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Profiling and quantification of regioisomeric caffeoyl glucoses in berry fruits Maria Alexandra Patras, Nikolai Kuhnert, Rakesh Jaiswal, and Gordon J. McDougall J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02446 • Publication Date (Web): 13 Oct 2017 Downloaded from http://pubs.acs.org on October 17, 2017

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Journal of Agricultural and Food Chemistry

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Profiling and quantification of regioisomeric caffeoyl glucoses in berry

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fruits

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Maria A. Patras a, Rakesh Jaiswal a1 , Gordon J. McDougallb and Nikolai Kuhnerta* a

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Department of Life Sciences & Chemistry, Jacobs University Bremen, Campus Ring 1, 28759, Bremen, Germany Present address: Doehler Group SE, Riedstrasse, 64295, Darmstadt, Germany

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b

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Scotland, UK

Environmental and Biochemical Sciences Group, James Hutton Institute, Invergowrie, Dundee, DD2 5DA,

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*

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Ring 1, 28759, Bremen, Germany. Tel. 0049 421 200 3120 Email: [email protected]

Correspondence to Nikolai Kuhnert, Department of Life Sciences & Chemistry, Jacobs University Bremen, Campus

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Abstract:

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Based on a recently developed tandem MS based hierarchical scheme for the identification of

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regioisomeric caffeoyl glucoses, selected berry fruits were profiled for their caffeoyl glucose

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ester content. Fresh edible berries profiled included strawberries, raspberries, blueberries,

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blackberries, red and black currants, lingonberries, gooseberries, cranberries, juice of

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elderberries, goji berries, chokeberries, cranberries, açai berries, sea buckthorn berries,

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Montmorency sour cherries, and pomegranate were investigated. 1-caffeoyl glucose was found to

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be the predominant isomer in the majority of samples with further profiling revealing the

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presence of additional hydroxycinnamoyl glucose esters and O-glycosides with p-coumaroyl,

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feruloyl and sinapoyl substituents. A quantitative LC-MS based method was developed and

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validated and all caffeoyl glucose isomers were quantified for the first time in edible berries.

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1. INTRODUCTION

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The impact of the diet on human health has been highlighted by numerous studies in the past

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decades. Diets rich in fruits and vegetables have been found to be associated to a multitude of

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health benefits, a fact which has drawn the consumer attention towards the so-called “functional”

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foods or dietary supplements rich in nutraceuticals.1 Berries in particular are gaining increasing

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popularity as rich sources of nutraceuticals as new research investigating their health promoting

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properties is constantly accumulating.2-8 Berries are only seasonally present in the human diet in

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their fresh form; however, they are widely available all year round in their processed forms such

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as frozen or dried fruits, juices, purees, jams, etc.,9 and more frequently in recent years in the

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form of extracts to be used as dietary supplements.3 The most notable health benefits of berries

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are preventative effects on degenerative and cardiovascular diseases, cancer and ageing.10-12

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These properties are mostly attributed to the high levels of phenolics in berries, which act through

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complex mechanisms like gene expression modulation and enzyme induction.4,5,13-16

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The two major classes of phenolics reported in berries are flavonoids and phenolic acids.

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Flavonoids appear as three main subclasses; anthocyanins (e.g. cyandin, delphinidin and malvidin

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derivatives) flavonols (e.g. quercetin, myricetin and kaempferol derivatives) and flavanols

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(catechin and epicatechin derivatives). Phenolic acids are dominated by hydroxycinnamic acids

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(HCAs) (e.g. caffeic acid, ferulic acid, p-coumaric acid derivaties) being present either in their

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free form or connected to various polyols like quinic and shikimic acids and simple or complex

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carbohydrates.17-19 The most abundant and well investigated derivatives of hydroxycinnamic

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acids (HCAs) are the hydroxycinnamoyl quinates, referred to as chlorogenic acids (CGAs),

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which have shown a multitude of beneficial health effects.20,21 Although not as abundant as the

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CGAs, conjugates of HCAs with carbohydrates have also been frequently reported in various



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vegetables, fruits and in particular in berries, although compounds have never been identified to

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regioisomeric levels.22,23

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distinguished for each HCA-hexose combination, namely O-esters, C-glycosides and O-

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glycosides. Representative structures are shown in Figure 3.

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For each hexose O-ester of a particular HCA, five regioisomers are possible, each existing in

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equilibrium as a pair of α- and β- anomers. Multiple studies have reported the presence of various

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isomeric HCA-hexose conjugates, observed by LC−MS in a wide variety of plants.23-36 However,

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these are mostly reported without any assignment of regio- and stereochemistry and without any

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quantification, as isolation of individual isomers from complex food matrices is a demanding

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process, frequently failing to provide pure compounds fit for structure elucidation.

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Recently, Jaiswal et al. introduced a hierarchical scheme using pure synthetic standards, which

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allowed for the individual identification of all ten caffeoyl glucose (CGs) isomers by a HPLC-

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tandem-MS based technique that yielded unique fragment spectra for all five regio-isomeric

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CGs.37 This approach allows unambiguous assignment of each individual isomer in any food

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source based on their retention time and distinct MS fragmentation patterns, omitting compound

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isolation. Although no biological data are as yet available for CGs, due to lack of authentic

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reference standards and difficulties in compound isolation and characterization, we assume that

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CG derivatives share many beneficial health properties of their CA relatives. It can be expected

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that HCs share similar gut micro floral metabolic pathways being substrates to bacterial esterases

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and glycosidases producing free hydroxycinnamic acids that undergo further bacterial and liver

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metabolism producing identical bioactive metabolites if compared to CGAs.20, 21

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This in depth contribution investigates the identity of HCA-hexose conjugates with a particular

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focus on caffeoyl glucoses (CGs) present in a series of berries including strawberries, raspberries,

Three structural subclasses of constitutional isomers are to be


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blueberries, blackberries, red currants, black currants, lingonberries and gooseberries, and

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provides both qualitative profiling data as well as quantitative data on this so far neglected class

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of dietary constituents. With their very frequent abundance in dietary plants (anecdotal note: we

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have observed HC derivatives in more than 50% of 800 plant extracts investigated in our research

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group over the last decade) and potential health benefits in mind HCs form an important class of

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dietary secondary metabolites requiring scientific attention.

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2. MATERIALS AND METHODS:

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2.1 Chemicals and reagents:

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LC-MS grade methanol was purchased from Carl-Roth (Karlsruhe, Germany). Formic acid and

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Hesperetin were purchased from Sigma-Aldrich (Steinheim, Germany).

