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

Screening of matrix reagents for negative MALDI-MS detection of monomeric and condensed catechins ...16 3.2.. However, themechanism of 1,5-DAN as MALDI matrix remains undetermined in the

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 1

Introduction 1

Chapter II 11

Enhanced matrix-assisted laser desorption/ionization mass spectrometry detection of polyphenols

11 1 Introduction 11

2 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

3 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

3.5 Potential of nifedipine as matrix reagent for polyphenol detection 34

4 Summary 39

Chapter III 40

Application of matrix-assisted laser desorption/ionization mass spectrometry-imaging technique for intestinal absorption of polyphenols

40 1 Introduction 40

2 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

3 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

3.5 Visualized detection of metabolites of monomeric and condensed catechins during intestinal

absorption 60

4 Summary 68

Chapter IV 71

Conclusion 71

References 77

Acknowledgement 88

Abbreviations

 ABC, ATP-binding cassette

 ADME, absorption, distribution,

metabolism, and excretion

 AMPK, adenosine monophosphate

activated-protein kinase

 ANOVA, analysis of variance

 BCRP, breast cancer resistance

 MCT, monocarboxylic transporter

 MeOH, methanol

 MRP2, multidrug resistance protein2

 Nd:YAG, neodymium-dopedyttrium aluminum garnet

 OATP, organic anion transportingpolypeptides

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 andpolymerization reactions occur in leaves to form oligomeric catechins, such as

theasinensins and theaflavins (TFs) including theaflavin (TF), gallate (TF3G), theaflavin-3’-O-gallate (TF3’G), and theaflavin-3-3’-di-O-

theaflavin-3-O-gallate (TF-33’diG) [1] To date, extensive studies have been performed onhealth-benefits of tea polyphenols, and showed their potential in preventingcardiovascular diseases [2], diabetes [3], and cancers [4]

Irrespective to the evidences on their preventive effects, it must beessential to know absorption, distribution, metabolism, and excretion (ADME)behavior, since the understanding of ADME is indispensable for elucidating thebioactive mechanism(s) and effective dosage of polyphenols in our body Ingeneral, polyphenols are thought to be absorbed into the circulation system,following distribution at organs, and/or excretion into urine and fecal viametabolism [1] Among catechins, EC and EGC have been reported to be highlybioavailable, compared to gallate catechins such as ECG and EGCG [5] Inhuman 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 teacatechins (EC,

36.54 mg; EGC, 15.48 mg; ECG, 31.14 mg; EGCG, 16.74 mg) [6] Anotherhuman study also revealed the absorption of not only catechins, but also theirconjugates in plasma at >50 ng/mL [7] They also clarified that ECG and EGCGwere absorbed in their intact form, while EC and EGC were susceptible tometabolism to produce conjugated forms [7] Another research group reportedhigh stability of EGCG during absorption process in human [8] In cell-lineexperiments 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) werepredominantly accumulated in cells at 3037 ± 311 and 2335 ± 446 pmol/mgprotein, respectively [10]

There were few researches on absorption of black tea TFs In humanstudy, even at high dose intake of 700 mg TFs, plasma and urine levels of TFswere as low as 1 and 2 ng/mL, respectively [11] In urine, TFs were not detectedafter consumption of 1000 mg of TF extract [12] Non-absorbable property of TFswas also confirmed by Caco-2 cell transport study, in which TF3’G was notdetected in basolateral side after 60-min transport [13] Irrespective to poorabsorption or low bioavailability of TFs, it was reported that they havepotential in the regulation of intestinal absorption route(s); in turn, TFs mayexert physiological function at the small intestine [14] However, the absorptionbehavior of TFs still remains unclear whether they could be incorporated intointestinal membrane or not.

