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

To date, attempts have been made to detect metal adducts of polyphenols in positive mode by MALDI-MS using conventional matrices such as DHB and CHCA [34][35] or to use some matrices suc

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

3.4 Proton-abstractive reaction of nifedipine in flavonol skeleton 32

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

 1,5-DAN, 1,5-diaminonaphthalene

 9-AA, 9-aminoacridine

 ABC, ATP-binding cassette

 ADME, absorption, distribution,

metabolism, and excretion

 AMPK, adenosine monophosphate

activated-protein kinase

 ANOVA, analysis of variance

 BCRP, breast cancer resistance

 ESI, electrospray ionization

 FA, formic acid

 IAA, trans-3-indoleacrylic acid

 ITO, indium-tin oxide

 OATP, organic anion transporting polypeptides

 PA, proton affinity

 PepT1, peptide transporter 1

 P-gp, P-glycoprotein

 S/N, signal-to-noise ratio

 SA, sinapinic acid

 SD rat, Sprague-Dawley rat

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), 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 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] 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 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 1 and 2 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 II metabolism, namely, methylation, sulfation, and glucuronidation [15][16] 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

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 [5] In absorption studies of EGCG in mice [15] or ECG in Wistar rats [22], 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 binding cassette (ABC) transporters such as multidrug resistance protein 2 (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]

ATP-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 cell-line 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),

generally known as “soft” ionization that can produce intact pseudomolecular ion species without fragmentation is currently used for simultaneous and selective detection of targets even in complex matrices including low and high molecular compounds [34] The advantages of MALDI-MS are high sensitivity and selectivity by selected mass units, as well as high speed and tolerance for impurities [34][35] Therefore, MALDI-MS has been used for diverse ionizable compounds such as proteins, lipids, and drugs [36] Currently, development of MALDI-MS-aided imaging technique receives much attention, since the extensive technique can provide not only the detection of targets presented in sample tissues, but also the distribution or localization in them [33] The combinational 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 for analyses with wide mass ranges of peptides, proteins, lipids, drugs, and food compounds [31][37][38][39] It is suggested that the MALDI-MS imaging technique has 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 slides and mounted onto indium-titanium oxide (ITO)-coated glass slides Sliced sections 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 desorb and 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 research was to apply MALDI-MS imaging technique to elucidate intestinal absorption and metabolism(s) of polyphenols with less active in MALDI In order to achieve 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 considering molecular property of neutral polyphenols, a subtraction reaction of proton from polyphenol molecule was targeted in this study According to this strategy, matrix reagents that possess molecular property capable for the formation of nitrosophenyl pyridine moiety when an ultraviolet (UV) is irradiated in MALDI process were examined Under the optimal MALDI conditions, 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 transport system 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-MS imaging As a result, both targets were successfully detected or visualized in

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

Chapter II

Enhanced matrix-assisted laser desorption/ionization mass

spectrometry detection of polyphenols

1 Introduction

Polyphenols, which are polyhydroxyl aromatic compounds, naturally occurring in a variety of fruits and vegetables, could exhibit preventive effects against cardiovascular diseases [2], diabetes [3], and cancers [4] However, their ADME behavior remains unclear by diverse metabolism such as methylation, sulfation, and glucuronidation [16][42] In order to get insight into the bioavailability 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 of standards for each metabolite In contrast, MADLI-MS ionization allows simultaneous 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 for labeling preparation [15][40] Thus, MALDI-MS imaging are becoming increasingly applied for the visualization of targets in organs, such as distribution and metabolism of erlotinib, an anti-cancer drug, in the lungs of tumor-bearing mice [44]

Irrespective to such advantages of MALDI-MS, matrix-dependent ionization limits its application and requires efforts to develop new matrices adequate for target compounds In general, matrix reagent for MALDI requires following properties: strong absorption at laser irradiation wavelength; good compatibility of analytes with matrix solvent; good vacuum stability and low vapor pressure; and participation in some kind of photochemical reactions such

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

[46] The use of 9-aminoacridine (9-AA) as a matrix for negative ionization mode

shows high sensitive detection of low-molecular weight compounds such as carboxylic acids, amines, alcohols, and phenols owing to its high gas-phase basicity which readily abstracts a proton from analytes [47] A strong base, 1,8-bis(dimethyl-amino)naphthalene (DMAN) allows detection of low molecular weight 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, could deprotonate analytes during MALDI ionization [48] The matrix 1,5-diaminonaphthalene (1,5-DAN) has been reported to visualize polyphenols and their metabolites in rat liver and kidney in negative mode [15] However, the mechanism of 1,5-DAN as MALDI matrix remains undetermined in the research

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

in positive mode by MALDI-MS using conventional matrices such as DHB and CHCA [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, a strategy for screening adequate matrix in this study lies in a compelling subtraction reaction of a proton from polyphenol molecule In this Chapter II, a photobase generator of chemicals (nifedipine in this study) was targeted, since a photobase 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 MALDI-irradiation

