Construction and Application of a Database for a Five Dimensional

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New Analytical Methods

Construction and Application of a Database for a Five Dimensional Identification of Natural Compounds in Garcinia species by means of UPLC-ESI-TWIMS-TOF-MS–Introducing Gas Phase Polyphenol Conformer Drift Time Distribution Intensity Ratios Timo D. Stark, Josef Ranner, Benedikt Stiglbauer, Patrick Weiss, Sofie Stark, Onesmo B. Balemba, and Thomas Hofmann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06157 • Publication Date (Web): 21 Dec 2018 Downloaded from http://pubs.acs.org on January 2, 2019

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Journal of Agricultural and Food Chemistry

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Construction and Application of a Database for a Five

2

Dimensional Identification of Natural Compounds in

3

Garcinia species by means of UPLC-ESI-TWIMS-TOF-

4

MS–Introducing Gas Phase Polyphenol Conformer Drift

5

Time Distribution Intensity Ratios

6 7

Timo D. Stark,§* Josef Ranner,§ Benedikt Stiglbauer,§ Patrick Weiss,§

8

Sofie Stark,§ Onesmo B. Balemba,$ and Thomas Hofmann§

9 10

§Lehrstuhl

11

Universität München, Lise-Meitner-Str. 34, 85354 Freising, Germany

12

$Department

für Lebensmittelchemie und Molekulare Sensorik, Technische

of Biological Sciences, University of Idaho, Moscow, ID, USA

13 14 15 16 17 18 19

*

To whom correspondence should be addressed

20

PHONE

+49-8161-71-2911

21

FAX

+49-8161-71-2949

22

E-MAIL

[email protected]

23

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ABSTRACT

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34 reference compounds from G. buchananii were analyzed by means of

27

UPLC-ESI-IMS-TOF-MS to build a database consisting of retention time,

28

accurate m/z of precursors and fragment ions as well as rotationally averaged

29

collision cross-sectional area (CCS).

30

compounds analyzed in bark extract in different concentrations and solvent

31

systems showed excellent intra- as well as interday precision (RSD ≤0.9%).

32

The established database was applied on different organs of G. buchananii as

33

well as G. kola, G. mangostana and G. cambogia enabling a fast and reliable

34

identification of these natural bioactives. For several compounds more than

35

one drift time species could be highlighted which we propose to be hydrogen

36

bond stabilized rotational isomers transferred from solution to gas phase. We

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used all CCS values of one compound, and we propose to add also the

38

intensity ratio of the conformers as a new and additional characteristic

39

compound

40

applications to reduce dereplication and false positives and strengthen the

41

identification.

parameter

in

The CCS value of six selected

compound

identification/screening/database

42 43 44

KEYWORDS:

Ion

mobility,

CCS,

CCS

intensity

45

polyphenols, GB-2 7''-O-β-D-glucopyranosyl-6''-malonic acid, UNIFI database,

46

drift time

47

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ratios,

conformers,

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INTRODUCTION

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Ion mobility spectrometry (IMS) is a rapid and sensitive technique to separate

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complex mixtures of (bio)molecules prior to mass spectrometry (MS). Strong

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benefits arise between these two complementary methods as the information

53

about gas phase ions were significantly increased. IM is able to separate ions

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from small molecules (< 500 Da) up to megadalton protein complexes based

55

on their differential mobility through a buffer gas, thus, resolve ions that may

56

be indistinguishable by MS alone, or to determine structural information like

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rotationally averaged collision cross-sectional area (CCS). Moreover, IM-MS

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offers the chance to obtain insights into the conformational dynamics of a

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system, sharing unique means of characterizing flexibility and folding

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changes.1,2

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Quite a few publications deal with the power of IM-MS in positive ion mode for

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structural characterization and the study of conformational dynamics of

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proteins, the relationship between protein crystal structures and their CCS in

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the gas phase. As nonnative protein conformations are rarely isolated in

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solution, they are often stable in the gas phase in varying charge states.

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Differences in the structures of nonnative conformations in the gas phase are

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often large enough to allow different charge states and shapes enabling

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separation because of differences in their mobilities through a gas.2-4

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Recently, IM-MS has been recognized for having significant research/applied

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industrial potential and comprises multi-/cross-disciplinary areas of science,

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the applications and impact from decades of research are only now beginning

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to be utilized for small molecule species.5 CCS was used as additional

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identification point in pesticide analysis,6,7 IM-MS to investigate different

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conformers of 25-hydroxyvitamin D3,8 positional isomers of drug metabolites

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and glucuronide positional isomers,9,10 isomeric carotenoids,11 regioisomers of

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methylenedianiline,12 and protonation isomers.13,14 Interestingly, most of the

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described applications also used the positive ion mode for small molecules.

78

Only few publications deal with the negative ion mode, e.g. the structural

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characterization of the deprotonated leucine-enkephaline peptide anion or

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glucuronide positional isomers of naringenin as well as β-estradiol.10,15

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The genus Garcinia, familiy Guttiferae, exists in about 400 species in which

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extracts and pure isolates causes different forms of bioactivity such as anti-

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inflammatory,16 antimicrobial,17 anticancer,18 and antioxidant properties.19-21

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Stem and root bark extracts of G. buchananii are traditionally used in Africa to

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treat various conditions associated with HIV/AIDS such as herpes zoster,

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cryptococcal meningitis, tuberculosis and chronic diarrhea, diabetes and

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cardiovascular diseases as well as illnesses including diarrhea and pain.22-25

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In recent years, G. buchananii has attracted interest as its stem bark extract

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exhibit anti-diarrheal and antinociceptive as well as antioxidant effects.26-30

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Activity-guided fractionation of stem and root bark as well as

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phytochemical analysis of leaf extract of G. buchananii lead to the discovery

92

of

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polyisoprenylated benzophenones,32,33 xanthones,32,33 the seco-xanthone 1,5-