Once being absorbed into the circulation system or organs, polyphenolsundergo phase II metabolism, namely, methylation, sulfation, andglucuronidation [15][16] Phase II enzymes catalyzing the methylation, sulfation,

and glucuronidation are catechol-O-methyltransferase, sulfotransferase, and

uridine diphosphate-glucuronosyltransferase, respectively [17] These metabolicenzymes were found not only in the intestine, but also in the liver and thekidneys [18][19][20] It has been reported that higher absorbable catechins such as

EC and EGC were more susceptible to such metabolic reactions, compared togallate catechins (ECG and EGCG) [7] For EC absorption, a predominantsulfate conjugate of EC were effluxed from the enterocytes back to theintestinal perfusate, while glucuronide conjugate was absorbed into blood, bile

and urine [21] When 500 mL of green tea was given to 10 volunteers, only intactECG and

EGCG were found in human plasma, whereas glucuronide, methyl-glucuronide,and methyl-sulfate conjugates of EC and EGC were detected [5] In absorptionstudies of EGCG in mice [15] or ECG in Wistar rats [22], their sulfate andglucuronide conjugates were found in blood, liver, and kidney, suggesting thatoverall absorption study is still required for further understanding of polyphenolbioavailability.

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 binding cassette (ABC) transporters such as multidrug resistance protein 2(MRP2) and P-glycoprotein (P-gp), which are located in the apical side [23] InCaco-2 cell transport experiments of monomeric catechin (EC), inhibition ofMRP2 route by MK-571, an inhibitor of MRP2, significantly reduced theeffluxes of EC and its sulfate conjugates to the apical compartment [24] InMRP2 transfected and P-gp transfected cells, it was demonstrated that thecellular accumulation of ECG was significantly increased by both MRP2 and P-

ATP-gp efflux inhibitors, suggesting the involvement of ECG in both ABCtransporters [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 expresstransport systems as in small intestine [27] By using Caco-2 transport system incombination with transporter inhibitors, investigations on transport routes ofpolyphenols have been widely performed [10][28] Irrespective to easy set of cell-line experiments, Caco-2 cell model remains some disadvantages such asdifferent 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 ofmembrane permeability using rat and human intestine mounted onto the UssingChamber 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 areavailable To date, liquid chromatography-mass spectrometry (LC-MS) inelectrospray ionization (ESI) mode is commonly used for absorption study ofpolyphenols [32], since LC-MS system could detect not only target polyphenols,but also metabolites simultaneously or one-in-run assay Irrespective to its highsensitivity and throughput characteristics, LC-based method remains somedrawbacks; it requires tedious pre-treatments such as preparation and extractionsteps, and could not obtain the localization of analytes in biological tissues [33]

On the other hand, matrix-assisted laser desorption/ionization MS (MALDI-MS),

generally known as “soft” ionization that can produce intact pseudomolecularion species without fragmentation is currently used for simultaneous andselective detection of targets even in complex matrices including low and highmolecular compounds [34] The advantages of MALDI-MS are high sensitivityand selectivity by selected mass units, as well as high speed and tolerance forimpurities [34][35] Therefore, MALDI-MS has been used for diverse ionizablecompounds such as proteins, lipids, and drugs [36] Currently, development ofMALDI-MS-aided imaging technique receives much attention, since theextensive technique can provide not only the detection of targets presented insample tissues, but also the distribution or localization in them [33] Thecombinational information of MS detection with spatial distribution, thus, opens

a novel scientific field regarding food and drug delivery system [33] Thus far,there have been some reports on the application of MALDI-MS imaging foranalyses with wide mass ranges of peptides, proteins, lipids, drugs, and foodcompounds [31][37][38][39] It is suggested that the MALDI-MS imaging techniquehas potential for elucidating the ADME of drugs and food compounds [40][41]

As illustrated in Figure 1-1, target tissues are cryosectioned into µm-thick slidesand mounted onto indium-titanium oxide (ITO)-coated glass slides Slicedsections are, then, sprayed and coated with MALDI matrix reagents Upon

irradiated by e.g., neodymium-doped yttrium aluminum garnet (ND:YAG)

laser, the sprayed matrix reagent absorbs the light energy at 355 nm to desorband ionize the analytes in the matrix plume The analytes are detected at mass-

to-charge (m/z)

value (for 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 points, the aim of the present researchwas to apply MALDI-MS imaging technique to elucidate intestinal absorptionand metabolism(s) of polyphenols with less active in MALDI In order toachieve the objective, the following works have been conducted.