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2.2. Sample preparation:

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2.2.1. Extraction:

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Fresh plant material of various commercial origins was purchased from local markets in different

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countries (e.g. Germany, Italy & Romania). Some berries were also obtained from a local garden

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(Bremen, Germany), as mentioned in Table 2. 200g of each fresh plant sample was subjected to

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homogenization with a food blender, followed by immediate freezing and subsequent freeze-

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drying. The resulting freeze dried plant material was further used for extraction.

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Five strawberry samples from different cultivars (Adria, Anoi, Elsanta, Romina and Sveva

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cultivars) were received in freeze-dried form from the James Hutton Institute (Scotland).

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0.5g of each freeze dried plant material was extracted with 10mL of methanol/water 70/30 (v/v)

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by 15 minutes of initial sonication followed by stirring at room temperature for 12 hours. After

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extraction, the suspensions were centrifuged for 10 minutes at 4400 rpm (3000 g). The 5 ACS Paragon Plus Environment


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supernatant was filtered through a CHROMAFIL® Xtra PTFE syringe filter with a pore size of

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0.45µm (Macherey Nagel, Düren, Germany) and transferred into a glass test tube. The solid

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residue was washed twice with 10mL of methanol/water (70/30 v/v) and centrifuged after each

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washing step. The supernatants resulting from each washing step were as well filtered through the

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PTFE syringe filter and combined with the crude extract into the glass test tube. The solvent in

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the glass test tube was removed under N2 gas using a TurboVap concentration work station

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(BIOTAGE, Uppsala, Sweden). In order to ensure complete removal of water, the crude extracts

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were subsequently freeze dried for 12 hours. The mass of each extract was recorded.

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2.2.2. SPE purification:

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The crude dry extracts were subjected to SPE pre purification. Chromabond C18ec 15mL/2000

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mg cartridges (Macherey Nagel, Düren, Germany) were used as stationary phase. Cartridge

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conditioning was performed by washing with 20mL of methanol followed by 20 mL of water.

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The crude extracts were dissolved in water in order to be loaded on the SPE cartridge, the amount

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of water depending on the mass of each extract, such that the total amount of extract contained in

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1mL (volume loaded on each cartridge) of water solution did not exceed 200mg. The washing

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was performed with 20mL of water followed by 20mL of 20% methanol/water (v %). The 20%

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methanol fraction was further used for HPLC-MS analysis. The SPE method was optimized using

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a methanolic extact of Ilex Paraguariensis (Maté) which was reported to contain all 10 isomers of

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CG. Sample concentration prior to the HPLC-MS analysis was performed by total evaporation of

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the solvent in the 20% methanol fraction, followed by dissolution of each individual sample into

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a specific amount 70% MeOH. Optimization of the sample concentration was performed such

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that the compounds of interest showed an intensity fitting into the linearity range of the

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calibration curve. 6 ACS Paragon Plus Environment

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2.2.3 Sample preparation from directly pressed juices:

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Commercially available direct juices of berries (see Section 3.1) were purchased from local

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stores in Bremen, Germany. Each sample was filtered through a CHROMAFIL® AQ Polyamide

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syringe filter with a pore size of 0.45µm (Macherey Nagel, Düren, Germany) and diluted 1:10

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with 70% methanol/water (v/v). The resulting solutions were used directly for HPLC-ion trap and

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HPLC-micrOTOF analysis.

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2.3. HPLC:

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Separation was achieved on a 250 × 3 mm i.d. C18 amide packing column with 5 µm particle size,

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with a 5 mm × 3 mm inner-diameter guard column (Varian, Darmstadt, Germany). Solvent A was

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water/formic acid (1000:0.005 v/v), and solvent B was methanol. Solvents were delivered at a

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total flow rate of 500µL/minute. The gradient profile used was: starting with 5% B, increasing to

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10% B in 3.7 minutes, isocratic 10% until 10 minutes, increasing to 15% in 13,5 minutes,

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isocratic 15% B until 15 minutes, increasing to 32,2% B until 19 minutes, increasing to 35% B

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until 30 minutes, followed by increasing at 80% B until 40 minutes, followed by washing with

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80% B until 55 minutes and decreasing to 5% B until 60 minutes followed by column re-

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equilibration at 5% B until 70 minutes.

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2.4 HPLC −Ion Trap MSn:

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The LC equipment (1100 Series, Agilent, Karlsruhe, Germany) comprised a binary pump, an

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auto sampler with a 100 µL loop, and a DAD detector with a light-pipe flow cell, recording at

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320 nm and scanning from 200 to 600 nm.

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For the profiling analysis, the LC system was interfaced with an ion-trap mass spectrometer

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fitted with an ESI source (HCT-Ultra, Bruker Daltonics, Bremen, Germany) operating in full 7 ACS Paragon Plus Environment


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scan, auto MSn mode for generating fragment ions. Tandem mass spectra were acquired in

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negative ion mode using the auto-MSn mode (smart fragmentation) using a ramping of the

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collision energy. Maximum fragmentation amplitude was set to 1 V, starting at 30% and ending

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at 200%. The MS operating conditions (negative ion mode) were: capillary temperature of

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365°C, drying gas flow rate of 10 L/minute, and a nebulizer pressure of 50 psi.

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When needed, targeted fragmentation experiments were performed, focusing only on compounds

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producing a [M-H]- ion of m/z 341.

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2.5 LC-microTOF:

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High resolution MS data was acquired using the same the same HPLC equipment described in

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Section 2.4 coupled with a high-resolution mass spectrometer (MicroTOF Focus, Bruker

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Daltonics, Bremen, Germany) fitted with an ESI source, and internal calibration was achieved

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with a 0.1 M sodium formate solution injected through a six-port valve prior to each

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chromatographic run. Calibration was carried out using the enhanced quadratic mode, and the

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mass error for the generated molecular formulae was below 5 ppm.

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2.6 Caffeoylglucose quantification:

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An LC-MS method was developed for the quantitative analysis of the caffeoyl glucose isomers

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using 6-Caffeoylglucose synthetic standard prepared and characterized by Jaiswal et al. 37 (see

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Section 3.3 for detailed discussion). Nine point calibration curves were obtained from serial

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dilutions of synthetic standards using extracted ion chromatograms at high mass resolution (m/z

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341.0878.+/- 0.002 Da). The lower limit of detection was defined as the concentration for which

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the signal/noise ratio was 5. The LOD was found to be 0.1µg/mL. The calibration curve was

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constructed from data points corresponding to concentrations in the range 0.5-40 µg/mL, with a 8 ACS Paragon Plus Environment

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Pearson correlation coefficient of 0.9978. The values for each data point were obtained as the

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average of 3 measurements, with relative standard deviation values below 7% (Supporting

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information section). Multiple intra and inter day measurements were performed at the LOD.