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dimethoxyajacareubin,32 the

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glycosides,30,35 (3α8'') and (3α6'')-linked biflavonoids, mainly biflavanones

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and -glycosides.30,32-38 We discovered that (2R,3S,2''R,3''R)-manniflavanone

97

(17, numbering refers to Table 1) is the major compound of G. buchananii

98

bark extract and also prominent in root and leaf extract and exhibits very

numerous

compounds

including

benzoyl

depsidone

4

glucuronosyl

glycerols,31

garcinisidone-G,32 flavanone-C-

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potent in vitro antioxidant activities.30,32,35 Biological tests revealed that 17

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protects proliferating skeletal muscle cells against oxidative stress, stimulates

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myotube formation and causes a relaxation of gut smooth muscle by

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modulating calcium mobilization including the influx by means of L-type

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calcium channels.39,40

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To date, neither chemical nor bioactivity investigations have been performed

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on extracts from branch bark, fruit rind, fruit flesh and seed of G. buchananii.

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Therefore, this study had four aims: (i) to build a database under UNIFI

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informatics platform from Waters Corporation consisting of retention time,

108

accurate m/z of the corresponding precursors, accurate m/z of the most

109

prominent fragment ions and CCS with 34 isolated pure reference compounds

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from G. buchananii, (ii) to demonstrate the “proof of concept” using various so

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far not investigated organs of G. buchananii, (iii) to demonstrate the potential

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for future applications by applying the database to other Garcinia species (G.

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mangostana, G. kola and G. cambogia), and (iv) to characterize deprotonated

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polyphenol anions in the gas phase yielding in more than one CCS value of a

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compound and, therefore, in a new characteristic compound parameter, the

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drift time distribution intensity ratio.

117 118

MATERIALS AND METHODS

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Chemicals. H2O for chromatographic separations was purified with a

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Milli-Q Gradient A10 system (Millipore, Schwalbach, Germany), and solvents

121

used were of HPLC-MS-grade (Merck, Darmstadt, Germany). Deuterated

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solvents were obtained from Euriso-Top (Saarbrücken, Germany).

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Plant Material. Leaves, stem and root bark from G. buchananii trees in

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their natural habitats in Masheshe, Karagwe, Tanzania. In addition, root bark,

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stem bark, branch bark, leaves and fruit from G. buchananii were collected

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from trees in their natural habitats in Kamagambo, Karagwe, Tanzania. Fruits

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were peeled to obtain fruit rind, flesh and seeds. All samples were dried and

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grounded to powder. A voucher specimen of the powders were deposited at

129

the University of Idaho Stillinger herbarium (voucher # 159918). Ethanolic G.

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kola seed extract (origin South Africa) was obtained from PepsiCo (New York,

131

USA) and G. mangostana fruit and fruit juice (Hanoju Bio Mangosteen Saft

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Premium 100%) were purchased in a local supermarket (Freising, Germany).

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The Hanoju Bio Mangosteen juice was freeze-dried (90 ml) to yield 10.7 g

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powder. The fruit was opened and separated into juice, fruit bark, flesh,

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seeds, fruit shaft and sepal and freeze-dried, respectively. G. cambogia whole

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fruit capsules (500 mg per capsule) as food supplement were purchased via

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Amazon.

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Extraction and Isolation. Extraction of G. buchananii seed powder and

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MPLC separation was performed as described for root, leaf and bark

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powder.30-33 14 fractions (M1-M14) were collected, concentrated under

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reduced pressure and freeze-dried. 12 was isolated from fraction M5 and 13

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from M6. Chromatography of MPLC fraction 5 was performed using a RP

143

column (10  250 mm, phenylhexyl, 5 μm; Phenomenex, Aschaffenburg,

144

Germany) as the stationary phase. The effluent (4.2 ml/min) was monitored at

145

270 nm and an isocratic gradient consisting of a mixture (53/47, v/v) of

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aqueous HCO2H (0.1% in H2O) and MeOH was applied. Collected fractions

147

were concentrated under reduced pressure and freeze-dried twice, affording 6

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(2R,3S,2''R,3''R)-GB-2 7''-O-β-D-glucopyranosyl-6''''-malonic acid (12, Figure

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1): colorless powder; UV (MeOH) λmax = 335, 288, 227, 206 nm; CD (MeOH,

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0.66 mmol/L): λmax (ΔƐ) = 351 (+1.4), 309 (-3.2), 283 (+11.6), 236 (-5.8), 232 (-

151

4.9), 217 (-11.6), 203 (-0.2); HRESIMS m/z 821.1568 [M-H]- (calcd for

152

C39H33O20, 821.1565); 1H NMR (DMSO-d6, 27 °C, 500 MHz) data, see Table

153

1; 13C NMR (DMSO-d6, 27 °C, 125 MHz) data, see Table 2.

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13 was obtained from M6 by semipreparative HPLC (4.2 ml/min, 270 nm)

155

using a ThermoHypersil ODS (10  250 mm, 5 μm; Kleinostheim, Germany)

156

column as the stationary phase and a mixture (62/38, v/v) of aqueous HCO2H

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(0.1% in H2O) and MeOH, and the MeOH content was linearly increased to

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41% within 20 min. Collected fractions were concentrated under reduced

159

pressure and freeze-dried twice, affording (2R,3S,2''R,3''R)-GB-1 7''-O-β-D-

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glucopyranoside (13, Figure 1): colorless powder; UV (MeOH) λmax = 338,

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289, 226 nm; CD (MeOH, 0.49 mmol/L): λmax (ΔƐ) = 369 (+2.0), 315 (-4.3),

162

287 (+9.1), 249 (-4.2), 243 (-4.0), 230 (-10.7), 226 (-10.3), 222 (+11.9), 204

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(+0.1), 202 (-0.7); HRESIMS m/z 719.1625 [M-H]- (calcd for C26H31O16,

164

719.1612);

165

Information (SI) Table S1;

166

Table S2 (SI).

