How to distinguish between cinnamoylshikimate esters and chlorogenic acid lactones by liquid...

Research Article

Received: 27 April 2011 Accepted: 15 July 2011 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jms.1972

How to distinguish betweencinnamoylshikimate esters and chlorogenicacid lactones by liquid chromatography–tandem mass spectrometryRakesh Jaiswal,a Marius Febi Matei,a Frank Ullrichb and Nikolai Kuhnerta*

In this study, liquid chromatography–tandem mass spectrometry (LC–MSn; n=2–3) has been used to characterize and distin-guish chlorogenic acid lactones from cinnamoylshikimate esters. This is the first time when an LC–MSn method has been de-veloped to distinguish between these two isomeric classes of compounds formed in particular in food processing fromchlorogenic acids at elevated temperature through loss of water. The structures of regioisomeric chlorogenic acid lactonesand shikimate esters have been assigned on the basis of LC–MSn patterns of fragmentation, relative hydrophobicity, and frag-mentation analogy with the synthetic standards of dimethoxycinnamic, ferulic, and caffeic acid containing monoacyl chloro-genic acid lactones and shikimate esters. Copyright © 2011 John Wiley & Sons, Ltd.

Keywords: chlorogenic acid lactone; caffeoylshikimate ester; feruloylshikimate ester; tandem mass spectrometry; coffee

* Correspondence to: N. Kuhnert, School of Engineering and Science, Chemistry,Jacobs University Bremen, Campus Ring 8, 28759 Bremen, Germany E-mail: [email protected]

a School of Engineering and Science, Chemistry, Jacobs University Bremen,Campus Ring 8, Bremen 28759, Germany

b Kraft Food R&D, Unterbiberger Str. 15, München 81737, Germany

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INTRODUCTION

Classically, chlorogenic acids (CGAs) are a family of esters formedbetween quinic acid and certain trans-cinnamic acids, most com-monly caffeic, p-coumaric, and ferulic acids[1–3] and sometimesdimethoxycinnamic, trimethoxycinnamic, and sinapic acids.[4,5]

Representative structures are shown in Fig. 1. In the IUPAC sys-tem, (�)-quinic acid is defined as 1 L-1(OH),3,4/5-tetrahydroxycy-clohexane carboxylic acid, but Eliel and Ramirez[6] recommend1a,3R,4a,5R-tetrahydroxycyclohexane carboxylic acid. CGAs arewidely distributed in plants,[2,3] but the coffee bean is remarkablyrich, containing at least 72 CGAs, at an average level of 200mgper cup of coffee, that are not acylated at the C1 of the quinicacid moiety.[4,5,7–10]

The immense interest in CGAs originates from their consider-able dietary burden, which is estimated at a level of 2 g per hu-man per day. Most edible plants contain various levels of CGAs,and detailed figures have been reviewed by Clifford.[2,3] There isincreasing epidemiological evidence that this class of com-pounds has a series of health benefits.[11–13] Furthermore, severalpharmacological activities of CGAs have been demonstratedin vitro and in vivo, including the antioxidant activity,[14–16] theability to increase hepatic glucose utilization,[17–21] the inhibitionof the HIV-1 integrase,[22–24] the antispasmodic activity,[25] andthe inhibition of the mutagenicity of carcinogenic compounds,[26]

have been revealed by studies so far.Previous work has established the presence of CGAs in a vari-

ety of fruits,[27–29] vegetables,[30–32] and plant-based beveragessuch as green tea,[33] maté,[5] or green coffee beans.[4,7–9,34,35]

Most such dietary materials are, however, not consumed in theirnative state but are subjected to extensive food processing. Suchfood processing usually involves treatment of the plant raw ma-terial at elevated temperatures, for example, baking, roasting, or

J. Mass. Spectrom. 2011, 46, 933–942

frying of vegetables, baking or cooking of fruits, or roasting ofcoffee. The secret behind achieving the desired aroma and tasteof coffee, for instance, lies in the roasting of the green coffeebeans because of the formation of CGAs derivatives and Maillardreaction products.[36–38] CGAs and hence their uncharacterizedderivatives formed in this Maillard process are known to contrib-ute to the acidity, the astringency, and the bitterness of the finalbeverage.[39–44]