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Relative standard deviations obtained for intra and inter day analytical replicates were below 6%

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(Supporting information). In order to calculate the reproducibility of the extraction and

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subsequent quantification procedure, triplicate analysis was performed on one representative of

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each sample type. The extraction as well as the quantification yielded relative standard deviation

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values below 10% (Supporting information). Hesperetin was used as internal standard for all the

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investigated samples and integration values normalized to the internal standard, to account for

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time dependent variations of the detector response.

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3. RESULTS AND DISCUSSION:

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3.1 Profiling of Caffeoylglucoses and Caffeic acid-O-glycosides

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Methanolic crude extracts of selected dietary berries were obtained, purified by SPE and

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subjected to targeted LC-ESI-tandem-MS profiling of hexose conjugates of caffeic acid, among

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them the ester derivatives 1-10. The representative edible berries examined were strawberries

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(Fragaria ananassa), raspberries (Rubus idaeus), blueberries (Vaccinium corymbosum),

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blackberries (Rubus fruticosus), red currants (Ribes rubrum), black currants (Ribes nigrum),

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lingonberries (Vaccinium vitis-idaea) and gooseberries (Ribes uva-crispa). Additionally directly

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pressed juices of purple chokeberry (Aronia melanocarpa), elderberry (Sambucus melanocarpa),

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cranberry (Vaccinium oxycoccos), goji berry (Lycium chinense), sea buckthorn (Hippophae

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rhamnoides), açai berry (Euterpe oleracea), sour cherry (Prunus cerasus) and pomegranate

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(Punica granatum) were also investigated. The optimized LC-tandem MS method employed

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provided base line separation of all ten regioisomeric caffeoyl glucoses 1-10 using MS detection 9 ACS Paragon Plus Environment


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in the negative ion mode. In all the investigated samples, assignment of the individual

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stereoisomeric caffeoyl glucose (CG) structures was carried out on the basis of their high

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resolution MS data (m/z 341.0878.+/- 0.002 Da) indicating the molecular formula [C15H17O9]- for

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the [M-H]- ion and respective fragmentation patterns and relative elution times, previously

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described.37

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3.1.1 1-O-Caffeoylglucose

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Our previous study37 found that the first eluting anomer of 1-CG (available as synthetic mixture

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of both α- and β- anomers) generated upon MS2 fragmentation, the deprotonated caffeic acid of

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m/z 179 as the base peak which upon further MS3 fragmentation generates the decarboxylated

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caffeic acid anion of m/z 135 as a single peak. This was speculated to be the α-anomer on the

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basis of the fragmentation mechanism arguments developed in the study, as the axial orientation

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of the anomeric hydroxyl group is in favor of the loss of the caffeoyl moiety, due to the anomeric

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effect. The later eluting anomer generated an MS2 base peak at m/z 203 and another high intensity

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peak at m/z 161. Based on the same fragmentation mechanisms, this was speculated to be the β-

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anomer, as only the equatorial orientation of the anomeric OH is in favor of the hexose chair

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inversion and subsequent sugar ring fission fragmentation leading to the formation of the ketene

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acetal of m/z 203. The mechanisms have been discussed in detail by Jaiswal et al.37 However, the

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unambiguous assignment of each anomer could not be made, as it was made for the other eight

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isomers, for which pure synthetic standard compounds were available.

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In order to verify the speculation that the first eluting isomer was most likely the α-anomer,

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theoretical dipole moment calculations were performed for the two anomers using the Gaussian

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software. Theoretical dipole moment values retrieved by the software were 7.4 Debye and 8.1

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Debye for 1-β-CG and 1-α-CG respectively. Based on these results, the more polar α-anomer is 10 ACS Paragon Plus Environment

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expected to elute first from a reversed phase chromatographic column, which confirms our

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previous speculation that the first eluting 1-CG can tentatively be assigned as 1-α-CG (2), which

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was found to be present in relatively higher amounts than other CG isomers in strawberry,

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raspberry, blueberry, red currant, black currant, gooseberry, lingonberry, aronia puree, cranberry

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juice, Montmorency sour cherry juice, goji berry juice, pomegranate juice, and absent from

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blackberry, acai berry juice, elderberry juice and sea buckthorn juice. The specific fragmentation

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pattern of the polar 1-β-CG (1) was not found in any of the investigated samples.

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Both anomers of 2-CG (3-4) were found in extracts of gooseberry, raspberry, aronia puree,

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Montmorency sour cherry and goji berry juice. The 2- α -CG was detected in black currant as a

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single anomer.

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Both anomers of 3-CG (5-6) were found in gooseberry in very low amounts.

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in low amounts in elderberry juice, but was absent from all other samples.

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Both anomers of 4-O-Caffeoylglucose (7-8) were found in gooseberry in very low amounts but

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absent from all other samples.

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Both anomers of 6-O-Caffeoylglucose (9-10) were found in gooseberry, strawberry, aronia puree,

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Montmorency sour cherry juice, elderberry juice, Goji berry juice, and pomegranate juice. The

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6-α-CG (10) was detected as single anomer in lingonberry and black currant.

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3.1.6 Caffeic acid-3-O-glucose and caffeic acid-4-O-glucose

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Caffeic acid-4-O-β-D-glucose and caffeic acid-3-O-β-D-glucose were reported in kiwi fruits after

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isolation by preparative chromatography and NMR identification.30 Therefore, a methanolic 11 ACS Paragon Plus Environment

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extract of kiwi was used as a surrogate standard38 for the identification of these two glycosides in

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all the berry samples analyzed. The extracted ion chromatogram at m/z 341.0878.+/- 0.002 Da

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(m/z 341 recorded with the ion trap spectrometer), corresponding to the molecular formula of

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[C15H17O9]- for the [M-H]- ion showed 5 peaks, all of which upon MS2 fragmentation generated

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the deprotonated caffeic acid of m/z 179 as the base peak. Further MS3 fragmentation of this peak

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generated the decarboxylated caffeic acid anion of m/z 135 as a single peak. In the absence of

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authentic standards, given that all isomers have identical MS2 fragmentation patterns, caffeic

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acid-4-O-β-D-glucose and caffeic acid-3-O-β-D-glucose were considered to correspond to the

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two most intense peaks (since they were reported exclusively after preparative HPLC) and the

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individual structures were tentatively assigned to each peak on the basis of their relative elution

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order from a reversed phase column. Dipole moment calculations were performed using the

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Gaussian software and the values retrieved were 5.45 Debye and 5.20 Debye for caffeic acid-3-

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O-β-D-glucose and caffeic acid-4-O-β-D-glucose respectively. Therefore, the first eluting peak

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was tentatively assigned as caffeic acid-3-O-β-D-glucose (11) and the second eluting isomer as

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caffeic acid-4-O-β-D-glucose (12) (Figure 3). The two regioisomers were found in many of the

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investigated samples, as summarized in Table 1.