167

Ultra

1H

NMR (DMSO-d6, 27 °C, 500 MHz) data, see Supporting

Performance

13C

NMR (DMSO-d6, 27 °C, 125 MHz) data, see

Liquid

Chromatography



Electrospray

168

Ionisation-Ion Mobility-Time-of-Flight Mass Spectrometry (UPLC-ESI-IM-

169

TOF MS).

170

Plant material (G. buchananii and mangostana, 5 mg each) were suspended

171

in a mixture of acetonitrile and water (1:1, v/v, 1 ml), vortexed for 1 min,

172

ultrasonicated (10 min) and centrifuged (5000 rpm, 5 min). The obtained

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extracts were membrane filtered and diluted to a final volume of 1 ml. Fruit

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and fruit juice extracts were prepared in a tenfold higher concentration.

175

Ethanolic G. kola seed extract was prepared in a concentration of 2 mg/ml

176

(acetonitrile/water 1:1, v/v). G. cambogia whole fruit capsule extracts were

177

prepared as described above (1 and 10 mg/ml) and, additionally, a solid

178

phase extraction (Chromabond C18ec, 1g, 60 Å, 45 µm, Macherey-Nagel,

179

Düren, Germany) with whole fruit capsule (1.2 g) was performed. The SPE

180

column was activated, loaded with the sample, washed with 1% acetonitrile

181

and flushed with MeOH (70 ml), the MeOH fraction collected, evaporated and

182

dissolved in MeOH (4 ml).

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Aliquots (8 µl) of the extracts were analyzed by means of UPLC-ESI-TWIMS-

184

TOF MS on a Waters Vion HDMS mass spectrometer (Waters, Manchester)

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coupled to an Acquity i-class UPLC system (Waters, Milford) equipped with a

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2 x 150 mm, 1.7 µm, BEH C18 column (Waters) consisting of a binary solvent

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manager, sample manager and column oven. Operated with a flow rate of 0.4

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ml/min at 45 °C, the following gradient was used for chromatography: starting

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with a mixture (10/90, v/v) of aqueous HCO2H (0.1% in H2O) and MeCN

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(0.1% HCO2H), the MeCN content was increased to 40% within 8 min, to 80%

191

within 1 min, to 90% within 2 min, to 100% within 1 min, kept constant for 1

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min, decreased to 10% within 1 min and finally kept constant for 1 min at 10%.

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Scan time for the HDMSe method was set to 1.0 sec. Analyses were

194

performed in ESI sensitivity mode using the following ion source parameters:

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capillary voltage 2.3 kV in negative mode and 3.0 kV in positive mode, source

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temperature 120 °C, desolvation temperature 550 °C, cone gas flow 50 l/h

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and desolvation gas 900 l/h. Data processing was performed by using UNIFI

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1.8 (Waters). All data were lock mass corrected on the pentapeptide leucine

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enkephaline (Tyr-Gly-Gly-Phe-Leu, m/z 554.2615, [M-H]-) in a solution (50

200

pg/µl) of ACN/0.1% HCO2H (1/1, v/v). Scan time for the lock mass was set to

201

2.0 s with an interval of 0.5 min. Calibration of the Vion in the range from m/z

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50 to 1200 was performed using a solution of the MajorMixTM (Waters). The

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UPLC and Vion systems were operated with UNIFITM software (Waters).

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Collision energy ramp for HDMSe was set from 20 to 30 eV. Further details of

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the Vion IMS qTof instrument and processing and detection parameters for

206

application of the database are listed in the SI (Table S3).

207 208

RESULTS AND DISCUSSION

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By means of UPLC-UV-ESI-TOF-MS we detected two biflavanone

210

glycosides as major constituents next to manniflavanone in G. buchananii

211

seed extract, therefore, compounds 12 and 13 were isolated and

212

characterized.

213

Compound 12 (Figure 1) was isolated as a colorless powder and

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showed a pseudomolecular ion peak m/z 821.1568 [M-H]- (calcd. 821.1565) in

215

the HR-ESI-MS (Figure S1, SI), corresponding to the molecular formula of

216

C39H34O20. The UV spectrum was very similar to reported biflavanones30,32-38

217

and the loss of 44 as well 162 Da in the high-collision energy mass spectrum

218

(MSe) was representative for a decarboxylation and hexose unit which yielded

219

the elemental composition of GB-2 or buchananiflavanone. However, further

220

characteristic mass fragments of m/z 447, 429, 419, 403, 269, and 125

221

indicated a GB-2 aglycone (Figure S2, SI).38

222

The 1H NMR spectrum of compound 12 (Figure S4, SI) in DMSO-d6 at

223

27 °C showed sharp signals and the typical signal doubling as known from

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literature for biflavananones,30,32,33,35,38 indicating rotational conformers in a

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ca. 3:1 ratio. The sets of aliphatic C-ring protons H-3 and H-2 as well as H-3''

226

and H-2'' coupling with each other were observed substantiating the GB-2

227

biflavananone aglycone structure. The coupling constants of J=12 Hz of these

228

protons verified their diaxial (trans) orientation. The COSY,

229

as well as 1H-13C HMBC NMR spectra (Figures S5-S8, SI) indicated the

230

presence of two carboxyl (δC 167.2, 168.1) and two carbonyl carbons (δC

231

195.5, 196.8, 197.9), 19 methine carbons, two methylene, and 14 further

232

carbons and were quite similar to that of (2R,3S,2''R,3''R)-GB-2 7''-O-β-D-

233

glucopyranoside.38 One methylene and two carboxyl groups could be

234

additionally detected which highlighted a malonic acid residue. The complete

235

assignments of 1H and

236

Tables 1 and 2. The fingerprint correlation in the 1H-13C HMBC spectrum

237

(Figure S8, SI) of both conformers between the proton H-3 and neighboring

238

carbon atom C-8'', C-8a'', and C-7'' demonstrated the intramolecular (38'')-

239

linkage of the two flavanone constituent units. The β-D-glucopyranosyl moiety

240

in both conformers was located at C-7'' as evidenced by the HMBC correlation

241

of the anomeric proton with C-7''. The couplings of the diastereotopic protons

242

H-6'''' to the carboxyl C-1''''' disclosed the linkage of the malonic acid unit to C-