It has been unequivocally shown that the large majority ofCGAs in the crude plant material do not survive these harsh con-ditions (food processing) but are chemically transformed intoother structures. For coffee, it has been demonstrated by Sheareret al.[45] that CGA lactones (CGLs) are formed in the roasting pro-cess (Fig. 2). Along with CGAs, CGLs also contribute to coffee fla-vor,[46,47] and despite their low concentrations, their effect on thefinal cup quality may be significant. CGLs have also been studiedfor their potential hypoglycemic effects[45] and for their actions atopioid and adenosine brain receptors.[48,49]

Very little is known about alternative reaction products gener-ated at elevated temperatures from the ubiquitous CGAs, but itcan be anticipated that many more are formed to explain the dis-appearance of CGAs from chromatographic data after foodprocessing. In the formation of CGL by the loss of water, an intra-molecular ester bond is formed; however, other mechanisms

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Figure 1. Structures, numbering, nomenclature, and substituents of CGAs and their derivatives.

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involving loss of water are eminently feasible, for example, theelimination of water from C1 of the quinic acid moiety to formtwo isomeric shikimic acid ester derivative, with the new doublebond connecting C1 and C2 (naturally occurring form) or con-necting C1 and C6. The shikimates described here are the naturalform, and further investigation will be required to determinewhether the other isomer is produced during food processing.It should be noted that caffeoylquinic acid lactone (CQL) and caf-feoylshikimic acid (CSA) are constitutional isomers, therefore dis-playing identical m/z values in their mass spectra with similarpolarities and a resulting similar chromatographic behavior.The aim of this contribution is to provide an analytical framework

to allow for a detailed study of the dehydration products of theCGAs, including CQLs and CSAs. The novel analytical methodologyaddresses unambiguously the questioned presence of the hydroxy-cinnamoyl shikimates in roasted coffee and offers a straightforwardway to discriminate between them and the CGLs by liquid chroma-tography–tandem mass spectrometry (LC–MSn).Because CGAs exist in plants as a complex mixture of many

regioisomeric structures, regioisomers can as well be expected forCQLs and CSAs, necessitating in an LC–MS method that allows forunambiguous differentiation between the two constitutional iso-meric groups of compounds and all of their possible regioisomers.Recently, LC–MSn has been used to characterize cinnamoyl-aminoacid conjugates and [50] monoacyl caffeoylshikimates[5] and todiscriminate between individual isomers of monoacyl, diacyland triacyl CGAs.[4,5,7–9,34,35] The MS fragmentation patterns intandem MS spectra, UV spectrum, retention time, relative hydro-phobicity, and synthetic standards have been used to develop

Figure 2. Formation of CGA derivatives by loss of a H2O molecule during th

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structure-diagnostic hierarchical keys for the identification ofCGAs and shikimates. In the present study, we applied thesemethods to distinguish between CGLs and shikimates.

EXPERIMENTAL

Chemicals

All the chemicals (Analytical grade) were purchased from Sigma-Aldrich (Bremen, Germany). 4-Caffeoylquinic acid was purchasedfrom PhytoLab (Vestenbergsgreuth, Germany).

LC–MSn

The LC equipment (Agilent 1100 series, Karlsruhe, Germany) com-prised a binary pump, an auto sampler with a 100-mL loop, and aDAD detector with a light-pipe flow cell (recording at 320 and254 nm and scanning from 200 to 600 nm). This was interfacedwith an ion-trap mass spectrometer fitted with an ESI source(Bruker Daltonics HCT Ultra, Bremen, Germany) operating in full-scan Auto-MSn mode to obtain fragment ion m/z. Tandem massspectra were acquired in Auto-MSn mode (smart fragmentation)using a ramping of the collision energy. Maximum fragmentationamplitude was set to 1 V, starting at 30% and ending at 200%.MS operating conditions (negative mode) had been optimizedusing 5-caffeoylquinic acid with a capillary temperature of365 �C, a dry gas flow rate of 10 L/min, and a nebulizer pressureof 10 psi. High-resolution LC–MS was carried out using the same

e roasting of coffee.