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 obtain theasinensin 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 an equal 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 manually spotted onto an ITO-glass slide (Bruker Daltonics, Bremen, Germany) After drying 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 equipped with 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, 20.00 kV; ion source 2, 18.80 kV; lens voltage, 7.50 kV; gain factor, 2.51, laser frequency, 200 Hz; laser power, 100%; offset, 60%; range 20%, laser focus range,

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 the whole spotted area using the batch analysis function in Flexanalysis 3.3 (Bruker Daltonics)

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 Software Inc., 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/z

715.1) were used as model polyphenol, since both polyphenols showed low ionization efficiency in MALDI-MS due to their neutral molecular property [15]and unexpected production of fragments upon MALDI-ionization [34] In this study, three commonly used matrices of CHCA [34], DHB [52], and THAP [34] were selected Additionally, according to previous reports on matrix regarding enhanced negative MALDI detection, IAA [49], 9-AA [47], and 1,5-DAN [15] were also targeted In this study, we newly selected nifedipine as possible matrix reagent for polyphenols, because it produce a proton-acceptor photobase by UV-irradiation; in turn, the rationale for the use of nifedipine as a negative MALDI matrix reagent is that it could act as a catalyst in UV-mediated cross-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, the wavelength of MALDI laser (355 nm) was adequate for the excitation of nifedipine (maximal UV-wavelength of 344 nm [54]), causing possible formation

of a proton-acceptor photobase Aiming at the selection of matrix reagent suitable for MALDI detection of neutral polyphneols, EGCG was targeted for the present MALDI-MS As summarized in Figure 2-1, among the seven matrix reagents, EGCG spotted at 20 pmol/spot was detected in negative MALDI-MS using 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], much higher MALDI detection was obtained in 1,5-DAN and nifedipine Although enhanced detection of EGCG by 1,5-DAN [15] has already been reported, this is the first finding that nifedipine can act as a novel MALDI matrix reagent and has 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 ratio) than that in IAA (14,964 ± 1,105; S/N ratio, 73 ± 10), and was comparable for 1,5-DAN (76,588 ± 3,839; S/N ratio, 491 ± 85) in the present experimental

conditions A potent negative MALDI-MS detection of TF3’G by nifedipine compared to other matrices were also observed in Figure 2-2 The intensity of

TF3’G in nifedipine (113,322 ± 2,513; S/N ratio, 3,158 ± 98) was 2.6-fold 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 from

the 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-MS detection by

subtracting proton from polyphenols

Matrix-related peaks often interfere the detection of target peaks at

low-mass ranges [55] In this study, even though 1,5-DAN caused a relevant detection

of EGCG at 20 pmol/spot with nifedipine (Figure 2-1), a significant and selective

detection of EGCG was diminished at lower 1 pmol/spot owing to overlapping

with contaminating or clustering matrix-related peaks (Figure 2-4A) In contrast,

nifedipine achieved a significant EGCG detection at 1 pmol/spot without any

interfering peaks from nifedipine-related matrix peaks (Figure 2-4A) According

to the pictures of matrix crystal formed onto ITO glass (Figure 2-4B), 1,5-DAN

formed heterogeneous matrix crystal, which may reduce MS resolution and

intensity by topographic effect [56], leading to a limited MALDI-MS detection of

analytes with <500 Da On the other hand, it was 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-MS measurements 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 of EGCG ([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-MS

measurements 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 of

TF3’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-MS measurements 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-MS measurements 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, 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-MS

measurements in negative mode Matrix reagents (IAA, 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 Crystal images were taken by a KEYENCE BZ-9000

microscope MALDI-MS conditions were the same as denoted in Figure 2-1

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 and TF3’G by negative MALDI-MS was examined over the range of 1 to 15 mg/mL Figures 2-5A and 2-5B showed that MS intensity of EGCG and TF3’G reached

a plateau at a concentration of ≥5 mg/mL Thus, further experiments were performed using 5 mg/mL of nifedipine Under the optimal concentration,

nifedipine-aided MALDI-MS could detect EGCG at >0.1 pmol/spot at S/N ratio

of 4.2 (Figure 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 (20 pmol/spot) were 1, 5, 10, and 15 mg/mL MALDI-MS conditions were same as denoted 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 were detected 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/spot were 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 nitrosophenyl pyridine photobase product upon UV irradiation [50] In order to get insight into the mechanism of nifedipine as a MALDI matrix, dihydropyridin derivatives such as nitrendipine, nimodipine, and amlodipine were used, together with nifedipine (each reagent at 5 mg/mL) in negative mode MALDI-MS of EGCG and TF3’G Figure 2-8 and Figure 2-9 clearly showed that nitrendipine, nimodipine and amlodipine did not adequately facilitate MS detection of EGCG and TF3’G It has been reported that the nitrosophenyl pyridine derivative was formed under UV-dehydration via an intramolecular proton transfer from