243

6'''' of the β-D-glucopyranosyl moiety. The absolute configuration of 12 was

244

defined by comparison of its experimental and the published ECD curves of

245

(2R,3S,2''R,3''R)-manniflavanone 7‘‘-O-β-D-glucp and (2R,3S,2''R,3''R)-GB-2

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7‘‘-O-β-D-glucp.38 The results showed that the ECD curve of 12 (Figure S9,

247

SI) was consistent with the ECD spectrum of the corresponding glycosides as

248

well as aglycones.30,35,38 On the basis of these data, compound 12 was

13C

13C, 1H-13C

HSQC

NMR of the two conformers at 27 °C are given in

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assigned as (2R,3S,2''R,3''R)-GB-2 7''-O-β-D-glucopyranosyl-6''''-malonic acid,

250

a

251

biflavanone glucopyranosyl malonic acid derivative.

new

compound,

an

ent-naringenin-(C3C8'')-dihydroquercetin-linked

252

Compound 13 (Figure 1) was isolated as a colorless powder and

253

showed a pseudomolecular ion peak m/z 719.1613 [M-H]- (calcd. 719.1612) in

254

the HR-ESI-MS (Fig. S10, SI), corresponding to the molecular formula of

255

C26H32O16. 1 and 2 D NMR of the major conformer, CD and UV spectroscopy

256

as well as mass spectrometry (Figures S11-S17, SI) were in line to

257

(2R,3S,2''R,3''R)-GB

258

glucopyranoside which was earlier described in unripe fruits of Clusia

259

paralicula.41

1

as

well

as

(2R,3S,2''R,3''R)-GB-1

7''-O-β-D-

260

34 isolated and fully characterized reference compounds from G.

261

buchananii were analyzed by means of UPLC-ESI-TOF-MS on a VION IMS-

262

QTOF mass spectrometer in the negative ion mode to build a database under

263

UNIFI informatics platform consisting of retention time, accurate m/z of the

264

corresponding precursors, accurate m/z of the most prominent fragment ions

265

and CCS (Table 3). It is interesting to mention that by means of IM and the

266

resulting drift time the different compound classes could be clearly separated:

267

the three xanthones showed the lowest CCS with 143-174 Å2, followed by the

268

two benzoyl glucuronosyl glycerols as well as six flavanone-C/O-glycosides or

269

flavonol-C-glycoside with CCS of 179-185 Å2 or 199-206 Å2, respectively. The

270

11 biflavonoids were evaluated with 219-225 Å2, the four polyisoprenylated

271

benzophenones with 249-250 Å2 and the four biflavanone–glycosides with

272

256-263 Å2. Only four compounds do not belong to one of the six compound

273

classes, the seco-xanthone 1,5-dimethoxyajacareubin (27) with a CCS of

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190.3 Å2, the depsidone garcinisidone-G (30) (183.2 Å2), the flavanone-

275

chromone biflavonoid preussianone (15) (194.1 Å2) and the diglycoside

276

apigenin-8-C-β-D-glcp-2''-O-L-rhap (9) (227.3 Å2) (Figure 2).

277

To check the stability of the CCS in a matrix sample, the intra- as well

278

as interday precision of the CCS of six selected compounds in a G.

279

buchananii bark extract was analyzed over a concentration range of three

280

magnitudes (Table 4). Additionally, the influence on the CCS area of different

281

solvents and formic acid ratio used for the chromatography, and therefore of

282

the ionization process, was investigated. In summary, the standard deviation

283

(SD) ranged between 0.1 and 0.9% and the average over 129 data points is

284

0.5%, which represents excellent precision of the CCS area. Consequently,

285

the CCS value is a compound specific parameter like m/z or fragments and

286

independent from the chromatographic conditions which could also be

287

combined with direct MS infusions. This is a very big advantage compared to

288

the retention time of a compound which changes with other column chemistry,

289

length, column age, flow rate, temperature as well as solvents and buffers.

290

In a next step, the established database was applied on different

291

organs of G. buchananii, including stem, branch, fruit and root bark, as well as

292

leaves, seeds and fruit flesh and the peak targeting is based on four fulfilled

293

criteria (Table 3): retention time (±0.12 min), accurate m/z of the

294

corresponding precursors (± 10 ppm), accurate m/z of the most prominent

295

fragment ions (± 10 mDa) and CCS (± 1.2 %). To the best of our knowledge,

296

branch and fruit bark as well as seeds and fruit flesh have never been

297

investigated so far. 22 compounds were detected in stem bark, mainly

298

biflavanoid

glycosides

and

their

corresponding

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flavonoid

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glycosides, polyisoprenylated benzophenones as well as the xanthone

300

jacareubin (29) (Table S4, SI). In root and branch bark the same number and

301

a very similar composition could be verified like in stem bark, with just minor

302

differences in 15 as well as ulmoside A (5), helicioside A (7) and isoprenyl

303

tetra hydroxyxanthone (28) (Table S5-S6, SI). In leaves, 25 compounds could

304

be identified, similar components to the barks mentioned so far, but no

305

xanthones instead 30 as well as the two benzoyl glucuronosyl glycerols (3, 6)

306

(Table S7, SI). The analysis of the seeds showed 16 identified compounds

307

and to be a rich source of isogarcinol (34) and mainly biflavanoid glycosides

308

and their corresponding aglycones as well as flavonoid glycosides (Table S8,

309

SI). It is interesting to mention that among others the edible part of G.