J. Mass. Spectrom. 2011, 46, 933–942Wiley & Sons, Ltd.

Figure 3. Synthesis of 1-caffeoyl-1,5-quinide (1-CQL) (4) and 4-caffeoyl-1,5-quinide (1-CQL) (6).

LC–MSn of chlorogenic acid lactones and shikimates

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high-performance liquid chromatography (HPLC) equipped with aMicrOTOF mass spectrometer (Bruker Daltonics, Bremen, Germany)fitted with an ESI source, and internal calibration was achieved with10mL of 0.1M sodium formate solution injected through a six portvalve before each chromatographic run. Calibration was carried outusing the enhanced quadratic mode.

High-performance liquid chromatography

Separation was achieved on a 150� 3mm i.d. column containingdiphenyl 5mm, with a 5 � 3mm i.d. guard column (Varian, Darm-stadt, Germany). Solvent A was water/formic acid (1000:0.005 v/v)and solvent B was methanol. Solvents were delivered at a totalflow rate of 500 mL/min. The gradient profile was from 10% B to70% B linearly in 60min followed by 10min isocratic and a returnto 10% B at 90min and 10min isocratic to reequilibrate.

Synthesis of the mixtures of regioisomers of CSA (1–3), feru-loylshikimic acid (FSA) (7–9), and dimethoxycinnamoylshikimicacid (DSA) (13–15)

Pyridine (4mL) and 4-acetylferuloyl/3,4-diacetylcaffeic/dimethoxy-cinnamic acid chloride (0.52mmol) were added into a solution of

J. Mass. Spectrom. 2011, 46, 933–942 Copyright © 2011 John

shikimic acid (100mg, 0.52mmol) and DMAP (8mg, 0.06mmol) inCH2Cl2 (10mL) at room temperature. The reaction mixture wasstirred for 12 h and acidified with 1M HCl (pH� 2) and stirredfor 30min to remove the acetyl protecting group. The layers wereseparated, and the aqueous phase was reextracted with CH2Cl2(1 � 20mL) and EtOAc (2 � 20mL). The combined organic layerswere dried over Na2SO4 and filtered, and the solvents were re-moved in vacuo. The resulting esters were analyzed by HPLC–MS.

Synthesis of the mixtures of the regioisomers of CQLand feruloylquinic acid lactone (FQL) (4–6 and 10–12,respectively)

Pyridine (4mL) and 3,4-diacetylcaffeic/4-acetylferulic/dimethoxy-cinnamic acid chloride (0.52mmol) were added into a solution of1,5-quinide (100mg, 0.52mmol) and DMAP (8mg, 0.06mmol) inCH2Cl2 (10mL) at room temperature. The reaction mixture wasstirred for 12 h and acidified with 1M HCl (pH� 2) and stirredfor 30min to remove the acetyl protecting group. The layers wereseparated, and the aqueous phase was reextracted with CH2Cl2(1 � 20mL) and EtOAc (2 � 20mL). The combined organic layerswere dried over Na2SO4 and filtered, and the solvents were re-moved in vacuo. The resulting esters were analyzed by HPLC–MS.

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Table 1. Negative ion MS[2], MS[3], and MS[4] fragmentation data for the cinnamoylshikimate esters and CGLs

No. Compound MS[1] MS[2] MS[3]