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3.2 Assignment of other compounds by tandem MS

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All the investigated samples showed a higher complexity than previously reported of isomers

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with [M-H]- ions at 341.0878.+/-0.002 ([C15H17O9]-), m/z 325.0929.+/-0.002 ([C15H17O8]-), m/z

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355.1035.+/-0.002

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hexose conjugates of caffeic, p-coumaric, ferulic and sinapic acids respectively. The following

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section presents compound assignment within chosen samples with a subsequent discussion of

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fragment spectra allowing such assignment. (MSn data from the ion trap spectrometerfor all peaks

([C16H19O9]-) and m/z 385.1140.+/-0.002 ([C17H21O10]-) corresponding to


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with precursor ions at m/z 341, m/z 325, m/z 355 and m/z 385 for individual samples is presented

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in the Supporting information section).

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3.2.1 Caffeic acid conjugates with hexoses other than glucose (C15H18O9)

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The optimized HPLC method achieved good separation of a large number of isomers

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corresponding to the molecular formula C15H18O9 confirmed by high resolution m/z values of

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341.0878.+/-0.002 for their [M-H]- ions. The largest isomeric complexity was found for

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gooseberry, with 20 peaks in the extracted ion chromatogram at m/z 341.0878.+/-0.002 (m/z

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values of 341 +/- 0.5 in ion trap spectra). Peaks with retention times below 10 minutes were not

293

taken into account as they correspond to isomeric disaccharides such as sucrose (with molecular

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formula of C12H22O10 confirmed by high resolution m/z values of 341.1089.+/-0.002 for their

295

[M-H]- ions and characteristic disaccharide fragmentation patterns.39

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Six of the peaks presented a fragmentation pattern characteristic to caffeic acid O-glycosides,

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generating in MS2 from the [M-H]- ion at m/z 341 a single peak of m/z 179 ([C9H7O4]-) through

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the loss of the hexosyl unit of 162 Da. Further MS3 fragmentation of the peak at m/z 179

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generated a single peak at m/z 135, through the loss of a carbon dioxide molecule, which is

300

indicative of the caffeic acid moiety in the parent structure. Therefore, the peaks giving this

301

specific fragmentation can be tentatively identified as O-glycosides of caffeic acid with various

302

hexoses (e.g. mannose, galactose or others), with each combination generating in theory 4

303

possible isomers. The remaining peaks show a fragmentation pattern resembling that of the

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caffeoyl glucose esters (fragments of m/z 323, 281, 251, 233, 203 etc.). Therefore, these peaks

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can be tentatively identified as caffeoyl esters of a hexose different from glucose, with each

306

combination giving in theory ten possible isomers.

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3.2.2 p-coumaric acid conjugates with gluconic acid (C15H18O9)

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The gooseberry, red currant and black currant extracts show six isomeric compounds with the

310

same molecular formula as the caffeic acid hexose conjugates, C15H18O9-confirmed by high

311

resolution m/z values of 341.0878.+/-0.002 (m/z 341 recorded with the ion trap spectrometer) for

312

their [M-H]- ions- but with different fragmentation behavior compared to both caffeoyl glucose

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esters and caffeic acid-O-glycosides. The first eluting isomer generated upon MS2 fragmentation

314

of the

315

(C6H10O6). MS3 fragmentation of the base peak at m/z 163 generated a single peak of m/z 119, by

316

the loss of carbon dioxide molecule, which is indicative of a p-coumaric acid moiety in the parent

317

structure. The remaining four isomers generated upon MS2 fragmentation a base peak of m/z 195

318

([C6H11O7]-, by a neutral loss of 146 Da (p-coumaroyl moiety C9H6O2) and another high intensity

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peak of m/z 163 ([C9H7O3]-) by a neutral loss of 178 Da (C6H10O6). Further MS3 fragmentation

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of the base peak of m/z 195 gave a base peak of m/z 159 ([C6H11O7-2H2O]-), another high

321

intensity peak of m/z 129 ([C6H11O7-2H2O-CH2O]- and smaller intensity peaks of m/z 177

322

([C6H11O7-H2O]-), m/z 149 ([C6H11O7-H2O-CO]-), m/z 141 ([C6H11O7-3H2O]-), and m/z 111

323

([C6H11O7-3H2O- CH2O]-). MS4 fragmentation of the peak of m/z 159 ([C6H7O5]-), gave the base

324

peak of m/z 129 ([C6H7O5-CH2O]-) and a low intensity peak of m/z 97 ([C6H7O53-H2O-CO2]-).

325

This fragmentation behavior which is characteristic to sugars,39 together with high resolution MS

326

data which indicates the molecular formula of C6H12O7, strongly suggests that this moiety is

327

gluconic acid, which has been previously reported in its free form in various fruits including

328

berries.40,41 Therefore, the compounds are tentatively assigned as p-coumaric acid conjugates of

329

gluconic acid. Selected representative structures are presented in Figure 3, Structures 13-14. The

330

regiochemistry and nature of the linkages remain unclear.

[M-H]- ion of m/z 341 a base peak of m/z 163 ([C9H7O3]-) by a neutral loss of 178 Da

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3.2.3 p-coumaric acid O-glycosides and p-coumaroyl glucoses (C15H18O8)

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Isomeric p-coumaric acid-hexose conjugates with molecular formula of C15H18O8 (confirmed by

334

the high resolution m/z values of 325.0929.+/-0.002 for their [M-H]- ions) have been found in

335

most of the investigated samples (the number of isomers present in each sample is given in Table

336

1; high resolution extracted ion chromatograms at m/z 325.0929.+/-0.002 for individual samples

337

are given in Supporting Information).

