Liquid chromatographic–electrospray ionization mass spectrometric analysis of neutral and charged...

transcript

UNCORRECTED PROOFS

JMS541

JOURNAL OF MASS SPECTROMETRYJ. Mass Spectrom. 2003; 38: 000–000Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.541

Liquid chromatographic/electrospray ionization massspectrometric studies of proanthocyanidins in foods

Liwei Gu,1 Mark A. Kelm,2 John F. Hammerstone,2 Ze Zhang,3 Gary Beecher,4

Joanne Holden,4 David Haytowitz4 and Ronald L. Prior1,5∗

1 Arkansas Children’s Nutrition Center, 1120 Marshall Street, Little Rock, Arkansas 72202, USA2 Analytical and Applied Sciences Group, Mars Incorporated, 800 High Street, Hackettstown, New Jersey, 07840, USA3 Bruker Daltonics, Manning Park, Billerica, Massachusetts, 01821, USA4 USDA–ARS, Food Composition Laboratory, Beltsville Human Nutrition Center, 10300 Baltimore Avenue., Beltsville, Maryland, 20705, USA5 US Department of Agriculture, Agriculture Research Servicež Q1

Received 18 July 2003; Accepted 6 September 2003

The proanthocyanidins in three foods (pinto beans, plums and cinnamon) were studied with electrosprayionization (ESI) mass spectrometry (MS) in the negative mode following separation by normal-phasehigh-performance liquid downatography. The MS/MS analysis demonstrated that the major ions derivedfrom heterocyclic ring fission and retro-Diels–Alder reaction of flavan-3-ol provided information aboutthe hydroxylation pattern and type of interflavan bond. The connection sequence of the oligomers wasidentified through diagnostic ions derived from quinone methide (QM) cleavage of the interflavan bond.Novel heterogeneous B-type proanthocyanidins containing (epi)afzelechin as subunits were identified inpinto beans. Proanthocyanidins with interestingly different A-type linkages were identified in plums andcinnamon. In efforts aimed at extending the identification capacity of ESI-MS to polymers, we found thatthe polymeric procyanidins fragmented readily instead of forming multiply charged ions in the negativeESI mode. Fragmentation patterns were proposed based on our data obtained by ESI-MS/MS and ESItime-of-flight MS. Copyright 2003 John Wiley & Sons, Ltd.

KEYWORDS: catechin; propelargonidin; procyanidin; proanthocyanidins; tannins; foods; liquid chromatography/massspectrometry

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INTRODUCTION

Proanthocyanidins, better known as condensed tannins,are ubiquitous and present as the second most abundantclass of natural phenolic compounds after lignin.1 Proantho-cyanidins existing in many foods possess potential health-promoting effects, such as antioxidant, anti-carcinogenicand anti-inflammatory effects.2 Proanthocyanidins are mix-tures of oligomers and polymers composed of flavan-3-olunits, linked mainly through C4—C8 bonds; however,C4—C6 bonds also exist. The flavan-3-ol units can alsobe doubly linked by an additional ether bond between C2and O7. Proanthocyanidins containing the single interfla-van linkages are B-type, whereas those containing doubleinterflavan linkages are A-type. The size of the proantho-cyanidin molecule is described by its degree of polymeriza-tion (DP).3 The common flavan-3-ols in proanthocyanidinsare shown in Fig. 1. The proanthocyanidins that consistexclusively of (epi)catechin are procyanidins. Proantho-cyanidins containing (epi)afzelechin or (epi)gallocatechinas subunits are called propelargonidin or prodelphinidin,respectively. Propelargonidin or prodelphinidin are mostlyheterogeneous in their constituent units and co-exist withthe procyanidins.3

ŁCorrespondence to: Ronald L. Prior, Arkansas Children’sNutrition Center, 1120 Marshall Street, Little Rock, Arkansas,72202, USA. E-mail: PriorronaldL@uams.edu