Since there have been few reports on matrix reagents for significant

MALDI-MS detection of polyphenols, in Chapter II, appropriate matrix

reagents for MS detection of polyphenols were screened By consideringmolecular property of neutral polyphenols, a subtraction reaction of protonfrom polyphenol molecule was targeted in this study According to thisstrategy, matrix reagents that possess molecular property capable for theformation of nitrosophenyl pyridine moiety when an ultraviolet (UV) isirradiated in MALDI process were examined Under the optimal MALDIconditions, diverse polyphenols including flavonols, flavones, flavanones,flavonones, chalcone, stilbenoid, and phenolic acid were successfully ionized

Under the established MALDI conditions for polyphenols, in Chapter

III, novel in situ MALDI-MS imaging concept or analytical application was

proposed to elucidate intestinal absorption behavior of polyphenols on the basis

of visualization-guided evidences By using the Ussing Chamber transportsystem mounted with rat intestinal membranes, non-absorbable and absorbable

polyphenols were subjected to ex vivo transport experiments Membrane

segments after the transport were, then, analyzed by established MALDI-MSimaging As a result, both targets were successfully detected or visualized in

tissue segments by the technique In addition, with the aid of inhibitors that canblock influx/efflux transport routes, MALDI-MS imaging allow to clarify theabsorption mechanism visually, together with visualized metabolic degradationduring absorption process.

Chapter II

Enhanced matrix-assisted laser desorption/ionization mass

spectrometry detection of polyphenols

1 Introduction

Polyphenols, which are polyhydroxyl aromatic compounds, naturallyoccurring in a variety of fruits and vegetables, could exhibit preventive effectsagainst cardiovascular diseases [2], diabetes [3], and cancers [4] However, theirADME behavior remains unclear by diverse metabolism such as methylation,sulfation, and glucuronidation [16][42] In order to get insight into thebioavailability of polyphenols, LC-MS using ESI have been widely used, owing

to their high selective and high sensitive detection properties [11][43] However,the application of LC-MS for absorption study is limited due to the lack ofstandards for each metabolite In contrast, MADLI-MS ionization allowssimultaneous detection of low- to high-molecular-weight compounds [34][35].Currently, MALDI-MS imaging technique has been received much attention,since the technique provides not only information of compounds presented in

samples, but also their distribution in biological samples, without the need forlabeling preparation [15][40] Thus, MALDI-MS imaging are becomingincreasingly applied for the visualization of targets in organs, such asdistribution and metabolism of erlotinib, an anti-cancer drug, in the lungs oftumor-bearing mice [44].

Irrespective to such advantages of MALDI-MS, matrix-dependentionization limits its application and requires efforts to develop new matricesadequate for target compounds In general, matrix reagent for MALDI requiresfollowing properties: strong absorption at laser irradiation wavelength; goodcompatibility of analytes with matrix solvent; good vacuum stability and lowvapor pressure; and participation in some kind of photochemical reactions such

as protonation or deprotonation in gas phase [35] Rational design of MALDImatrix could be based on physicochemical properties of the expected matrix [45].Matrix reagents for negative MALDI mode should be a strong base, whereashigh acidity in gas phase seems to be an important characteristic of a matrix forpositive MALDI mode [45] Matrices such as α-cyano-4-hydroxycinnamic acid(CHCA), sinapinic acid (SA), and 2,5-dihydrobenzoic acid (DHB) have beenwidely used in both negative and positive MALDI detection of proteins,peptides, and lipids [46] However, due to some limitations of these conventionalmatrices e.g., many interfered signals at the low mass range in positive modeand low ionization efficiency in negative detection mode, efforts have beenmade to screen and develop high ionization efficiency matrices for negative