338

p-Coumaric acid-4-O-β-D-glucopyranose (13), synthesized and characterized by Galand et. al.,42

339

was used as a reference standard. The standard generated a high resolution m/z value of

340

325.0920. for its [M-H]- ion (theoretical m/z value of 325.0929, which confirms the molecular

341

formula of C15H18O8 (error of 2.7 ppm from the theoretical m/z value of 325.0929). MS2

342

fragmentation of the [M-H]- ion of m/z 325, generated the base peak of m/z 163 ([C9H7O3]-)

343

through the loss of the glycosyl unit of 162 Da, and a small intensity peak of m/z 119. Further

344

MS3 fragmentation of the MS2 base peak (m/z 163) generates a single peak of m/z 119, through

345

the loss of a carbon dioxide molecule, characteristic for p-coumaric acid moiety. This

346

fragmentation behavior is in agreement to that of the caffeic acid-O-glycosides. Some of the

347

investigated isomers presented this O-glycoside characteristic fragmentation pattern and some

348

isomers presented distinct fragmentation patterns, identical to the fragmentation patterns of their

349

caffeoyl glucose analogues. p-Coumaric acid-4-O-β-D-glucopyranose was successfully identified

350

in black currant, strawberry, lingonberry, blueberry, elderberry juice, sea buckthorn berry juice

351

and sour cherry juice, on the basis of high resolution MS data, fragmentation pattern and

352

retention time.

353

Two C15H18O8 isomers-confirmed by the high resolution m/z values of 325.0929.+/-0.002 for

354

their [M-H]- ions- found in red currant, black currant and strawberry samples, presented identical

355

MS2 fragmentation behavior as the two anomers of 6-CG, namely sugar ring fission 15 ACS Paragon Plus Environment


Page 16 of 32

356

fragmentations generating neutral losses of 60Da, 90Da and 120 Da and loss of the glycosyl unit

357

of 162 Da to generate the deprotonated acid. MS2 fragmentation of the [M-H]- ion of m/z 325

358

([C15H17O8]-) generated the base peak at m/z 265 ([C15H17O8-C2H4O2]-) by a neutral loss of 60

359

Da, another high intensity peak at m/z 235 ([C15H18O8-C3H6O3]-) by a neutral loss of 90 Da and

360

smaller intensity peaks at m/z 205 ([C15H17O8-C4H8O4]-) by a neutral loss of 120 Da and m/z 163

361

([C15H17O8-C6H10O6]-) through the loss of the glycosyl unit of 162 Da.

362

fragmentation patterns are identical to the MS3 fragmentation patterns (Figure 2) of the

363

analogous caffeoyl species. Therefore, the two isomers were tentatively assigned as the two

364

anomers of

Further MS3

6-O-p-coumaroyl-glucose (16-17).

365 366

3.2.4 Ferulic acid-O-glycosides (C16H20O9)

367

Ferulic

368

355.1035.+/-0.002 for their [M-H]- ions) were found to be present in most of the investigated

369

samples. The number of isomers present in each sample is given in Table 1. Ferulic acid-4-O-β-

370

D-glucopyranoside (16) synthesized and characterized by Galland et al.,42 was used as a reference

371

standard. The standard generated a high resolution m/z value of 355.1028 for its [M-H]- ion

372

(theoretical m/z value of 355.1035, which confirms the molecular formula of C16H20O9 (error of

373

1.9 ppm from the theoretical m/z value of 355.1035). Ion trap MS2 fragmentation of the [M-H]-

374

ion of m/z 355 generating a single peak of m/z 193 (deprotonated ferulic acid [C10H9O4]-) through

375

the loss of the glycosyl unit of 162 Da. Further MS3 fragmentation of the MS2 base peak (m/z

376

193) generated a base peak of m/z 149 ([C10H9O4-CO2]-), through the loss of a carbon dioxide

377

molecule, and another peak at m/z 177 ([C10H9O4-CH3]-) through the loss of a methyl group.

378

Further MS4 fragmentation of the peak at m/z 149 ([C10H9O4-CO2]-) gave a single peak at m/z 134

379

([C10H9O4-CO2-CH3]-) through the loss of a methyl group. All the MS2-4 spectra confirmed the

acid-hexose

conjugates

with

molecular

formula


C16H20O9

(m/z

values

of

Page 17 of 32


380

presence of the ferulic acid moiety in the parent structures.43 Therefore, based on the high

381

resolution MS data as well as fragmentation data and retention time ferulic acid-4-O-β-D-

382

glucopyranoside (18) was successfully identified in Goji Berry.

383 384

3.2.5 Sinapic acid hexose conjugates (C17H22O10)

385

Sinapic acid-hexose conjugates

386

385.1140.+/-0.002 for their [M-H]- ions) were found to be present in most of the investigated

387

samples. The number of isomers present in each sample is given in Table 1. The black currant

388

sample investigated showed a high intensity peak at m/z 385.1135 (m/z 385 recorded with the ion

389

trap spectrometer). MS2 fragmentation of the [M-H]- ion generated the deprotonated acid of m/z

390

223 ([C11H11O5]-), through the loss of the glycosyl unit of 162 Da, which is a fragmentation

391

characteristic to O-glycosides, confirmed during our study by all the O-glycosides investigated.

392

caffeic acid-, p-coumaric acid- and ferulic acid-O-glycosides. In the absence of reference

393

standards, the identity of the hexose remains unclear. MS3 fragmentation of this ion generated a

394

base peak at m/z 208 ([C11H11O5-CH3]-) by the loss of the first methyl group and lower intensity

395

peaks at m/z 179 ([C11H11O5-CO2]-) by the loss of the carbon dioxide molecule and m/z 164

396

([C11H11O5-CO2-CH3]-) from the loss of the carbon dioxide and the second methyl group. MS4

397

fragmentation of the peak at m/z 208 gave a base peak at m/z 164 ([C11H11O5-CH3-CO2]-) and

398

lower intensity peaks at m/z 193 ([C11H11O5-2CH3]-) and m/z 149 ([C11H11O5-2CH3-CO2]-).

399

Therefore, this isomer is preferentially losing the methyl group over the carboxylate group from

400

the carboxylate ion ([C11H11O5]-). The fragmentation of deprotonated sinapic acid however,

401

previously reported for 3-sinapoylquinic acid, occurs preferentially through the loss of a carbon

402

dioxide molecule, generating a base peak of m/z 179.44

with

molecular formula C17H22O10 (m/z


values

of


Page 18 of 32

403

The same difference in fragmentation behavior has been previously reported for ferulic and

404

isoferulic acid, with ferulic acid preferentially losing a carbon dioxide molecule from the

405

deprotonated ion and isoferulic acid preferentially losing the methyl group.43

406

C17H22O10 isomer from black currant shows the fragmentation pattern specific to isoferulic acid,

407

it was tentatively assigned as an O-hexoside of 3,4-dimethoxy-5-hydroxy-cinnamic acid, with

408

two different anomeric structures being possible. Representative structures are presented in

409

Figure 3. A summary of the profiling results is given in Table 1.