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Owing to the structural complexity of proanthocyani-dins, mass spectrometric (MS) studies of them are limitedin comparison with other polyphenols.4 Moreover, most ofthese studies have focused on either monomers or homo-geneous B-type procyanidins.5 – 8 Very few papers have dis-cussed the heterogeneous proanthocyanidins and the A-typeproanthocyanidins.9,10 Elucidation of the structures of proan-thocyanidins has relied on strenuous purification steps fol-lowed by spectral and chemical degradation analysis (such asthiolytic degradation). However, most of these studies wereconfined to the level of dimers or trimers because of technicaldifficulties.3 The present studies demonstrate the capacityof liquid chromatography/electropray ionization tandemmass spectrometry (LC/ESI-MS/MS) as a rapid and sensi-tive method to determine the proanthocyanidins in extractsof food samples after separation using normal-phase high-performance liquid chromatography (HPLC). The negativeion mode, which had been demonstrated by other researchersto be more sensitive and selective than the positive mode forproanthocyanidins,6,10 was used in these studies.

EXPERIMENTAL

Reagents and samplesMethanol, methylene chloride and acetic acid (HPLC grade)were purchased from Fisher Scientific (Boston, MA, USA).Sephadex LH-20 is a product of Sigma Chemical (St. Louis,

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2 L. Gu et al.

Flavan-3-ols R1 R2 R3 R4 R5Afzelechin H H OHOH H

H OH HOH HH H OHOH OHH OH HOH OH

OH H OHOH OHOH OH HOH OH

EpiafzelechinCatechinEpicatechinGallocatechinEpigallocatechin

Figure 1. Structures of the flavan-3-ol units inproanthocyanidins.

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MO, USA). Food samples were in the form of freeze-dried powders. They were collected and processed in theDepartment of Biochemistry of Virginia Technical University(Blacksburg, VA, USA). The samples were shipped frozenon dry-ice and kept at �70 °C before use.

Reference compounds(�)-Epicatechin and (C)-catechin were purchased fromSigma Chemical. Seven partially pure procyanidin oligomers(dimers through octamers), which contain procyanidinhomologues with the same DP, have been described before.11

A polymeric procyanidin fraction with an average DPof 36.1 was used as a polymer standard. This polymerfraction doses not contain procyanidins with DP � 10. Itwas fractionated from blueberries on a Sephadex LH-20column. Characterization of this polymer fraction has beendescribed.12

Sample extraction and purificationThe sample (1 g) was extracted in a 15 ml screw-cappedtube with 10 ml of mixed solvent (acetone–water–aceticacid, 70 : 29.5 : 0.5 v/v/v). After adding solvent, the tubewas vortex mixed for 30 s followed by sonication at 37 °Cfor 10 min. The tube was kept at room temperature for50 min. At the end of extraction, the tube was centrifugedat 3500 rpm for 15 min. Part of the supernatant (7.5 ml) waspipetted out and the acetone was evaporated at 25 °C ina SpeedVac (SC210A; Thermo, Marietta, OH, USA) undervacuum (1.5 Torr (1 Torr D 133.3 Pa)). The residue afterevaporation of acetone was dissolved in ¾6 ml of 30%(v/v) aqueous methanol and loaded on a Sephadex LH-20 column. The column (6 ð 1.5 cm i.d.) was packed with3 g of Sephadex LH-20, which was equilibrated with 30%(v/v) aqueous methanol for >4 h before use. After loadingthe sample, the column was washed with 40 ml of 30% (v/v)aqueous methanol to remove sugars and other phenols.Proanthocyanidins were recovered from the column byeluting with 70 ml of 70% (v/v) aqueous acetone. Effluentswere evaporated to dryness under vacuum in a SpeedVac at25 °C. The dried substance was dissolved in the extractionsolvent and transferred to a volumetric flask. The finalvolume was brought up to 5 ml. The solution was centrifuged

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at 14 000 rpm, then 10 µl were injected for normal-phaseLC/MS/MS analyses.