Parent ion Base peak Secondary peak Base peak Secondary peak

m/z m/z int m/z int m/z int m/z m/z int m/z int m/z int

1 3-CSA 335.1 178.9 178.5 50 134.9 7 85.3 127.0 71 172.9 67

2 4-CSA 335.1 178.9 178.9 60 190.8 20 135.0 9 93.2 111.0 48

3 5-CSA 335.1 178.9 178.5 5 135.0 15 85.2 126.9 66 172.9 27

4 1-CQL 335.1 160.8 172.8 67 132.8 14 132.8

5 3-CQL 335.1 160.8 134.8 82 132.8

6 4-CQL 335.1 160.8 134.8 17 132.9

7 3-FSA 349.1 192.9 155.0 10 148.9 177.9 73 134.0 78

8 4-FSA 349.1 192.9 174.9 24 154.9 24 137.0 13 148.9 177.9 63 134.0 75

9 5-FSA 349.0 192.9 155.0 27 148.9 177.9 81 134.0 71

10 1-FQL 349.0 172.7 192.7 56 175.0 88 159.7 19 93.1 159.7 22 110.9 32

11 3-FQL 349.0 174.7 192.7 42 148.7 65 133.8 32 159.7

12 4-FQL 349.1 174.7 192.7 41 148.7 13 159.7 20 159.7

13 3-DSA 363.1 206.8 154.8 70 136.8 45 294.7 35 115.0 130.8 93

14 4-DSA 363.1 154.8 206.8 15 136.7 50 136.8 111.0 12 93.0 15

15 5-DSA 363.1 154.8 206.8 50 136.7 50 110.9 16 136.8 111.0 15 93.0 20

16 1-DQL 363.1 206.8 154.8 17 132.8 191.8 19 162.8 66 148.9 20

17 3-DQL 363.1 206.8 148.8 190.8 23 162.8 32 134.8 34

18 4-DQL 363.1 206.8 148.8 191.8 52 130.8 67

int, intensity.

Figure 4. Proposed fragmentation pathways of the regioisomers of caffeoyl-1,5-quinide (CQL) (4–6).

R. Jaiswal et al.

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Figure 5. Extracted ion chromatograms (EIC) at m/z 335 and MS[3] spectra for CQLs (4–6) in negative ion mode.


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Synthesis of 1-caffeoyl-1,5-quinide (1-CQL) (4)

Triethylamine (10mL) and 3,4-di-O-allyl-caffeic acid chloride(1400mg, 5.02mmol)[52] were added into a solution of 3,4-isopropylidene-1,5-quinide (717mg, 3.35mmol)[51] and DMAP(123mg, 1.01mmol) in CH2Cl2 (35mL) at room temperature.The reaction mixture was refluxed for 24 h and acidified with1M HCl (pH� 2). The layers were separated, and the aqueousphase was reextracted with CH2Cl2 (3 � 30mL). The combinedorganic layers were dried over Na2SO4 and filtered, and the sol-vent was removed in vacuo. The resulting ester, 1-O-(3,4-di-O-allylcaffeoyl)-3,4-isopropylidene-1,5-quinide, was purified bycolumn chromatography (petroleum ether–ethyl acetate 8:2)to obtain a pale yellow solid (1042mg, 2.28mmol).

1H-NMR (CDCl3): d 7.62 (d, 1H, CAr-CH), 7.08 (d, 1H, CArH), 7.05(s, 1H, CArH), 6.86 (d, 1H, CArH), 6.26 (d, 1H, CAr-CH=CH), 6.06(m, 2H, CH2 = CH), 5.44 (t, 1H, CHH=CH), 5.40 (t, 1H, CHH=CH),5.31 (d, 1H, CHH=CH), 5.28 (d, 1H, CHH=CH), 4.80 (dd, 1H, C5H),4.63 (tt, 4H, CH2-O), 4.55 (dt, 1H, C4H), 4.33 (d, 1H, C3H), 3.10(m, 1H, C6H), 2.62 (d, 1H, C2H), 2.51 (ddd, 1H, C6H), 2.42 (dd, 1H,C2H), 1.53 (s, 3H, CH3), and 1.33 (s, 3H, CH3).

13C-NMR (CDCl3): d 173.76 (C1-COO), 165.32 (COO-C1), 151.12(CAr-OPr), 148.64 (CAr-OPr), 146.71 (CAr-CH), 133.09 (CH2 =CH),132.89 (CH2 =CH), 127.21 (CAr-CH), 123.22 (CAr), 118.13 (CH2 =CH),118.07 (CH2= CH), 114.48 (CH-COO), 113.40 (CAr), 112.78 (CAr), 110.05(CH3-CH), 76.13 (C1), 75.51 (C4), 72.58 (C3), 71.28 (C5), 70.04 (CH2-O),69.78 (CH2-O), 35.78 (C6), 30.86 (C2), 27.08 (CH3), and 24.44 (CH3).