Since the

410 411

3.3 Quantification of CGs

412

Prior to quantification, the caffeoyl glucose derivatives from the methanolic crude extracts were

413

enriched using a C18 SPE cartridge using a water wash step and elution with 20% MeOH, as

414

described in section 2.2.2. The more sensitive LC-MS technique was chosen for further

415

quantification work, as the extracts showed a satisfactory signal in the UV chromatogram at

416

320 nm only for the major isomer 1-α-CG. From our previous work, synthetic reference standards

417

were available for 6-CG, 3-CG and 1-CG. 6-CG was found to be by far the most stable

418

derivative showing a small degree of epimerization and no acyl migration chemistry after

419

dissolution in aqueous solution.37 All other derivatives were less stable, at least 10% of acyl

420

migration products after 20 min in aqueous solution. For this reason we related all quantifications

421

to a 6-CG calibration curve, assuming a response factor of unity for all isomers quantified here.

422

This assumption is supported by comparison of the slope of different calibration curves obtained

423

for all derivatives (three available as authentic reference standards) and by the observation that in

424

isomeric mixtures obtained through acyl migration, the sum of all LC-MS peak areas remained

425

constant (+/- 5 %) within acceptable boundaries.


Page 19 of 32


426

Since all samples were thoroughly desiccated prior to extraction, the results are normalized to

427

100g of dry weight (DW). Quantitative data for caffeoyl glucoses are provided for the first time

428

for all the berry samples investigated. CG were quantified in nineteen samples (Table 2). For

429

calculating the reproducibility of the quantification procedure, triplicate extraction was performed

430

on one representative of each sample type (namely samples 7, 10, 12, 15, 18 and 19-Table 2)

431

serving as technical replicates and each extract was analyzed per duplicate of injection, serving as

432

analytical replicates. For all the other samples, single extracts were analyzed per duplicate of

433

injection. Table 2 presents the average value resulting from the duplicate injections. Relative

434

standard deviations were always below 5 %.

435

1-α-CG was the most abundant CG isomer in all the investigated samples. The highest

436

concentration of CGs was found for the lingonberry sample (35 mg/100g DW), followed by

437

gooseberry samples (13 mg/100 g DW). The lowest concentration was found for blueberry

438

samples, with an average content of 1.5 mg/100 g DW. Five different cultivars of strawberry

439

were investigated. No outstanding differences were observed between samples. The highest CG

440

content was found for the cultivar Romina (4.33 mg/ 100g DW) and the lowest content was

441

found for Elsanta (2.65 mg/ 100g DW). The Food and Agriculture Organization of the United

442

Nations (FAO) reports a continuous increase in the world production of strawberry, raspberry and

443

blueberry, over the last decade, with 8114373 tons of strawberry, 612571 tons of raspberry and

444

525620 tons of blueberry produced worldwide in 2014. Given the average contents of CGs found

445

for these 3 main berry crops, we have estimated an annual world natural production of 25 tons of

446

CGs from only these fruit sources.

447 448 449 19 ACS Paragon Plus Environment


450

4. Abbreviations used:

451

CG-caffeoyl glucose

Page 20 of 32

452 453

5. Acknowledgements:

454

IT support from Dr. Abhinandan Shrestha and technical support from Ms Anja Müller is greatly

455

acknowledged. Gordon McDougal is grateful for support from the Scottish Government’s Rural

456

and Environment Science and Analytical Services Division. We thank Prof O. Dangles for the

457

provision of authentic reference samples.

458 459

Statement: This research did not receive any specific grant from funding agencies in the public,

460

commercial or not-for-profit sectors.

461 462

Supporting information: The supporting information document contains high resolution MS

463

data as well as fragmentation data for individual samples. It also contains statistical information

464

regarding the quantification method.


Page 21 of 32


465

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466 467

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468 469

2. Nile, S.H.; Park, S.W. Edible berries: Bioactive components and their effect on human health. Nutrition 2014, 30, 134-144.

470 471 472 473

3. Seeram, N.P.; Adams, L.S.; Zhang, Y.; Lee, R.; Sand, D.; Scheuller, H.S.; Heber, D. Blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts inhibit growth and stimulate apoptosis of human cancer cells in vitro. J. Agric. Food Chem. 2006, 54, 9329-9339.

474 475 476

4. Paredes-Lopez, O.; Cervantes-Ceja, M.L.; Vigna-Perez, M.; Hernandez-Perez, T. Berries: Improving Human Health and Healthy Aging, and Promoting Quality Life-A Review. Plant Foods for Human Nutrition 2010, 65, 299-308.

477 478

5. Seeram, N.P.; Heber, D. Impact of Berry Phytochemicals on Human Health: Effects beyond Antioxidation. Antioxidant Measurement and Applications 2007, 956, 326-336.

479 480

6. Seeram, N.P.; Shukitt-Hale, B. Advances in berry research: The sixth Biennial Berry Health Benefits Symposium 1. J. Berry Res. 2016, 6, 93-95.

481 482

7. Seeram, N.P. Berries and human health: Research highlights from the fifth biennial berry health benefits symposium. J. Agric. Food Chem. 2014, 62, 3839-3841.

483 484

8. Joseph, S.V.; Edirisinghe, I.; Burton-Freeman, B.M. Berries: Anti-inflammatory effects in humans. J. Agric. Food Chem. 2014, 62, 3886-3903.

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12. Heinonen, M. Antioxidant activity and antimicrobial effect of berry phenolics - a Finnish perspective. Molecular Nutrition & Food Research 2007, 51, 684-691.

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13. Nile, S.H.; Park, S.W. Edible berries: Bioactive components and their effect on human health. Nutrition 2014, 30, 134-144.

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15. Seeram, N.P. Berry fruits: compositional elements, biochemical activities, and the impact of their intake on human health, performance, and disease. J. Agric. Food Chem. 2008, 56, 627-629.

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16. Basu, A.; Rhone, M.; Lyons, T.J. Berries: emerging impact on cardiovascular health. Nutr. Rev. 2010, 68, 168-177.

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17. Hakkinen, S.H.; Karenlampi, S.O.; Heinonen, I.M.; Mykkanen, H.M.; Torronen, A.R. HPLC method for screening of flavonoids and phenolic acids in berries. J. Sci. Food Agric. 1998, 77, 543-551.