HPLC and ESI-MS/MSChromatographic separation was performed on an Agilent1100 HPLC system consisting of a binary pump, a quaternarypump, a solvent degasser, an autosampler, a thermostat col-umn compartment, a diode-array detector and a fluorescencedetector (Agilent Technologies, Wilmington, DE, USA). Sep-aration was carried on a 250 ð 4.6 mm i.d. Luna Silica (2)column (Phenomenex, Torrance, CA, USA) with a particlesize of 5 µm at a column temperature of 37 °C. The ter-tiary mobile phase consisted of (A) methylene chloride, (B)methanol and (C) acetic acid–water (1 : 1 v/v). The 70 mingradient was as follows: 0–20 min, 14.0–23.6% B linear;20–50 min, 23.6–35.0% B linear; 50–55 min, 35.0–86.0% Blinear; 55–65 min, 86.0% B isocratic; 65–70 min, 86.0–14.0%B linear, followed by 10 min of re-equilibration of the col-umn before the next run. A constant 4.0% of C was keptthroughout the gradient. UV detection was carried out at280 nm (8 nm bandwidth) versus a reference wavelength at650 nm (30 nm bandwidth). For fluorescence detection, theexcitation and emission wavelengths were 276 and 316 nm,respectively.

The eluting stream �1 ml/min�1� from the HPLC appa-ratus was introduced into a Bruker Esquire-LC ion trapmass spectrometer (Bruker Daltonics, Billerica, MA, USA).Enhancement of ionization of procyanidins using post-column addition of 10 mmol/l�1 ammonium acetate has beendescribed previously.13 We optimized the mass spectrome-ter parameters using the procyanidin oligomers, and theseparameters were applied to detect the proanthocyanidins.The nitrogen pressure and flow-rate on the nebulizer were50 psi and 10 l/min�1, respectively, with a drying gas tem-perature of 350 °C. The capillary voltage was 3.5 kV. The scanrange was set at m/z 150–2200. All other parameters wereadjusted according to the smart mode of the mass spectrom-eter using the Esquire Control software (version 4.5). Theywere expressed as compound stability level and ion traplevel, respectively. Settings were 50 and 25% for monomer,50 and 90% for dimers, 30 and 110% for trimers and 80and 110% for tetramers through decamers. The [M � H]�

ions of monomers through heptamers were set as targetmasses for the ion trap in their detection segment to increasesensitivity. The doubly charged ions �[M � 2H]2�� were setas target masses for octamers (m/z 1153), nonamers (m/z1297) and decamers (m/z 1441). The parameters for decamerswere applied to polymers. For direct injection MS analyses,samples were dissolved in 70% (v/v) aqueous acetone at0.5 mg ml�1 and delivered by a syringe pump (Cole-Parmer,Vernon Hills, IL, USA) at a flow-rate of 100 µl min�1. Heliumwas applied as the collision gas in the ion trap at 1 ð 10�6 barto facilitate collision-induced dissociation (CID). A collisionenergy level of 100% was applied.

ESI-TOFMS of the polymersThe procyanidin polymer standard was dissolved in 70%(v/v) aqueous acetone and diluted to a concentration of4 µg ml�1. This solution was injected into an ESI time-of-flight

UNCORRECTED PROOFS

LC/MS of proanthocyanidins in foods 3

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(TOF) mass spectrometer (Bio-TOF II, Bruker Daltonics)at a flow-rate of 2 µl min�1. Mass spectra were recordedin the negative ionization mode. The voltages applied onhigh-voltage electrodes were �2200, �4000 and �4500 Vfor cylinder, end plate and capillary, respectively. Voltageson the capillary exit, trap and lens were 140, 45 and50 V, respectively. A single flight pass detection mode wasemployed.

RESULTS AND DISCUSSION

Tandem mass spectrometric studies of B-typeproanthocyanidinsUsing normal-phase HPLC, proanthocyanidin monomersthrough decamers (oligomers) were separated accordingto their DP. Other proanthocyanidins with DP > 10(polymers) were eluted as a single peak after 54.7 min.12

The proanthocyanidin profile of pinto beans is shown inFig. 2(A). Two kinds of dimers were detected in pinto beansat m/z 577 and 561. The m/z 577 ion is indicative of B-typeprocyanidin dimers. The m/z 561 ion is a minor componentwith 16 Da less than m/z 577. The product ion spectrum ofm/z 561 is shown in Fig. 3(A). The major ions containing thestructural information are m/z 435.0, 425.0, 289.0 and 271.0.