1-O-(3,4-di-O-allylcaffeoyl)-3,4-isopropylidene-1,5-quinide(963mg, 2.11mmol) was suspended in 60mL of methanol/water


(9:1) mixture, and PTSA.H2O (84mg, 0.44mmol) was added.The reaction mixture was put under a N2 atmosphere, and10% Pd/C (840mg) was added slowly after which themixture was refluxed (60 �C–70 �C) for 48 h. The reaction mixturewas then cooled to room temperature and filtered, and themethanol was removed in vacuo. A volume of 30mL of distilledwater was added, and the aqueous reaction mixture wasextracted with ethyl acetate (3 � 50mL). The combined organiclayers were dried over Na2SO4 and filtered, and the solvent wasremoved in vacuo. The crude product was purified by columnchromatography (petroleum ether–ethyl acetate 8:2) to give 1-O-caffeoyl-3,4-isopropylidene-1,5-quinide as a white powder (113mg,0.30mmol).[53]

1H-NMR (acetone-D6): d 8.35 (br, 2H, 2xCAr-OH), 7.58 (d, 1H,CAr-CH), 7.17 (d, 1H, CArH), 7.06 (dd, 1H, CArH), 6.86 (d, 1H, CArH),6.28 (d, 1H, CAr-CH=CH), 4.81 (dd, 1H, C5H), 4.62 (dt, 1H, C4H), 4.34(m, 1H, C3H), 3.08 (m, 1H, C6H), 2.53 (d, 1H, C2H), 2.44 (ddd, 1H,C6H), 2.31 (dd, 1H, C2H), 1.48 (s, 3H, CH3), and 1.31 (s, 3H, CH3).

13C-NMR (acetone-D6): d 173.09 (C1-COO), 164.94 (COO-C1),148.38 (CAr-OH), 146.67 (CAr-CH), 145.52 (CAr-OH), 126.50 (CAr-CH),122.18 (CAr), 115.62 (CAr), 114.63 (CH-COO), 113.60 (CAr), 109.51(CH3-CH), 75.99 (C1), 75.24 (C4), 72.58 (C3), 71.17 (C5), 35.74 (C6),30.27 (C2), 26.42 (CH3), and 23.73 (CH3).

1-O-caffeoyl-3,4-isopropylidene quinide (79mg, 0.21mmol)was dissolved in a trifluoro acetic acid (TFA) 80% solution(3.75mL), and the solution was stirred for 3 h at room tempera-ture. The solvents were removed in vacuo to afford the target paleyellow product, 1-O-caffeoyl-1,5-quinide (70mg, 0.21mmol),without further purification.


Figure 6. Proposed fragmentation pathways of regioisomers of FSA (7–9).

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1H-NMR (acetone-D6): d 7.56 (d, 1H, CAr-CH), 7.16 (s, 1H, CArH),7.06 (d, 1H, CArH), 6.85 (d, 1H, CArH), 6.27 (d, 1H, CAr-CH=CH), 4.85(t, 1H, C5H), 4.06 (t, 1H, C4H), 3.81 (td, 1H, C3H), 3.02 (m, 1H,C6H), 2.56 (d, 1H, C2H), 2.13 (d, 1H, C6H), and 2.02 (p, 1H, C2H).

13C-NMR (acetone-D6): d 172.27 (C1-COO), 164.97 (COO-C1),148.34 (CAr-OH), 146.48 (CAr-OH), 145.53 (CAr-CH), 126.51 (CAr-CH), 122.12 (CAr), 115.59 (CAr), 114.59 (CH-COO), 113.77 (CAr),76.59 (C1 + C4), 66.10 (C3), 65.79 (C5), 37.04 (C6), and 32.73 (C2).

Synthesis of 4-caffeoyl-1,5-quinide (4-CQL) (6)

Toluene (10mL) and p-toluenesulfonic acid (0.2mg) were addedinto a solution of 4-caffeoylquinic acid (0.2mg) in DMSO(0.2mL) at room temperature. The reaction mixture was heatedat 90 �C for 10 h and neutralized by adding solid sodiumbicarbonate (5mg). The solvents were removed in vacuo, andthe residue was dissolved in acetone and filtered. The filtratewas dried in vacuo and used for HPLC–MS analysis.