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19. Häkkinen, S.H.; Törrönen, A.R. Content of flavonols and selected phenolic acids in strawberries and Vaccinium species: influence of cultivar, cultivation site and technique. Food Res. Int. 2000, 33, 517-524.

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20. Clifford, M.N. Chlorogenic acids and other cinnamates - nature, occurrence and dietary burden. J. Sci. Food Agric. 1999, 79, 362-372.

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21. Clifford, M.N. Chlorogenic acids and other cinnamates - nature, occurrence, dietary burden, absorption and metabolism. J. Sci. Food Agric. 2000, 80, 1033-1043.

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22. Schuster, B.; Herrmann, K. Hydroxybenzoic and Hydroxycinnamic Acid-Derivatives in Soft Fruits. Phytochemistry 1985, 24, 2761-2764.

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23. Koeppen, B.H.; Herrmann, K. Flavonoid Glycosides and Hydroxycinnamic Acid-Esters of Blackcurrants (Ribes Nigrum) - Phenolics of Fruits 9. Zeitschrift Fur LebensmittelUntersuchung Und-Forschung 1977, 164, 263-268.

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24. Jaiswal, R.; Karakoese, H.; Ruehmann, S.; Goldner, K.; Neumueller, M.; Treutter, D.; Kuhnert, N. Identification of Phenolic Compounds in Plum Fruits (Prunus salicina L. and Prunus domestica L.) by High-Performance Liquid Chromatography/Tandem Mass Spectrometry and Characterization of Varieties by Quantitative Phenolic Fingerprints. J. Agric. Food Chem. 2013, 61, 12020-12031.

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25. Alakolanga, A.G.A.W.; Siriwardane, A.M.D.A.; Kumar, N.S.; Jayasinghe, L.; Jaiswal, R.; Kuhnert, N. LC-MSn identification and characterization of the phenolic compounds from the fruits of Flacourtia indica (Burm. F.) Merr. and Flacourtia inermis Roxb. Food Res. Int. 2014, 62, 388-396.

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26. Vallverdu-Queralt, A.; Jauregui, O.; Di Lecce, G.; Andres-Lacueva, C.; LamuelaRaventos, R.M. Screening of the polyphenol content of tomato-based products through accurate-mass spectrometry (HPLC-ESI-QTOF). Food Chem. 2011, 129, 877-883.

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27. Vallverdu-Queralt, A.; Jauregui, O.; Medina-Remon, A.; Andres-Lacueva, C.; LamuelaRaventos, R.M. Improved characterization of tomato polyphenols using liquid chromatography/electrospray ionization linear ion trap quadrupole Orbitrap mass 22 ACS Paragon Plus Environment

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28. Du, Q.; Xu, Y.; Li, L.; Zhao, Y.; Jerz, G.; Winterhalter, P. Antioxidant constituents in the fruits of Luffa cylindrica (L.) Roem. J. Agric. Food Chem. 2006, 54, 4186-4190.

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29. Chu, N.; Clydesdale, F.; Francis, F. Isolation and Identification of some Fluorescent Phenolic Compounds in Cranberries. J. Food Sci. 1973, 38, 1038-1042.

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30. Fiorentino, A.; D'Abrosca, B.; Pacifico, S.; Mastellone, C.; Scognamiglio, M.; Monaco, P. Identification and Assessment of Antioxidant Capacity of Phytochemicals from Kiwi Fruits. J. Agric. Food Chem. 2009, 57, 4148-4155.

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31. Winter, M.; Herrmann, K. Esters and Glucosides of Hydroxycinnamic Acids in Vegetables. J. Agric. Food Chem. 1986, 34, 616-620.

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32. Marin, A.; Ferreres, F.; Tomas-Barberan, F.A.; Gil, M.I. Characterization and quantitation of antioxidant constituents of sweet pepper (Capsicum annuum L.). J. Agric. Food Chem. 2004, 52, 3861-3869.

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33. Clifford, M.N.; Wu, W.; Kirkpatrick, J.; Kuhnert, N. Profiling the chlorogenic acids and other caffeic acid derivatives of herbal chrysanthemum by LC-MSn. J. Agric. Food Chem. 2007, 55, 929-936.

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34. Jaiswal, R.; Sovdat, T.; Vivan, F.; Kuhnert, N. Profiling and Characterization by LC-MSn of the Chlorogenic Acids and Hydroxycinnamoylshikimate Esters in Mate (Ilex paraguariensis). J. Agric. Food Chem. 2010, 58, 5471-5484.

557 558 559 560

35. Harbaum, B.; Hubbermann, E.M.; Wolff, C.; Herges, R.; Zhu, Z.; Schwarz, K. Identification of flavonoids and hydroxycinnamic acids in pak choi varieties (Brassica campestris L. ssp. chinensis var. communis) by HPLC-ESI-MSn and NMR and their quantification by HPLC-DAD. J. Agric. Food Chem. 2007, 55, 8251-8260.

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36. Harborne, J.B.; Corner, J.J. Plant polyphenols. 4. Hydroxycinnamic acid-sugar derivatives. Biochem. J. 1961, 81, 242-250.

563 564 565

37. Jaiswal, R.; Matei, M.F.; Glembockyte, V.; Patras, M.A.; Kuhnert, N. Hierarchical Key for the LC-MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose. J. Agric. Food Chem. 2014, 62, 9252-9265.

566 567

38. Clifford, M.N.; Madala, N.E. Surrogate Standards: A Cost-Effective Strategy for Identification of Phytochemicals. - J. Agric. Food Chem. 2017,

568 569 570

39. Gao, Q.; Nilsson, U.; Ilag, L.L.; Leck, C. Monosaccharide compositional analysis of marine polysaccharides by hydrophilic interaction liquid chromatography-tandem mass spectrometry. Anal. Bioanal. Chem. 2011, 399, 2517-2529.

571 572 573

40. Zhang, J.; Wang, X.; Yu, O.; Tang, J.; Gu, X.; Wan, X.; Fang, C. Metabolic profiling of strawberry (Fragaria×ananassa Duch.) during fruit development and maturation. J. Exp. Bot. 2011, 62, 1103-1118. 23 ACS Paragon Plus Environment


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41. Ramachandran, S.; Fontanille, P.; Pandey, A.; Larroche, C. Gluconic acid: Properties, applications and microbial production. Food Technol. Biotechnol. 2006, 44, 185-195.

576 577 578

42. Galland, S.; Mora, N.; Albert-Vian, M.; Rakotomanomana, N.; Dangles, O. Chemical synthesis of hydroxycinnamic acid glucosides and evaluation of their ability to stabilize natural colors via anthocyanin copigmentation. J. Agric. Food Chem. 2007, 55, 7573-7579.