To describe the structure of a dimer, we define the twoflavan-3-ol units in the dimers as the top unit (1) and base unit(2) (Fig. 4). The m/z 425.0 ion is derived from m/z 561 after a136 Da neutral loss. Elimination of ring B from the flavan-3-olthrough retro-Diels–Alder (RDA) reaction of ring C led tothis ion.7 The RDA reaction could take place on the top unitor the base unit of the dimer. However, elimination of ringB on the top unit gives rise to a fragment ion with a larger� � � hyperconjugated system than RDA on the base unit.Hence it is more energetically favorable. The RDA cleavageof dimers on the top unit has been reported previously.9 Lossof 136 Da indicates that ring B of the top unit has a singlehydroxyl group. The m/z 425.0 ion loses a water molecule,most likely from the free 3-OH, to form a more stable ion atm/z 407.0. The m/z 435.0 ion ([M � H � 125]�� is formed afterthe elimination of ring A from the dimer. This fragmentationpathway differs from that of the RDA reaction.

The MS/MS study of catechin provided clues for thefragmentation mechanism of this dimer.8 This mechanismis designated as heterocyclic ring fission (HRF) and isillustrated in Fig. 4. The m/z 435.0 ion is formed after HRFon the top unit of the dimer. HRF on the base unit of thedimer is not favored for the same reason as RDA. Loss of126 Da indicates that the A ring of the top unit has a 1,3,5-trihydroxybenzene structure. Formation of the m/z 435.0 ionalso indicates that a single hydroxyl group is located at the40 position of the B ring, because absence or methylationof OH on 40 would inhibit the formation of m/z 435.0 ionby blocking the HRF pathway. Hence the top unit of thisdimer is identified as (epi)afzelechin. Because the chiralityof C-3 on the flavan-3-ols cannot be differentiated by MS,(epi)afzelechin represents either afzelechin or epiafzelechin.The connection sequence of this dimer has been deduced to be(epi)afzelechin–(epi)catechin. The unusual (epi)afzelechinhas not been detected in pinto beans before.

0 20 40 600

min15 17.5 20 22.5

561577

577451

833865

561577

B4B5 B6 B7

Polymers

B6 B7 B8 B9

575561

A6 A7 A8

Figure 2. Normal-phase HPLC fluorescence trace of theproanthocyanidins from (A) pinto beans, (B) plums and(C) cinnamon. The labels B1–B9 on the peaks indicate thedegree of polymerization of B-type procyanidins. A1–A6indicate the degree of polymerization of proanthocyanidinswith an A-type linkage. The regions of dimers and trimers inpinto beans and cinnamon are shown as insets. The [M � H]�

(m/z) of the peaks in the inset and also in the chromatogram ofcinnamon are listed.

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Identification of this sequence was confirmed by the frag-ment ions at m/z 271.0 and 289.0. These two ions were formedfrom the top and the base unit after quinone methide (QM)cleavage of the interflavan bond.7 Its isomeric dimers withan (epi)catechin–(epi)afzelechin connection would fragmentinto two different ions, at m/z 287.0 and m/z 273.0. QMcleavage of interflavan bonds produces the diagnostic ionsto identify the connection sequence of other heterogeneousoligomers. One minor peak of the trimer in pinto beansshowed [M � H]� at m/z 833.0 (Fig. 2(A)). The fragmentions with m/z 561.2, 543.1, 289.0 were observed in theproduct ion spectrum (Fig. 3(B)). The m/z 543.1 ion andits conjugate ion at m/z 289.0 were produced after QMcleavage of the interflavan bond between the middle unitand base units. Hence the base unit was identified as(epi)catechin. The cleavage of the interflavan bond betweenthe top and middle units gave rise to the m/z 561.2ion (Fig. 3(B)). The connection of this trimer was iden-tified to be (epi)afzelechin–(epi)afzelechin–(epi)catechin.