MALDI-MS [46] The use of 9-aminoacridine (9-AA) as a matrix for negativeionization mode

shows high sensitive detection of low-molecular weight compounds such ascarboxylic acids, amines, alcohols, and phenols owing to its high gas-phasebasicity which readily abstracts a proton from analytes [47] A strong base, 1,8-bis(dimethyl-amino)naphthalene (DMAN) allows detection of low molecularweight compounds (fatty acids, amino acids, plant and animal hormones,vitamins and small peptides) in negative MALDI-MS [48] The basic center,formed by steric position of two dimethyl-amino groups in DMAN, coulddeprotonate analytes during MALDI ionization [48] The matrix 1,5-diaminonaphthalene (1,5-DAN) has been reported to visualize polyphenols andtheir metabolites in rat liver and kidney in negative mode [15] However, themechanism of 1,5-DAN as MALDI matrix remains undetermined in theresearch.

To date, attempts have been made to detect metal adducts of polyphenols

in positive mode by MALDI-MS using conventional matrices such as DHB andCHCA [34][35] or to use some matrices such as trans-3-indoleacrylic acid (IAA)

[49] and 1,5-DAN [15] in negative MS detection mode However, poor

MALDI-MS detection of neutral polyphenols still remains problematic due to their lack

of proton-removal or -additional groups In order to detect polyphenols, astrategy for screening adequate matrix in this study lies in a compellingsubtraction reaction of a proton from polyphenol molecule In this Chapter II, aphotobase generator of chemicals (nifedipine in this study) was targeted, since aphotobase can produce a proton-acceptor moiety upon UV-irradiation [50]

EGCG and TF3’G, most common polyphenols in green and black tea, respectively,

were selected to evaluate proton subtraction property of matrices under irradiation.

MALDI-2 Materials and methods

2.1 Materials

CHCA, DHB, 2’,4’,6’-trihydroxyacetophenone (THAP), nimodipine, EC,

EGCG, naringin, and 5-O-methylnaringenin were obtained from Sigma-Aldrich

(St Louis, MO, USA) 1,5-DAN and resveratrol was purchased from Tokyo Chemical Ind (Tokyo, Japan) Nitrendipine, amlodipine, TF3’G, hesperidin,

sakuranetin (7-O-methylnaringenin), luteolin, acacetin, curcumin, and quercetin

were purchased from Nacalai Tesque Co (Kyoto, Japan) Nifedipine, IAA, kaempferol, and ellagic acid were obtained from Wako Pure Chemical Ind (Osaka, Japan) 9-AA was obtained from Merck Millipore (Darmstadt,

Germany) Procyanidin B2 and isosakuranetin (4’-O-methylnaringenin) were

obtained from Extrasynthese (Lyon, France) Theasinensin A was synthesized according to the method by Tanaka et al [51] Briefly, EGCG was oxidized with Japanese pear homogenate to obtain dehydrotheasinensin A Ascorbic acid, then,was added to dehyrotheasinensin A, followed by elution with methanol (MeOH)

in MCI-gel CHP 20P column (Mitsubishi Chemical Co., Tokyo, Japan) to obtaintheasinensin A Naringenin was purchased from MP Biomedicals LLC (Santa Ana, CA, USA) All other chemicals were of analytical grade and were used without further purification

2.2 Sample and matrix preparations

Polyphenols used in this study were dissolved in MeOH/water (50/50, v/v) Matrix reagents were dissolved in acetonitrile/water (3/1, v/v) solution.Polyphenol solution at concentrations of 0.1 ‒ 200 µmol/L was mixed with anequal volume of matrix solution (2 ‒ 30 mg/mL) An aliquot (0.2 µL,corresponding to 0.01 ‒ 20 pmol each) of the polyphenol solution was manuallyspotted onto an ITO-glass slide (Bruker Daltonics, Bremen, Germany) Afterdrying in air at room temperature, optical images of the spotted matrix crystals

on ITO-coated glass slide were taken by a microscope BZ-9000 (KEYENCE,Osaka, Japan)