310

buchananii fruit contains the most components, namely 26. All compound

311

classes

312

benzophenones and the recently in leaves identified 1-O-4-hydroxybenzoyl-

313

3-O-α-D-glucuronosylglycerine (3). The fruit rind extract revealed 25

314

compounds with similar composition to the fruit in which garcicowin C (32),

315

garcinol (33) and 15 could not be observed, but found 27 and quercetin-6-C-

316

β-D-glcp (1).

are

represented,

highlighting

several

polyisoprenylated

317

In summary, the extracts of different organs of G. buchananii tree

318

principally showed a similar chemical composition pattern, as some

319

compounds are ubiquitous, like e.g. GB-1, GB-2, manniflavanone, -O-

320

glycoside, buchananiflavanone, isogarcinol but there were also clear

321

differences. For example: 3, 9, 10, 23 and 25 are markers for leaves,31,33 fruit

322

and fruit rind. Unique organ markers are euxanthone (26) for fruit, 6 and 30 for

323

leaves, 12 for seeds, 27 for fruit bark and 28 for branch bark. For the proof of

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324

principle, the database constructed with compounds isolated from seeds,

325

leaves, stem and root bark was applied on the corresponding extracts and on

326

additional organs not investigated so far, namely, branch bark, fruit rind and

327

fruit extract and yielded fast and very reliable results based on four fulfilled

328

criteria.

329

To strengthen the benefits of the constructed database, we applied it to

330

other Garcinia species, namely, G. kola (seeds), G. mangostana (juice, pulp,

331

seeds, sepal, fruit shaft, exo- and mesocarp) and G. cambogia (whole fruit

332

capsules). 14 compounds could be identified in an ethanolic extract of G. kola

333

seeds (Table S11, SI), whereas 7, (2R,3S,2‘‘S)-buchananiflavanon (11), 9, 26,

334

32-34 could be detected for the first time. The application of the database on

335

different organs of G. mangostana revealed six different compounds, thereof

336

five (3, 4, 6, 10 and 29) were identified for the first time in this species (Table

337

S12, SI). In contrast, the analysis of the whole fruit capsules of G. cambogia,

338

also additionally enriched via SPE, did not show any hit. This is surprising as

339

at least 33 along with other polyisoprenylated benzophenones as well as

340

xanthones have already been described in G. cambogia fruits.42

341

Positive ESI in combination with TWIM-MS could resolve or indicate

342

protomers of small molecules or different confomers or proteins,2,13,14 in

343

contrast, in the negative ESI mode such investigations are rarely found, like

344

deprotomers of ortho, meta, and para hydroxybenzoic acid and a

345

deprotonated porphyrin derivative.43,44

346

By means of NMR spectroscopy we observed rotational conformers in

347

solution for compounds 8 and 11-25,30,32,33,35,38 and, therefore, had a closer

348

look on the TWIM-MS data of these compounds. Exemplified for 17 (Figure

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3A), two different drift time species could be detected, which showing identical

350

retention time, accurate mass and fragments, but a CCS of 224.1 and 309.1

351

Å2 (Table 3) and an intensity ratio of 95.3/4.7%. To clarify if the detected drift

352

time species could be attributed to either deprotomers, rotational isomers or in

353

general to gas phase conformers, the nine fold methylated analogue of 17

354

was analyzed, only highlighting one drift time species and proposing that the

355

hydrogen bond stabilized rotational isomers 17a and 17b were transferred

356

from solution to gas phase (Figure 3B).

357

positive ion mode revealed very similar CCS values and, especially, a drift

358

time intensity distribution of 94.7/5.3%, which further strengthen our

359

suggestion. We have exemplary checked the intra- and interdaily stability of

360

the intensity of the drift time distribution of the manniflavanone (17)

361

conformers under our conditions as well as different solvents listed in Table 5.

362

Intraday a ratio of 95.2/4.8%±0.1% RSD, interday of 96.0/4.0%±0.2%RSD

363

and with different solvents and acid concentration of 96.4/3.6%±0.5% RSD

364

was determined. The mean over all (21) analyses is 96.0/4.0±0.6%RSD which

365

seems to be quite robust. Other compounds showed comparable data. The

366

data for the diverse drift time intensity distributions is summarized in Table 6.

367

With the exception of 8 and 12, basically, the denser drift time species is

368

dominating, at least 72% for 2, normally >80-90%. It is very interesting to

369

mention, that vice versa for the corresponding glycosides 8 and 12 the higher

370

CCS is dominating. This observation was further strengthened by the

371

glycosides 11 (data not shown) and 13 which showed the same shift (Figure

372

4). We propose to use all CCS values of one compound, and also the intensity

373

ratio of the conformers as a new characteristic compound parameter next to

15

UPLC-ESI-TWIMS of 17 in the

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m/z, fragments, Rt and CCS in compound identification/screening/database

375

applications to reduce false positives and strengthen the identification. By

376

plotting the mass vs. the CCS values of the major rotational conformers (in

377

case of two or more drift time species) of all compounds the strong linear

378

correlation between molecular weight and CCS can be depicted which is in

379

line with literature (Figure 2).5,7 By trend, increasing molecular weight reflects

380

in increasing CCS whereas to keep in mind that this plot was performed with

381

the major conformer. In TWIMS, bigger ions roll over the wave, whereas

382

smaller ions “surfing” on the traveling wave exciting the gas-filled mobility cell

383

earlier. By comparing manniflavanone (17) with its 7’’-O-glucoside (8), and

384

therefore 162 Da heavier, the CCS of the major conformers (224.3  261.7

385

Å2) seems to follow the general trend with increasing molecular weight

386

(Figure 5). But if we directly compare the drift time distributions of the first and

387

the second conformers the CCS values are decreasing (309.0  261.7 and

388

224.3  212.6 Å2) with increasing m/z, respectively, or more precisely, by

389

glycosylation (Figure 5). The same results could be determined for 21 and 18

390

as well as their corresponding glycosides 11 and 13 and even more for 12, the

391

glucopyranosyl malonic acid derivative of GB-2 (Table 5). Further studies on

392

the CCS values of the isomers of manniflavanone 14, 17 and 19 revealed that

393

the major drift time species of these three compounds are sharing the same

394

CCS value of 224 Å2 although the discrimination is possible via the minor

395

conformer (no minor conformer for 14, 326.2 for 19 and 309.0 Å2 for 17) and

396

therefore enables the identification of the isomers in plant extracts by means

397

of direct infusion experiments without column chromatography (Table 3 and

398

5). Another example for the influence of the stereochemistry on the CCS

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value is the comparison of (2R,3R)-taxifolin-6-C-β-D-glucopyranoside (2) and

400

its diastereomer ulmoside A (5), differing in the configuration of the agycone.