579 580 581

43. Kuhnert, N.; Jaiswal, R.; Matei, M.F.; Sovdat, T.; Deshpande, S. How to distinguish between feruloyl quinic acids and isoferuloyl quinic acids by liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2010, 24, 1575-1582.

582 583 584

44. Jaiswal, R.; Patras, M.A.; Eravuchira, P.J.; Kuhnert, N. Profile and characterization of the chlorogenic acids in green Robusta coffee beans by LC-MSn: Identification of seven new classes of compounds. J. Agric. Food Chem. 2010, 58, 8722-8737.

585 586

587 588 589

590

591 592 593 594 595 596 597 598 599 600 601 602 603 604 24 ACS Paragon Plus Environment

Page 24 of 32

Page 25 of 32

605 606


Figure Captions

607 608

Figure 1: The extracted ion chromatogram at m/z 341 (negative ion mode) from an ion trap mass spectrometer

609

and MS2 spectra of labeled peaks from gooseberry extract. Numbering of peaks refers to structures shown in

610

Figure 3.

611 612

Figure 2: a. The extracted ion chromatogram at m/z 341 (negative ion mode) from an ion trap mass

613

spectrometer and MS2-4 spectra of 6-β-CG from strawberry;

614

(negative ion mode) from an ion trap mass spectrometer and MS2-4 spectra of the tentatively assigned 6-β-p-

615

coumaroylglucose from black currant;

616

Numbering of peaks refers to structures shown in Figure 3.

b. The extracted ion chromatogram at m/z 325

c. MS2 fragmentation pathways of the two analogous compounds.

617 618

Figure 3: Individual structures of the compounds 1-20 found in the investigated berry extracts



Page 26 of 32

Table 1: Summary of the profiling results for all the investigated berry extracts (‘yes’ indicates the presence of the respective isomer, ND-not detected-indicates the absence of the respective isomer)

Sample

1-α-CG (2)

2-CG (3-4)

3-CG (5-6)

4-CG (7-8)

6-CG (9-10)

Caffeic acid 3-O-βglucose

Caffeic acid 4-O-βglucose

No. of isomers m/z 341.0878. +/-0.002

No. of isomers m/z 325.0929. +/-0.002

Strawberry

yes

ND

ND

ND

yes

yes

yes

10

2

Raspberry

yes

yes

ND

ND

ND

yes

yes

9

0

0

0

Blueberry

yes

ND

ND

ND

ND

yes

ND

4

3

4

3

Blackberry

ND

ND

ND

ND

ND

ND

ND

5

3

0

4

Red currant

yes yes yes yes yes

ND

ND

ND

ND

ND

ND

11

3

0

2

yes yes yes yes

ND

ND

7

8

5

yes

20

5

2

3

ND

ND

ND

13

5

5

1

ND

ND

yes yes yes yes

7

yes

yes yes yes yes

ND

yes

ND

9

4

2

2

ND

ND

yes

ND

yes

yes

ND

13

3

4

5

yes

ND

ND

ND

ND

yes

yes

8

5

4

4

yes

yes

ND

ND

yes

yes

yes

9

7

7

4

ND

ND

ND

ND

ND

ND

ND

0

1

3

2

ND

ND

ND

ND

ND

ND

ND

0

4

3

6

Sour Cherry juice

yes

yes

ND

ND

yes

yes

yes

12

9

3

2

Pomegranate juice

yes

ND

ND

ND

yes

yes

ND

5

5

5

2

LingonBerry Gooseberry Black currant Aronia juice Elderberry juice Cranberry juice Goji Berry juice Açai Berry juice Sea buckthorn juice

26

ACS Paragon Plus Environment

No. of No. of isomers isomers m/z m/z 385.1140. 355.1035+/+/-0.002 0.002 1

1

Page 27 of 32


Table 2: Summary of the quantification results of individual isomers of CG in selected samples; results given in mg CG/100g of dry weight (DW). Analysis were performed in replicate. Mean values are stated.

No

Sample

1

Strawberry (c.v. Adria) Strawberry (c.v. Anoi) Strawberry (c.v.Romina) Strawberry (c.v. Elsanta) Strawberry (c.v.Sveva) Strawberry Italy Strawberry Romania Strawberry Romania Strawberry Germany Blueberry Germany Blueberry Morocco Raspberry Germany Raspberry Romania Red currant Germany Red currant Germany (garden) Black currant Germany (garden) Gooseberry Germany Gooseberry Germany (garden) Lingonberry

2 3 4 5 6 7 8 9 10 11 12 13 14 15

16

17 18 19

2-β-CG [mg/100g DW] (%STDEV)

2-α-CG [mg/100g DW] (%STDEV)


6-β-CG [mg/100g DW] (%STDEV)


Total [mg/100g DW]

ND

ND

2.13

0.39

0.42

2.94 +/- 0.14

ND

ND

2.31

0.40

0.42

3.13 +/-0.16

ND

ND

3.40

0.46

0.47

4.33 +/-0.22

ND

ND

1.56

0.53

0.54

2.63 +/- 0.13

ND

ND

ND

ND

2.42

0.40

0.40

3.22 +/- 0.16

2.46

0.72

0.72

3.89 +/-0.19

ND

ND

1.44 (2.39%)

0.38 (5.58%)

0.39 (3.04%)

2.22 +/- 0.12

ND

ND

1.58

0.44

0.43

2.45 +/- 0. 22

ND

ND

2.63

0.34

0.34

3.31 +/- 0.17

ND

ND

ND

ND

ND

ND

1.43 (2.97%) 1.68

ND

ND

ND

ND

ND

1.43 +/- 0.08 1.68 +/- 0. 09

ND

ND

ND

4.50 (5.17%) 4.29

ND

ND

ND

ND

3.40

ND

ND

ND

ND

5.20 (2.81%)

ND

ND

ND

ND

1.61

ND

ND

1.61 +/- 0.08

0.89

1.04

10.48

0.83

ND

13.23 +/- 0.62

0.38 (6.4%)

0.4 (6.09%)

8.81 (3.48%)

0.76 (3.79%)

ND

10.34 +/-0.51

ND

ND

15.8 (2.98%)

20.1 (3.65%)

ND

35.9 +/- 0.95


4.50 +/- 0.25 4.29 +/- 0.22 3.40 +/- 0.17 5.20 +/- 0.26


Figure 1


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Figure 2



Figure 3


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Table of Contents Graphic




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Profiling and quantification of regioisomeric ... - ACS Publications

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