2.3 MALDI-MS analyses

MALDI-MS analysis was performed using an Autoflex III MS equippedwith SmartBeam III (Bruker Daltonics) MS data were acquired in the range of

100–1000 m/z by summed signals from 100 consecutive laser pulses on the

spotted sample area The MS parameters were as follows: ion source 1, 20.00kV; ion source 2, 18.80 kV; lens voltage, 7.50 kV; gain factor, 2.51, laserfrequency, 200 Hz; laser power, 100%; offset, 60%; range 20%, laser focusrange,

100%; value, 6% Signal intensity and signal-to-noise (S/N) ratio were

calculated as a sum of MS data from 100 randomly selected positions on thewhole spotted area using the batch analysis function in Flexanalysis 3.3 (BrukerDaltonics)

2.4 Statistical Analyses

Data for intensity and S/N ratio were expressed as the mean ± standard

deviation (SD) The statistical differences between groups were analyzed using

one-way analysis of variance (ANOVA), followed by Tukey-Kramer’s t-test for

post-hoc analysis A P value of <0.05 was considered statistically significant.

All analyses were performed using a GraphPad Prism 5 (GraphPad SoftwareInc., San Diego, CA, USA)

3 Results and discussion

3.1 Screening of matrix reagents for negative MALDI-MS detection

of monomeric and condensed catechins

In order to clarify matrix reagents suitable for negative MALDI-MS

detection of polyphenols, EGCG ([M - H]-, m/z 457.1) and TF3’G ([M - H]-, m/

MALDI matrix reagent is that it could act as a catalyst in UV-mediatedcross-linking

reaction in polymerization by its proton-removal property [50][53] In addition,when Nd:YAG is used as a laser source of the present MALDI-MS system, thewavelength of MALDI laser (355 nm) was adequate for the excitation ofnifedipine (maximal UV-wavelength of 344 nm [54]), causing possible formation

of a proton-acceptor photobase Aiming at the selection of matrix reagentsuitable for MALDI detection of neutral polyphneols, EGCG was targeted forthe present MALDI-MS As summarized in Figure 2-1, among the seven matrixreagents, EGCG spotted at 20 pmol/spot was detected in negative MALDI-MSusing IAA, 1,5-DAN, and nifedipine, while other four matrix reagents could not

detect EGCG Although the detection of EGCG in IAA with >70 S/N ratio was

in agreement with the report on significant detection of procyanidin [49], muchhigher MALDI detection was obtained in 1,5-DAN and nifedipine Althoughenhanced detection of EGCG by 1,5-DAN [15] has already been reported, this isthe first finding that nifedipine can act as a novel MALDI matrix reagent andhas potential in ionizing neutral polyphenols with high ionization efficiency,compared to IAA and 1,5-DAN The intensity of EGCG in nifedipine (84,035 ±

4,598; S/N ratio, 3,669 ± 103) was 5.3-fold higher (50.6-fold higher in S/N

higher (35-fold higher in S/N) than that in IAA (43,143 ± 2,061; S/N ratio, 90

± 11),

being comparable for 1,5-DAN (62,088 ± 1,469; S/N ratio, 995 ± 100) Figure

2-1 and Figure 2-2 also demonstrated that no multiple-charged ions of EGCG ([M

- 2H]2-, m/z 228.1) and TF3’G ([M - 2H]2-, m/z 357.1) were produced by

nifedipine, suggesting that nifedipine may preferably remove one proton fromthe targets In positive mode of MALDI-MS, there was no peak of EGCG at [M+ H]+ of m/z 459.1 in nifedipine as well as other matrices (Figure 2-3),

indicating the preferential use of nifedipine for negative-mode MALDI-MSdetection by subtracting proton from polyphenols