401

Interestingly, both stereoisomers showed two drift time species, probably

402

gaseous conformers, whereas the CCS of the major conformer of 2 and of the

403

minor conformer of 5 is bigger and the values differed in 4 Å2, each. Also the

404

intensity ratios of the two drift time species differed clearly. Moreover, 2

405

highlighted the possibility of a third drift time species (Figure 6). Again, the

406

identification of 2 or 5 in plant extracts would be possible by means of direct

407

infusion experiments without column chromatography and TWIMS with the

408

different CCS values and intensity ratios of the drift time species (Table 3 and

409

5).

410

Although literature and our own studies showed a high correlation between

411

m/z and CCS trusting on m/z alone to predict CCS is not adequate as many

412

ions have the same m/z but different 3D structure as demonstrated for the

413

stereoisomers (2 and 5), and, especially glycosylation increases the molecular

414

weight by 162 Da but yielded in a more compact structure and smaller CCS

415

(8, 11-13). We demonstrated that the CCS value is a robust and precise

416

compound parameter and to use all CCS values of one compound, and

417

beyond that propose the intensity ratio of the conformers as a new

418

characteristic compound parameter next to m/z, fragments, Rt and CCS. The

419

constructed database could be used as a fast, robust and reliable

420

dereplication tool and the determined CCS values could be used in public,

421

growing databases.

422 423 424

ACKNOWLEDGEMENT

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425 426

We thank PepsiCo (New York, USA) for providing the G. kola seed extract.

427

ASSOCIATED CONTENT

428 429 430

Supporting Information

431

UPLC-UV-ESI-TOF MS, MSe, CD, 1- and 2D NMR spectra of compound 12

432

and 13, and Tables summarizing all COSY, HSQC and HMBC couplings of

433

13, detailed instrument parameters of the Vion IMS qTOF and processing

434

options of the database as well as compound identification in the different

435

plant organs of G. buchananii and G. mangostana and G. kola seed extract

436

using the in house database. This information is available free of charge via

437

the Internet at http: //pubs.acs.org.

438

Notes

439

The authors declare no competing financial interest.

440 441

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Table 1. 1H NMR data of the major and minor conformers of 12 at 27 ℃ (DMSO-d6, 500 MHz) δH in ppm (multiplicity, J in Hz) H no. Flavanone-unit (I) H no. Flavanone-unit (II) aA bB aA bB I-2 5.48 (d, 12.1) 5.71 (d, 12.1) II-2‘‘ 4.88 (d, 11.2) 4.91 (d, 11.9) I-3 5.09 (d, 12.1) 4.48 (d, 12.1) II-3‘‘ 4.25 (d, 11.2) 3.99 (d, 11.9) I-6 5.89 (d, 2.1) 5.84 (d, 1.6) II-6‘‘ 6.22 (s) 6.40 (s) I-8 5.77 (d, 2.1) 5.82 (d, 1.7) II-2‘‘‘ 6.81 (d, 2.0) 6.85 (d, 1.5) e I-2‘ 7.23 (d, 8.2) 7.09 (d, 8.3) II-5‘‘‘ 6.65 (d, 8.2) e I-3‘ 6.61 (d, 8.2) 6.74 (d, 8.3) II-6‘‘‘ 6.68 (dd, 2.0, 8.3) I-5‘ 6.61 (d, 8.2) 6.74 (d, 8.3) I-6‘ 7.23 (d, 8.2) 7.09 (d, 8.3) I-5-OH 12.08 (s) 11.97 (s) δH in ppm (multiplicity, J in Hz) H no. Glucopyranoside-unit H no. Malonic acid-unit aA bB aA bB d 1‘‘‘‘ 4.73 (d, 7.0) 5.02 (d, 7.8) 2‘‘‘‘‘ 3.34 (s) 3.35 2‘‘‘‘ 3.33c 3.12c 3‘‘‘‘ 3.33c 3.28c c 4‘‘‘‘ 3.17 3.13c 5‘‘‘‘ 3.67 (m) 3.67d e 6‘‘‘‘α 4.06 (dd, 7.4, 11.9) e 6‘‘‘‘β 4.37 (d, 10.9) Sets A and B are respectively in an intensity ratio of (~1:0.3) in 12. Additional OH signals as brs for 4', 3''' and 4''' at 11.71, 8.96, 5.75 and 4.70 ppm. aA represents major conformer of 12 at 27 ℃, bB represents minor conformer of 12 at 27 ℃, coverlapped with H O peak, assigned with COSY and HSQC 2 experiments. doverlapped with major signal, assigned with HSQC experiments. e not assignable, probably overlapped with signals of major conformer.