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4 L. Gu et al.

-MS/MS (561.0), 15.3 min (#424)

ns. ×

250 300 350 400 450 500 550 600

-MS/MS (833.0), 21.3 min (#588)

-MS/MS (849.0), 22.0 min (#606)0

300 400 500 600 700 800

519.1407.2

451.0407.3

Figure 3. Product ion spectra of a proanthocyanidin dimerand two trimers detected in pinto beans. The dimer has an(epi)afzelechin–(epi)catechin connection (A, m/z 561.0). Itsfragmentation pathway is illustrated in Fig. 4. The trimers are(epi)afzelechin–(epi)afzelechin–(epi)catechin (B, m/z 833.0)and (epi)afzelechin–(epi)catechin–(epi)catechin (C, m/z 849.0),respectively.

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Another trimer in pinto beans was identified to be(epi)afzelechin–(epi)catechin–(epi)catechin based on itsfragment ions of m/z 559.1 and 577.1 (Fig. 3(C)). For the trimer(epi)afzelechin–(epi)afzelechin–(epi)catechin, HRF on thetop and middle units produced ions at m/z 707.3 and 435.4,respectively. RDA on the top and middle units of the trimergave rise to m/z 679.6 and 407.2 ion (after water elimination).However, HRF on the top unit seems to produce the moreabundant ions for B-type proanthocyanidins than RDA.

Identification of the connection sequences of the hetero-geneous proanthocyanidins could be accomplished up totetramers and pentamers. These minor components weredistinguished from the predominant procyanidins by con-structing the extracted ion chromatograms from the total ionchromatograms (TIC). Four tetramers and two pentamershave been identified in pinto beans. Their [M - H]� andproduct ions are listed in Table 1. The low molecular massproanthocyanidins (DP < 5) are thought to be absorbed inthe human gastrointestinal tract.2 Detection of dimers inhuman blood has been reported recently after subjects hadconsumed a proanthocyanidin-rich diet.14 The capacity ofMS/MS to elucidate the detailed structures of these proan-thocyanidins is of great importance in order to evaluate theirabsorption and trace their metabolism.

Although the linkage sequence of the low proanthocyani-din oligomers could be deduced from their characteristicfragments, the position and stereochemistry of the interfla-van bond cannot be elucidated. Four peaks of procyanidindimers were detected in pinto beans at m/z 575, but theywere different stereoisomers that gave rise to the same tan-dem mass spectra. A similar observation has been madepreviously.15 The identification of the connection sequenceof the proanthocyanidin with DP D 6 was inconclusive onMS/MS because of the complexity of the fragmentation pat-terns. However, the proportion of constituent flavan-3-olscan be deduced for proanthocyanidins with DP D 6–10

-126 Da

m/z 561.1m/z 425.0

-136 Da

m/z 271.0

m/z 289.0

m/z 407.0

m/z 435.0

Figure 4. Fragmentation pathway of a proanthocyanidin dimer in pinto beans. The fragment mechanisms are RDA(retro-Diels–Alder), HRF (heterocyclic ring fission), and QM (quinone methide).

UNCORRECTED PROOFS

Table 1. Identification of tetramers and pentamers with heterogeneous subunits or A-type linkages

Source Connection sequence [M � H]� Product ions (m/z)

Pinto beans (epi)Afz–(epi)Afz–(epi)Cat–(epi)Cat 1121.3 849.0, 831.1, 577.2, 543.1(epi)Afz–(epi)Cat–(epi)Afz–(epi)Cat 1121.2 849.0, 831.1, 561.1(epi)Afz–(epi)Cat–(epi)Cat–(epi)Cat 1137.3 865.3, 577.2, 559.0(epi)Cat–(epi)Afz–(epi)Cat–(epi)Cat 1137.3 849.3, 847.3, 577.2, 559.1(epi)Afz–(epi)Afz–(epi)Cat–(epi)Cat–(epi)Cat 1409.3 1119.3, 865.0, 831.1, 577.1(epi)Afz–(epi)Cat–(epi)Cat–(epi)Cat–(epi)Cat 1425.3 1153.3, 1135.7, 865.1, 577.1