Matrix-related peaks often interfere the detection of target peaks at mass ranges [55] In this study, even though 1,5-DAN caused a relevant detection

low-of EGCG at 20 pmol/spot with nifedipine (Figure 2-1), a significant andselective detection of EGCG was diminished at lower 1 pmol/spot owing tooverlapping with contaminating or clustering matrix-related peaks (Figure 2-4A) In contrast, nifedipine achieved a significant EGCG detection at 1pmol/spot without any interfering peaks from nifedipine-related matrix peaks(Figure 2-4A) According to the pictures of matrix crystal formed onto ITOglass (Figure 2-4B), 1,5-DAN formed heterogeneous matrix crystal, which mayreduce MS resolution and intensity by topographic effect [56], leading to alimited MALDI-MS detection of analytes with <500 Da On the other hand, itwas clear that the formed crystal of nifedipine was homogeneous on ITO-glass(Figure 2-4B), which may allow high sensitive and selective detection of EGCG

in low-mass ranges (Figure 2-4A)

Figure 2-1 MALDI-MS detection of EGCG at 20 pmol/spot in negative mode by CHCA, DHB, THAP, IAA, 9-AA, 1,5-DAN, and nifedipine as matrix.

EGCG spotted onto ITO-glass (20 pmol/spot) was used for MALDI-MSmeasurements in negative mode Matrix reagents (CHCA, DHB, THAP, IAA,9- AA, 1,5-DAN, and nifedipine), each at 10 mg/mL in acetonitrile/water (3/1,v/v) was mixed with an equal volume of EGCG MALDI-MS measurements ofEGCG ([M - H]-, m/z 457.1) were performed in the range of 100–1000 m/z by

the sum of signals from 100 consecutive random laser pulses on the spotted

sample area Results are expressed as mean of S/N ± SD N.D.: not detected.

Figure 2-2 MALDI-MS detection of TF3’G at 20 pmol/spot in negative mode by CHCA, DHB, THAP, IAA, 9-AA, 1,5-DAN, and nifedipine as matrix.

TF3’G spotted onto ITO-glass (20 pmol/spot) was used for MALDI-MSmeasurements in negative mode Matrix reagents (CHCA, DHB, THAP, IAA,9- AA, 1,5-DAN, and nifedipine), each at 10 mg/mL in acetonitrile/water (3/1,v/v) was mixed with an equal volume of TF3’G MALDI-MS measurements ofTF3’G ([M - H]-, m/z 715.1) were performed in the range of 100‒1000 m/z by

the sum of signals from 100 consecutive random laser pulses on the spotted

sample area Results are expressed as mean of S/N ± SD N.D.: not detected.

Figure 2-3 Detection of EGCG at 20 pmol/spot in positive mode

MALDI-MS by CHCA, DHB, THAP, IAA, and nifedipine as matrix.

EGCG spotted onto ITO-glass (20 pmol/spot) was used for MALDI-MSmeasurements in positive mode The concentration of reagents (CHCA, DHB,THAP, IAA, and nifedipine) was 10 mg/mL in acetonitrile/water (3/1, v/v).Matrices were mixed with an equal volume of EGCG MALDI-MSmeasurements of EGCG ([M + H]+, m/z 459.1) were performed in the range of 100–1000 m/z by the sum of signals from 100 consecutive random laser pulses

on the spotted sample area Results are expressed as mean of S/N ± SD N.D.:

not detected

Figure 2-4 MALDI-MS detection of EGCG at 1 pmol/spot in negative mode using IAA, 1,5-DAN, and nifedipine (A), and the crystal images of IAA, 1,5- DAN, and nifedipine (B).

