] S H Hansen, A Olsson, J E Casanova Wortmannin, an inhibitor of phosphoinositide 3-kinase, inhibits transcytosis in polarized epithelial cells J Biol Chem 1995, 270, 28425 [71] M Roth, B N Timmermann, B Hagenbuch Interactions of green tea catechins with organic anion-transporting polypeptides Drug Metab Dispos 2011, 39, 920 [72] K Y Chan, L Zhang, Z Zuo Intestinal efflux transport kinetics of green tea catechins in Caco-2 monolayer model J Pharm Pharmacol 2007, 59, 395 [73] M Qadir, K L O Loughlin, S M Fricke, N A Williamson, W R Greco, H Minderman, M R Baer Cyclosporin A is a broad-spectrum multidrug resistance modulator Clin Cancer Res 2005, 11, 2320 [74] J Hong, J D Lambert, S H Lee, P J Sinko, C S Yang Involvement of multidrug resistance-associated proteins in regulating cellular levels of (-)epigallocatechin-3-gallate and its methyl metabolites Biochem Biophys Res Commun 2003, 310, 222 86 [75] P Matsson, J M Pedersen, U Norinder, C A S Bergström, P Artursson Identification of novel specific and general inhibitors of the three major human ATP-binding cassette transporters P-gp, BCRP and MRP2 among registered drugs Pharm Res 2009, 26, 1816 [76] N Kusunoki, K Takara, Y Tanigawara, A Yamauchi, K Ueda, F Komada, Y Ku, Y Kuroda, Y Saitoh, K Okumura Inhibitory effects of a cyclosporin derivative, SDZ PSC 833, on transport of doxorubicin and vinblastine via human P-glycoprotein Jpn J Cancer Res 1998, 89, 1220 [77] J L Donovan, V Crespy, C Manach, C Morand, C Besson, A Scalbert, C Rémésy Catechin is metabolized by both the small intestine and liver of rats J Nutr 2001, 131, 1753 [78] S Ganguly, T G Kumar, S Mantha, K Panda Simultaneous determination of black tea-derived catechins and theaflavins in tissues of tea consuming animals using ultra-performance liquid-chromatography tandem mass spectrometry PLoS One 2016, 11, e0163498 87 Acknowledgement First and foremost, I would like to express my sincere gratitude to my supervisor, Prof Toshiro Matsui for his continuous support during my Ph.D study and related research His intellectual guidance, innovative ideas, and patience efficiently helped me in all the time of my research It was my great honor and privilege to be a PhD student in laboratory of food analysis under his supervision My academic background and research experience have been improved significantly throughout the time studying in his laboratory I also would like to thank my committee members, Prof Mitsuya Shimoda and Prof Takahisa Miyamoto for their time, consideration, and valuable comments Their advices and suggestions have been a great help in completion of my doctoral dissertation I would like to offer my special thanks to Assistant Prof Mitsuru Tanaka for his technical training, advices, and motivation throughout my period of research His supports and caring is also valuable for my personal life in Japan I am also grateful to Ms Kaori Miyazaki for her technical and secretarial support Without her kind caring and support, my life in Japan would not be smooth as it was I would like to take this opportunity to thank all members of laboratory of food analysis for their help, support, and sharing during my research and my life in Japan A special gratefulness I would like to give to Seong-Min-Hong, Riho Koyanagi, Naoto Hirasaki, Vu Thi Hanh, Toshihiko Fukuda, and Kumrungsee Thanutchporn 88 I highly appreciated the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) for the financial support during my PhD research I also appreciated all Kyushu university staffs for their kind support Last, I express my deep appreciation to my parents, my wife, my children and my friends for their support, encouragement and always behind me during my journey in Japan 89 ... Chapter III Application of matrix- assisted laser desorption/ ionization mass spectrometry- imaging technique for intestinal absorption of polyphenols Introduction To date, MALDI-MS imaging has... MALDI-MS imaging application, they are visualized with ion density image) Figure 1-1 Schematic workflow of matrix- assisted laser desorption/ ionization mass spectrometry imaging According to the aforementioned... subtract one proton from the 5-position OH group in the A ring of the flavonoid skeleton Therefore, the enhancement of MALDI-MS detection of polyphenols by nifedipine might be possible for polyphenols,