Plums (epi)Cat–(epi)Cat–A–(epi)Cat–A–(epi)Cat 1149.3 861.4(epi)Cat–(epi)Cat–(epi)Cat–A–(epi)Cat 1151.2 863.5, 575.1(epi)Cat–(epi)Cat–(epi)Cat–(epi)Cat–A–(epi)Cat 1439.3 1151.3, 863.4, 575.2

Cinnamon (epi)Afz–(epi)Cat–A–(epi)Cat–(epi)Cat 1135.2 863.2, 847.2, 573.0(epi)Cat–A–(epi)Cat–A–(epi)Cat–(epi)Cat 1149.3 859.2(epi)Cat–(epi)Cat–A–(epi)Cat–(epi)Cat 1151.2 863.4, 861.2, 573.2(epi)Cat–A–(epi)Cat–(epi)Cat–(epi)Cat 1151.2 861.3, 573.1(epi)Cat–A–(epi)Cat–A–(epi)Cat–(epi)Cat–(epi)Cat 1437.4 1147.4, 859.5(epi)Cat–(epi)Cat–(epi)Cat–A–(epi)Cat–(epi)Cat 1439.4 1151.2, 1149.4, 863.2, 575.1, 573.1(epi)Cat–(epi)Cat–A–(epi)Cat–(epi)Cat–(epi)Cat 1439.4 1151.3, 1149.4, 861.5, 577.2, 575.2, 573.1

(epi)Afz and (epi)Cat are abbreviations for (epi)afzelechin and (epi)catechin. –A– represents an A-type interflavan linkage.

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based on their [M � H]� or [M � 2H]2� ions. Most of theoligomers in pinto beans are homogeneous B-type procyani-dins. About 8.2% of the oligomers contain various numbersof (epi)afzelechin as constituent units. A peak labeled withan asterisk in the chromatogram of pinto beans (Fig. 2(A))gave rise to [M � H]� at m/z 451.1 and a fragment ion at m/z289.0. It was identified as (epi)catechin glycoside.

Tandem mass spectrometric studies of A-typeproanthocyanidinsThe profiles of proanthocyanidins in plums and cinnamonare shown in Figs 2(B) and 3(C). The proanthocyanidinswith one A-type linkage were identified readily on MS bytheir [M - H]� ions being 2 Da less than those of the B-typeproanthocyanidins. The product ion spectrum of the A-typetrimers (m/z 863.0) detected in plums is shown in Fig. 5(A). Adifferent product ion spectrum was observed for an A-typetrimer (m/z 863.0) in cinnamon (Fig. 5(B)). The characteristicion at m/z 575.1 derived from QM cleavage of the interflavanbond indicated that the A-type interflavan linkage of thetrimers in plums was between the middle and base units(Fig. 6(A)). The conjugate ion at (m/z 287.0) derived from thetop unit was not observed. The fragment ion at m/z 573.3 andits conjugate ion at m/z 289.0 revealed that the A-type linkageof the trimer in cinnamon was between the top and middleunits (Fig. 6(B)). It seems that the A-type interflavan bondsdo not undergo observable QM cleavage in the presence of aB-type interflavan bond in the same molecule.

Comparison between these two A-type isomers facili-tated the identification of a novel A-type trimer in cinnamon.The product ion spectrum of this trimer (m/z 847, [M - H]�)shows m/z 557.0 derived from QM cleavage (Fig. 5(C)).Its conjugate ion at m/z 289.0 indicates the base unit ofthis trimer is (epi)catechin. It can be deduced that the A-type is between the top and middle units. The m/z 411.0ion was formed after cleavage of the middle unit throughthe HRF mechanism. The same ion has been observed in

411.0451.0

737.0693.3

811.20

327.0 449.3

737.0845.4

-MS/MS (863.0), 19.3 min (#625)

-MS/MS (863.0), 18.2 min (#643)

-MS/MS (847.0), 20.6 min (#650)

-MS/MS (861.0), 18.7 min (#598)

300 400 500 600 700 800 900

693.3 843.5

D721.0

ns. ×

Figure 5. Product ion spectra of four different A-typeprocyanidin trimers detected typically in (A) plums and (B–D)cinnamon. The structures for A, B and C and theircharacteristic fragmentation pathway are shown in Fig. 6. Thestructure of the trimer for D and its fragmentation pathway areillustrated in Fig. 7.