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Table 2. 13C NMR data of the major and minor conformers of 12 at 27 ℃ (DMSO-d6, 125 MHz) δC in ppm (C-Type) C no. Flavanone-unit (I) C no. Flavanone-unit (II) aA bB aA bB I-2 82.0 (CH) 81.3 (CH) II-2‘‘ 83.1 (CH) 82.8 (CH) I-3 46.7 (CH) 47.6 (CH) II-3‘‘ 72.1 (CH) 72.2 (CH) I-4 196.8 (C) 195.5 (C) II-4’’ 197.9 (C) 197.9 (C) I-4a 101.1 (C)c 101.4 (C) II-4a’’ 101.9 (C) 101.4 (C)c I-5 163.6 (C) 163.7 (C) II-5’’ 161.9 (C) 162.2 (C) I-6 95.9 (CH) 96.0 (CH) II-6’’ 96.2 (CH) 96.2 (CH) I-7 166.3 (C) 166.2 (C) II-7’’ 162.9 (C) 162.3 (C) I-8 94.8 (CH) 94.7 (CH) II-8’’ 102.4 (C) 102.5 (C) I-8a 162.8 (C) 162.7 (C) II-8a‘‘ 158.9 (C) 159.4 (C) I-1‘ 127.1 (C) 127.9 (C) II-1‘‘‘ 128.0 (C) 127.9 (C) d I-2‘ 129.8 (CH) 128.8 (CH) II-2‘‘‘ 115.1 (CH) 115.7 (CH)e I-3‘ 115.0 (CH) 114.8 (CH) II-3‘‘‘ 144.8 (C) 145.0 (C) I-4‘ 157.7 (C) 157.7 (C) II-4‘‘‘ 145.6 (C) 145.1 (C) I-5‘ 115.0 (CH) 114.8 (CH) II-5‘‘‘ 115.2 (CH)d 115.2 (CH)e I-6‘ 129.8 (CH) 128.8 (CH) II-6‘‘ 117.8 (CH) 119.0 (CH) δC in ppm (C-Type) C no. Glucopyranoside-unit C no. Malonic acid-unit aA bB aA bB f 1‘‘‘‘ 99.9 (CH) 99.7 (CH) 1‘‘‘‘‘ 167.2 (C) 2‘‘‘‘ 72.9 (CH) 73.5 (CH) 2‘‘‘‘‘ 41.9 (CH2) 41.6 (CH2) f 3‘‘‘‘ 75.9 (CH) 76.9 (CH) 3‘‘‘‘‘ 168.1 (C) 4‘‘‘‘ 69.9 (CH) 70.2 (CH) 5‘‘‘‘ 73.8 (CH) 74.0 (CH) f 6‘‘‘‘ 64.0 (CH2) *C-type was deduced from HSQC in combination with 1H experiment. aA represents major conformer at 27 ℃, bB represents minor conformer at 27 ℃, c,d,e

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interchangeable, fnot observable, probably overlapped

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Table 3. The database under UNIFI informatics platform consisting of retention time, accurate m/z of the corresponding precursors,

accurate m/z of the most prominent fragment ions and CCS of 34 isolated reference compounds of G. buchananii. compound

Quercetin-6-C-β-D-glcp (2R,3R)-Taxifolin-6-C-β-D-glcp (2R)-1-O-4-hydroxybenzoyl-3O-α-D-glucuronosyl-glycerol (2R,3R)-Aromadendrin-6-C-β-D-glcp Ulmoside A ((2S,3S)-taxifolin-6-C-β-Dglcp) (2S)-1-O-4-hydroxy-3-methoxybenzoyl-3O-α-D-glucuronosyl-glycerol Helicioside A (2R,3S,2''R,3''R)-Manniflavanone-7''-O-βD-glcp Apigenin-8-C-β-D-glcp-2''-O-L-rhap Vitexin (2R,3S,2''R,3''R)-GB-2 7''-O-β-D-glycp (2R,3S,2''R,3''R)-GB-2 7''-O- β-D-glcp-6''''malonic acid (2R,3S,2''R,3''R)-GB-1 7''-O-β-D-glycp (2R,3S,2''S,3''S)-Manniflavanone (2''R,3''R)-Preussianone (2R,3S)-Buchananiflavonol

nr

expected RT (min)

1

1.46

expected neutral mass (Da) 464.0955

2 3

1.47 1.84

466.1111 388.1006

4 5

1.86 1.86

450.1162 466.1111

6

1.95

418.1111

7 8

2.62 3.24

450.1162 752.1589

9 10 11 12

3.48 3.59 3.62 4.07

578.1636 432.1056 736.1639 822.1643

13 14 15

4.38 4.67 5.31

720.1690 590.1060 480.0693

16

5.10

588.0904

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expected fragment (m/z)

adduct

observed CCS (Å2)*

151.0037, 343.0459, 345.0616, 367.0459, 373.0565 167.0350, 345.0616, 375.0722 137.0244, 211.0612, 249.0616, 267.0722 301.0718, 329.0667, 359.0772 167.0350, 301.0718, 329.0667, 331.0823, 359.0772 108.0217,152.0115

-H

206.4

-H -H

198.6 177.8

-H -H

205.7 194.8

-H

185.2

259.0612, 269.0455 151.0037, 437.0878, 445.0565, 463.0671 293.0455, 311.0561, 413.0878 283.0612, 311.0561, 341.0667 403.0823, 429.0616, 447.0722, 447.0722, 429.0616, 777.1672

-H -H

205.5 263.3

-H -H -H -H

227.3 196.4 259.6 256.9

431.0772, 557.1089, 579.0933 435.0722, 445.0565, 463.0671 149.0244, 217.0142, 341.0667, 461.0514, 311.0561, 435.0722, 437.0878, 445.0565, 461.0514, 463.0671

-H -H -H

256.4 224.3 194.1

-H

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(2R,3S,2''R,3''R)-Manniflavanone (2R,3S,2''R,3''R)-Manniflavanone, conformer (2R,3S,2''R,3''R)-GB-2 (2R,3S,2''R,3''R)-Isomanniflavanone (2R,3S,2''S)-Buchananiflavanone (2R,3S,2''R,3''R)-GB-1 (2R,3S,2´´S)-GB-2a (2R,3S)-Morelloflavone (2R,3S,2''S)-GB-1a (2R,3S)-Volkensiflavone Euxanthone 1,5-Dimethyoxyajacureubin 2-Isoprenyl-1,3,5,6-tetrahydroxyxanthone Jacareubin Garcinisidone G Paucinone C Garcicowin C Garcinol Isogarcinol