RESULTS AND DISCUSSION

Preliminary assessment of data

All data for CGAs presented in this paper use the recommendedIUPAC numbering system,[1] and the same numbering system has


been adopted for CGLs and shikimates. Their structures are pre-sented in Fig. 1. When necessary, previously published data hasbeen amended to ensure consistency and avoid ambiguity.

In general, peak identities were consistent both within andbetween analyses. However, when the mass spectrum for a partic-ular compound included two ions of similar mean intensities,within-analysis experimental error dictated that in some individualMS scans, one would be more intense and for other scans the re-verse would be true. This phenomenon was encountered primarilywhen the signal intensity was lower, that is, with quantitativelyminor components and/or higher order spectra. For example,1-feruloyl-1,5-quinide lactone (1-FQL) (10) produces MS[2] ionsat m/z 173 and 175, which are essentially coequal in some spec-tra. However, in this particular case, the lower mass ion has beenassigned consistently as the base peak. Fragment ions with inten-sities <10% of the base peak have been reported only when theyare needed for comparison.

For the selective syntheses of 1-CQL (4) and 4-CQL (6), themethods reported in literature were followed with little modifica-tions and the third regioisomer (3-CQL 5) follows automatically ina mixture of all the three regioisomers (Fig. 3). Mixtures of theregioisomers of shikimates and lactones were synthesized by asimple condensation reaction between a cinnamoyl chlorideand a shikimic acid or quinic acid lactone. In these mixtures, theirelemental composition was confirmed by high-resolution mass


Figure 7. Extracted ion chromatogram (EIC) at m/z 349 and MS[3] spectra for FSAs (7–9) in negative ion mode.


spectrometry with the error being less than 5 ppm. The order ofelution for the cinnamoylshikimate esters and lactones were5> 4> 3 and 3> 4> 1, respectively; the data are in agreementwith the literature.[5,54]

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Characterization of CSAs and CQLs (Mr 336)

CSAs and CQLs showed identical MS[1] spectra with the parention at m/z 335. All the CSAs produced an MS[2] base peak atm/z 179 ([caffeic acid-H+]�) by the loss of the shikimate partTable 1), whereas the CQLs showed an MS[2] base peak at m/z161 ([caffeic acid-H2O-H

+]�) by the loss of the lactone part anda H2O molecule (Fig. 4 and 5). Consequently, they can be identi-fied and distinguished by their MS[2] spectra.

For the discrimination between CSAs and CQLs or even withinthe same class, MS[2] and MS[3] spectra were used. Recently, wereported on a tandem MS method for discriminating betweencaffeoylshikimates.[5]

The 1-CQL (4) produced the MS[2] base peak at m/z 161 andMS[2] secondary peaks at m/z 179 ([caffeic acid-H+]�), 173([quinide-H+]�) and 133 ([caffeic acid-H2O-CO-H

+]�). The 3-CQL(5) and 4-CQL (6) produced the MS[2] base peak at m/z 161 butMS[2] secondary peaks with different intensities at m/z 179([caffeic acid-H+]�) and 133 ([caffeic acid-H2O-CO-H

+]�) (Table 1).The MS[2] secondary peak at m/z 173 was absent in 3-CQL (5)and 4-CQL (6) (Fig. 5 and Table 1).


Characterization of FSAs (7–9) and FQLs (10–12) (Mr 350)

FSAs and FQLs produced the parent ion at m/z 349 and the MS[2]

base peaks at m/z 193 ([ferulic acid-H+]�) (Figs 6 and 7), m/z 173([quinide-H+]�), and m/z 175 ([ferulic acid-H2O-H

+]�) (Figs 8 and9), respectively. Consequently, FSAs and FQLs can be distin-guished by their MS[2] spectra.