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6 L. Gu et al.

411.0 451.0

411.0 435.0

289.0499.3

737.0 721.0

Figure 6. Structures of the A-type procyanidin dimers detected typically in (A) plums and (B, C) cinnamon. Their fragmentationpositions are shown.

m/z 285.0

m/z 575.1

m/z 571.1

m/z 289.3

m/z 861.0

m/z 735.3m/z 449.0

-126 Da

m/z 709.3

Figure 7. Fragmentation pathway of a novel procyanidin trimer detected in cinnamon.

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an analog A-type trimer in cinnamon (Fig. 6(B)). Its con-jugate ion at m/z 435.0 reveals that the B ring of themiddle unit has one hydroxyl group and the HRF path-way indicates that this hydroxyl group is at the 40-position.Hence the middle unit of this trimer was identified as(epi)afzelechin. RDA cleavage of one of the C rings inthis trimer gave rise to an abundant m/z 711.2 ion. TheA-type interflavan linkage would inhibit RDA cleavageon the top unit. In this case the RDA takes place on themiddle unit. The trimer has been tentatively identified as(epi)catechin–A–(epi)afzelechin–(epi)catechin, where –A–symbolizes an A-type linkage (Fig. 6(C)).

Another very interesting novel A-type trimer identifiedin cinnamon shows an ion at m/z 861.0 ([M - H]��. A slightlyhigher collision energy level (120% in comparison with 100%)

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was applied to obtain its product ion spectrum (Fig. 5(D)).The fragmentation pathway of this structure, which hasnever been studied before, is elucidated in Fig. 7. The QMcleavage of the interflavan bond is concomitant with thecleavage of the C2—O7 bond for the A-type interflavan link-age. The QM cleavage between the middle and base units isunique. It converts the hydroxyl group on C5 into a quinonein contrast to C7 being a preferred conversion site in allother instances. An equal chance of cleavage on both setsof A-type bonds gives rise to m/z 575.1 and 571.1 ions withsimilar abundances to the diagnostic ions (Fig. 5(D)). TheRDA cleavage of flavan-3-ol on the base unit, which has beendiscussed as an unfavorable pathway, takes place on thistrimer to produce an m/z 709.3 ion, because RDA on the topthe middle units are blocked by the A-type bonds.

UNCORRECTED PROOFS

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The fragmentation pathway and characteristic fragmentions of these different A-type trimers were applied to iden-tify A-type tetramers and pentamers in plums and cinnamon(Table 1). Two A-type tetramers and one pentamer wereidentified in plums. The presence of proanthocyanidins inplums has never been reported before. Four A-type tetramersand three pentamers were identified in cinnamon. Some ofthe A-type procyanidins identified in cinnamon have beenpurified by chromatographic methods.16 The base unit for aproanthocyanidin oligomer or polymer is called a terminalunit. All the flavan-3-ols connected above it are extensionunits. A close examination revealed that the A-type interfla-van linkages of proanthocyanidins in plums are above theterminal unit. This particular structure is designated as an A-type terminus. It can be seen that the A-type terminus is typ-ical for procyanidins in plums, whereas it is absent in proan-thocyanidins in cinnamon. This conclusion is supported bythiolytic degradation of the total proanthocyanidins in plumsand cinnamon (data not shown). The A-type procyanidinshave been suggested to prevent urinary tract infection, a com-mon health problem for women. The discovery of differentA-type procyanidins in plums and cinnamon raises a ques-tion about structure–activity relations for these compounds.