17a 17b

5.10 5.10

590.1060 590.1060

18 19

5.77 6.19

574.1111 590.1060

20

6.45

574.1111

21

6.51

558.1162

22 23 24 25 26 27 28 29

7.09 7.20 8.02 8.09 9.39 9.59 9.77 9.85

558.1162 556.1006 542.1213 540.1056 228.0423 356.1260 328.0947 326.0790

30 31 32 33 34

9.90 10.30 11.30 11.64 12.69

358.1053 634.3506 600.3451 602.3607 602.3607

285.0405, 435.0722, 463.0671 435.0722, 437.0878, 463.0671, 479.0620 296.0326, 419.0772, 447.0722 285.0405, 435.0722, 463.0671, 445.0565 285.0405, 311.0197, 421.0929, 447.0722 269.0455, 296.0326, 403.0823, 431.0772 269.0455, 295.0248, 431.0772 401.0667, 403.0823, 429.0616 295.0248, 389.1031, 415.0823 151.0037, 387.0874, 413.0667 210.0322 108.0217, 123.0452, 231.0663, 272.0326 267.0299, 293.0455, 307.0612, 309.0405 137.0244, 176.0843, 298.0847 109.0295, 497.3272 329.2122, 409.1657 465.3374, 481.3323, 555.3480 108.0217, 109.0295, 465.3374

* CCS for major conformer

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-H -H

224.2 309.1

-H -H

222.4 224.5

-H

221.9

-H

224.9

-H -H -H -H -H -H -H -H

220.4 218.5 220.9 219.1 143.2 190.3 169.9 173.6

-H -H -H -H -H

183.2 249.8 249.4 249.2 248.6

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Table 4. CCS determination of the major conformer of six selected compounds in G. buchananii bark extract and solvents used. analysis

compoud intradaya

CCS expected CCS observed 1 2 3 4 5 6 SD [%]

2 198.6 200.5 199.9 199.7 198.5 197.3 196.8 0.8%

21 224.9 226.1 225.3 224.7 223.7 223.1 223.4 0.6%

18 222.4 224.1 223.7 223.3 222.4 222.4 222.6 0.5%

17 224.2 224.9 224.2 224.5 224.0 222.9 223.2 0.4%

20 221.9 222.8 219.5 221.3 221.8 222.2 221.3 0.6%

34 248.7 250.5 250.4 250.2 249.9 248.5 248.8 0.6%

221.9 223.0 221.5 222.6 222.0 222.7 0.9%

220.6 220.4 220.1 221.0 220.4 220.5 0.7%

248.9 247.6 248.2 248.5 248.6 249.9 0.3%

interdayb

CCS observed 1 2 3 4 5 6 SD [%]

198.6 198.8 199.4 199.6 199.1 198.0 0.3%

223.4 223.5 223.7 223.8 223.1 223.8 0.7%

221.9 222.1 222.3 221.3 222.0 222.1 0.3%

concentrationsc

0.1 µl 1.0 µl 10.0 µl SD [%]

CCS observed 1 2 3 4 5 6 7 SD [%] run 1 2

n.d. 198.8 198.7 0.1%

199.0 198.0 197.2 198.4 196.9 196.7 198.4 0.6%

226.1 225.1 223.7 0.5%

224.2 224.5 223.7 223.2 224.0 223.6 224.1 0.5%

223.9 222.6 222.4 0.5%

223.9 223.9 224.2 0.1%

different

solventse

222.6 221.6 220.5 220.0 220.8 220.4 222.1 0.8%

223.7 224.0 223.1 221.9 222.7 222.2 223.3 0.7%

A 0.1% FA 0.1% FA

n.d. 222.1 221.8 0.1%

221.5 222.1 219.8 219.9 220.3 220.3 222.5 0.7% B ACN MeOH

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n.d. 249.7 248.9 0.4%

250.3 248.5 248.6 247.5 250.3 246.4 249.5 0.6%

Journal of Agricultural and Food Chemistry

3 4 5 6 7

H 2O 1% FA 1% FA H 2O 1% FA

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ACN ACN 1% FA in ACN MeOH 1% FA in MeOH

Standard Deviation, SD [%]; a six injections; b six injections 11 days later; c 0.1, 1 and 10 µl injections; d n.d. not detected; e the following solvents were used.

Table 5. CCS and ratio of CCS of selected compounds. compound ESI neg

CCS peak 1

response peak 1

ratio peak 1 %

CCS peak 2

response peak 2

ratio peak 2 %

21 18 23 4 17 22 20 16 8 19 12 1 5 2

223.7 222.4 218.9 205.1 224.3 221.1 221.8 221.1 212.6 223.6 205.1 204.8 194.3 198.7

2694701 6285892 1459946 1614750 7796276 583662 2495813 905930 122314 2086970 31488 1002479 282444 3499230

94.9 86.1 92.8 97.1 95.3 91.3 95.5 90.2 2.6 98.3 11.1 94.9 80.6 72.3

336.8 330.3 300.3 289.1 309.0 320.7 303.7 319.3 261.7 326.2 258.2 283.0 287.5 283.3

144546 1012608 113727 48675 382972 55604 117994 98850 4633952 35391 251342 54242 68039 1338972

5.1 13.9 7.2 2.9 4.7 8.7 4.5 9.8 97.4 1.7 88.9 5.1 19.4 27.7

ESI pos 17

226.37

5143482

94.7%

310.95

287531

5.3%

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FIGURE LEGENDS Figure 1. Chemical structures of compounds 12 and 13. Figure 2. Correlation between mass of G. buchananii compounds and the TWIMS-derived CCS values. Figure 3. Drift time distributions of (A) 17a and 17b and (B) nonamethly-17. Figure 4. Drift time distributions of (A) 13a and 13b, (B) 12a and 12b and (B) 8a and 8b. Figure 5. Drift time distributions of (A) 17a and 17b and (B) 8a and 8b. Figure 6. Drift time distributions of (A) 2a and 2b and (B) 5a and 5b.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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for TOC only

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