4-FSA (8) produced the MS[2] secondary peak atm/z 175, whichis absent in 3-FSA (7) and 5-FSA (Figs 6 and 7). 3-FSA producedMS[2] secondary peaks at m/z 178 ([ferulic acid-CH3-H

+]�), whichis absent in 5-FSA (9) (Fig. 7). Thus, on the basis of the abovearguments, FSAs were easily discriminated.

3-FQL (11) and 4-FQL (12) produced the common MS[2] basepeak at m/z 175 ([ferulic acid-H2O-H

+]�) and a secondary peakat m/z 160. 3-FQL produced MS[2] secondary peaks at m/z 149([ferulic acid-CO2-H

+]�) and 193 ([ferulic acid-H+]�), the latterbeing either absent or of very low intensity in 4-FQL. 1-FQL (10)produced the MS[2] base peak at m/z 173 and secondary basepeaks at m/z 175 and 193.

Characterization of DSAs (13–15) and dimethoxycinnamoylquinicacid lactones (16–18) (Mr 364)

DSAs produced the MS[2] base peak at either m/z 155 ([quinide-H2O-H

+]�) or m/z 207 ([dimethoxycinnamic acid-H+]�), whereasdimethoxycinnamoylquinic acid lactones (DQLs) produced theMS[2] base peak at m/z 207 ([dimethoxycinnamic acid-H+]�).


Figure 8. Proposed fragmentation pathways of regioisomers of feruloyl-1,5-quinide (FQL) (10–12).

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Consequently, they can be distinguished on the basis of theirMS[2] spectra.5-DSA (15) and 4-DSA (14) produced the MS[2] base peak atm/z

155 ([shikimic acid-H2O-H+]�) and a secondary base peak at m/z

137 ([shikimic acid-2H2O-H+]�) and 207 ([dimethoxycinnamic

acid-H+]�), respectively, but with different intensities (Table 1).In the MS[2] spectra, 5-DSA 15 favors the loss of shikimic acid partif compared with 4-DSA (Table 1). 5-DSA also produced MS[2]

secondary peaks atm/z 259, 301, and 345 (10% of the base peak),which are completely absent in 4-DSA and 3-DSA. 5-DSA and4-DSA produced the MS[3] base peak at m/z 137 ([shikimicacid-2H2O-H

+]�) and a secondary base peak at m/z 93 ([shikimicacid-2H2O-CO2-H

+]�) with comparable intensities. 3-DSA pro-duced the MS[2] base peak at m/z 207 and secondary peaks atm/z 155, 137, and 295 (absent in 4-DSA and 5-DSA) (Table 1).All the three DQLs produced MS[2] spectra at m/z 207

([dimethoxycinnamic acid-H+]�), but 3-DQL (17) did not produceany secondary ion (Table 1). 4-DQL (18) and 1-DQL (16) producedthe MS[2] secondary peak at m/z 295 and 155 ([quinide-H2O-H+]�), respectively. Both 3-DQL and 4-DQL produced the MS[3]

base peak at m/z 149 by the loss of a CO fragment and a HCHOmolecule (Table 1). 3-DQL produced MS[3] secondary peaks at


m/z 116 (23%) and 131 (32%), both being absent in 4-DQL. 1-DQL produced the MS[3] base peak at m/z 133 and secondarypeaks at m/z 163 and 192.

CONCLUSIONS

In this study, we were able to distinguish between the CGLs andthe cinnamoylshikimate esters on the basis of their behavior onreverse phase column and nonidentical tandem mass spectra.We were also able to discriminate between the individual typesof cinnamoylshikimate esters and CGLs. LC–MSn is not onlylimited to monoacyl CGLs and shikimates but might also proveuseful for the diacylated derivatives. These methods can beapplied for the identification of CGLs and shikimates in roastedcoffee as well as in other food materials.

Acknowledgments

The authors acknowledge funding from Kraft Food and JacobsUniversity Bremen gGmbH and excellent technical support fromMs Anja Müller.


Figure 9. Extracted ion chromatogram (EIC) at m/z 349 and MS[3] spectra for FQLs (10–12) in negative ion mode.


Supporting Information

Supporting information can be found in the online version of thisarticle.

REFERENCES

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