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

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 strongly indicates that the nitrosophenyl pyridine derivative, a photobase product from nifedipine under UV-irradiation, is crucial for nifedipine-induced MALDI-MS detection of polyphenols

In order to confirm the production of the active nitrosophenyl pyridine photobase 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

positive mode MALDI-MS (because of its poor MS intensity in negative mode)

Figure 2-10 showed a major peak at m/z 329.1 corresponding to [M - H2O + H]+

from the product ion of nifedipine ([M + H]+, m/z 347.1) This indicated the

dehydration of nifedipine to form nitrosophenyl pyridine photobase product under the present MALDI-MS conditions

Figure 2-8 Dihydropyridine structures (A) and detection of EGCG in

negative MALDI-MS using dihydropyridines (B)

EGCG spotted onto ITO-glass (20 pmol/spot) was used for negative

MALDI-MS measurements Dihydropyridines (nifedipine, nitrendipine, nimodipine, and

amlodipine), each at concentration of 5 mg/mL was used as matrix reagent to

facilitate MS detection of EGCG MALDI-MS conditions were the same as

denoted in Figure 2-1 Results are expressed as mean of S/N ± SD

Figure 2-9 Detection of TF3’G in negative MALDI-MS using dihydropyridines

TF3’G spotted onto ITO-glass (20 pmol/spot) was used for negative

MALDI-MS measurements Dihydropyridines (nifedipine, nitrendipine, nimodipine, and amlodipine), each at concentration of 5 mg/mL were used as matrix reagents to facilitate MS detection of TF3’G MALDI-MS conditions were the same as

denoted in Figure 2-1 Results are expressed as mean of S/N ± SD N.D.: not

detected

Figure 2-10 Laser irradiation reaction of nifedipine (A) and MALDI-MS spectra of nifedipine (5 mg/mL) in positive mode (B)

MALDI-MS measurement of nifedipine in positive mode was performed in the

range 100–500 m/z M: nifedipine

3.4 Proton-abstractive reaction of nifedipine in flavonol skeleton

Our further investigation to assess the proton-removal characteristics of nifedipine as a matrix for MALDI-MS of polyphenols was performed in this study Naringenin and commercially available naringenin analogues with

different methylation on the A and B rings (4’, 5, and 7-O-methylnaringenin)

(Figure 2-11A) were subjected to nifedipine-aided MALDI-MS detection ([M - H]-, m/z 271.1 for naringenin and m/z 285.1 for methylated naringenins) As

shown in Figure 2-11B, naringenin and its methylated analogues were detected

by nifedipine-aided MALDI-MS, indicating that the nitrosophenyl pyridine photobase could remove one proton from the OH group in the flavonoid skeleton

However, a significantly (P < 0.05) lower detection of 5-O-methylnaringenin

compared to those of other naringenins was observed, suggesting that the nifedipine-aided MALDI-MS detection of naringenins was influenced by the position of mono-methylation It led to the speculation that nifedipine might preferably 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, in which the OH group on the A ring is not modified by, e.g., methylation or glycosylation

Figure 2-11 The structure of naringenin and its methylated conjugates (A)

and detection of naringenins by nifedipine-aided MALDI-MS in negative

mode (B)

Naringenin and three methylated naringenins spotted onto ITO-glass (20

pmol/spot) were subjected to negative MALDI-MS using nifedipine (5 mg/mL)

MALDI-MS conditions were the same as denoted 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 Tukey-Kramer’s t-test

3.5 Potential of nifedipine as matrix reagent for polyphenol detection

The potential of nifedipine as a matrix reagent for negative MALDI-MS detection of polyphenols was evaluated using a variety of polyphenols, including flavonol, flavones, flavanones, flavonones, chalcone, stilbenoid, and phenolic acid Each polyphenol at 20 pmol/spot was subjected to nifedipine-aided MALDI-MS analysis MS intensities of these polyphenols in nifedipine were compared to those in IAA (5 mg/mL) to validate the enhancement of MS detection of polyphenols in nifedipine Table 2-1 clearly showed the potential of nifedipine as a MALDI matrix, since nifedipine provided significant MS

intensities and S/N ratios for the tested polyphenols, compared to those by IAA

As shown in Table 1 (MS spectra for each polyphenol are shown in Figure 12), the nifedipine-induced MS detection was not restrictive to flavonoids, as successful detection of a chalcone (curcumin) and a stilbenoid (resveratrol) were also achieved by nifedipine It has been reported that, proton affinity (PA) value, which is the enthalpy change of a compound upon deprotonation in the gas phase, correlates with the potential of a compound as a MALDI-matrix [58][59] The PA values for IAA (893.9 kJ/mol [58]) and pyridine (the moiety of the nitrosophenyl photobase) (930 kJ/mol [60]) suggested that nifedipine has higher proton abstraction potential than that of IAA, supporting the significant MS detection

2-of polyphenols by nifedipine, compared to those in IAA

Table 2-1 MALDI-MS detection of polyphenols in negative mode by IAA and nifedipine