EGCG spotted onto ITO-glass (1 pmol/spot) was used for MALDI-MSmeasurements in negative mode Matrix reagents (IAA, 1,5-DAN, andnifedipine), each at 10 mg/mL in acetonitrile/water (3/1, v/v) was mixed with

an equal volume of EGCG Crystal images were taken by a KEYENCE

BZ-9000 microscope MALDI-MS conditions were the same as denoted in Figure2-1

3.2 Effect of concentration of nifedipine on negative MALDI-MS detection of monomeric and condensed catechins

The effect of nifedipine concentration on the detection of EGCG andTF3’G by negative MALDI-MS was examined over the range of 1 to 15mg/mL Figures 2-5A and 2-5B showed that MS intensity of EGCG and TF3’Greached a plateau at a concentration of ≥5 mg/mL Thus, further experimentswere performed using 5 mg/mL of nifedipine Under the optimalconcentration, nifedipine-aided MALDI-MS could detect EGCG at >0.1 pmol/

spot at S/N ratio of 4.2 (Figure 2-6) and TF3’G at >0.01 pmol/spot at S/N ratio

of 10.0 (Figure

2-7) Considering the reported detection of EGCG by 1,5-DAN (>5 pmol/spot)

[15], nifedipine seems to show higher detection than 1,5-DAN as shown in Figure 2-

6 and Figure 2-7

Figure 2-5 Effect of concentration of nifedipine on EGCG (A) and TF3’G (B) detection by MALDI-MS in negative mode.

Concentrations of nifedipine in EGCG- and TF3’G-spotted ITO-glass (20pmol/spot) were 1, 5, 10, and 15 mg/mL MALDI-MS conditions were same asdenoted in Figure 2-1 Results are expressed as mean of intensity ± SD (n = 3)

Data without common letters are significantly different (P < 0.05) by Kramer’s t-test.

Tukey-Figure 2-6 Effect of concentration of EGCG on MALDI-MS detection in negative mode.

EGCG-spotted ITO-glass at concentrations of 0.1, 0.5, 1, and 5 pmol/spot weredetected by nifedipine (5 mg/mL)-aided MALDI-MS in negative mode.MALDI-MS conditions were the same as denoted in Figure 2-1

Figure 2-7 Effect of concentration of TF3’G on MALDI-MS detection in negative mode.

TF3’G-spotted ITO-glass at concentrations of 0.01, 0.05, 0.5, and 1 pmol/spotwere detected by nifedipine (5 mg/mL)-aided MALDI-MS in negative mode.MALDI-MS conditions were the same as denoted in Figure 2-1

3.3 Photobase reaction of nifedipine as matrix in MALDI

Nifiedipine is a photobase generator, which could produce nitrosophenylpyridine photobase product upon UV irradiation [50] In order to get insight intothe mechanism of nifedipine as a MALDI matrix, dihydropyridin derivativessuch as nitrendipine, nimodipine, and amlodipine were used, together withnifedipine (each reagent at 5 mg/mL) in negative mode MALDI-MS of EGCGand TF3’G Figure 2-8 and Figure 2-9 clearly showed that nitrendipine,nimodipine and amlodipine did not adequately facilitate MS detection of EGCGand TF3’G It has been reported that the nitrosophenyl pyridine derivative wasformed under UV-dehydration via an intramolecular proton transfer from

pyridine moiety to the nitro group occurs at the ortho-position of the phenyl

ring [50][57] In these dihydropyridines, only nifedipine (ortho-positioned nitro

group) produced the nitrosophenyl pyridine moiety, whereas nitrendipine,

nimodipine (meta-positioned nitro group), and amlodipine (no nitro group) did

not undergo the UV-dehydration [57] The adequate facilitation in MS detection

of EGCG and TF3’G by nifedipine, but not other dihyropyridines stronglyindicates that the nitrosophenyl pyridine derivative, a photobase product fromnifedipine under UV-irradiation, is crucial for nifedipine-induced MALDI-MSdetection of polyphenols

In order to confirm the production of the active nitrosophenyl pyridinephotobase from nifedipine during MALDI irradiation by a Nd:YAG laser at 355

nm, nifedipine spotted onto an ITO glass slide (5 mg/mL) was subjected to