Fragmentation of procyanidin polymers innegative ESIAttempts to identify proanthocyanidins with higher molec-ular masses using MS have been pursued vigorously. Guyotet al. demonstrated that high oligomers of procyanidins (DP8–16) tend to form doubly charged ions on ESI. A hep-tadecamer was detected based on its [M � 3H]3� ion in aprocyanidin fraction extracted from apples.17 A recent studyreported the detection of procyanidins with DP D 28 accord-ing to an [M - 3H]3� ion in grape seeds.18 These identifications

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were highly tentative because only mass spectrometers withlow or medium resolution were used.

It has been implied that polymeric procyanidins tend toform multiply charged ions. However, the structural featuresof the procyanidins make the judgment of multiply chargedions difficult. For example, if procyanidins with DP of 10, 15,20 and 50 lose 2, 3, 4 and 10 protons to form multiply chargedions, their mass-to-charge ratio would be m/z 1441.3, 1440.9,1440.8 and 1440.6, respectively. These ions cannot be resolvedby a mass spectrometer with medium resolution (such as theEsquire-LC). This problem was overcome by using a high-resolution ESI Bio-TOF mass spectrometer (resolution D15 000, 10% mass peak height, single flight pass mode). Thespectrum of the polymeric procyanidin standard obstainedon the TOFMS instrument is shown in Fig. 8. A similar spec-trum was also obtained on the Esquire-LC mass spectrom-eter, but without the detailed isotope information (data notshown). The intervals between the isotope peaks in Fig. 8 are1 Da, which indicates that all the major ions in the spectrumare singly charged. This suggests that instead of formingmultiply charged ions, polymeric procyanidins cleave exten-sively to give rise to a number of singly charged fragments.

We postulated that the facile cleavage of polymers innegative ESI would be caused by their tendency to losemultiple protons. The C4 of monomeric flavan-3-ols inthe procyanidins is an electrophile and is susceptible toattack by nucleophilic negative ions in the electrospraychamber. The deprotonation of the hydroxyl at 7-OH onthe monomeric unit enhances the electrophilic propertiesof C4 and accelerates the cleavage of the interflavanbond. Intermolecular repulsion also reduces the stabilityof multiply charged ions. Owing to the coordination ofthese forces, a multiply deprotonated polymer would cleavepromptly at more than one point through a QM mechanism(Fig. 9, cleavage 1).7 The extension unit cleaves into many

400 600 800 1000 1200 1400 1600 1800 2000 2200 2400

1313 1315 1317 1319 m/z 1437 1439 1441 1443 1445 m/z

Figure 8. ESI Bio-TOFMS of the polymeric procyanidin standard obtained by direct injection. The isotope patterns near m/z1313.68 and 1439.71 are expanded as insets.

UNCORRECTED PROOFS

8 L. Gu et al.

m/z 1439.71 (n = 3)

n-1Cleavage 2

-288 Da

m/z 1151.52 (n = 3)Cleavage 3

-126 Da

m/z 1313.68 (n = 3)

Cleavage 1

Terminalunit

m = 1~6

Figure 9. Cleavage pattern proposed for the polymeric procyanidins based on ESI ion trap MS/MS and Bio-TOFMS. Cleavage ofpolymer A through the QM mechanism (cleavage 1) gives rise to a number of B from extension units and a single C containing theterminal unit. B forms the major ions (m/z 575.0 C 288n). B cleaves through the same mechanism to D (cleavage 2), and also throughthe HRF mechanism to give E (cleavage 2). D contributes to the m/z 575.0 C 288n ions and E forms the m/z 737.2 C 288n ions.

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fragments having similar structures, which are observed asthe major ions (m/z 575.1 C 288n). This cleavage results inonly one ion having a different structure, which containedthe terminal unit. It is masked by the major ions andgave as very weak signals (m/z 577.1 C 288n). MS/MSstudies indicate that the singly charged 575.1 C 288n ionscleave further through the same mechanism into smaller575.1 C 288n ions and also through the HRF mechanismto give rise to 737.2 C 288n ions (Fig. 9, cleavages 2 and3). The isotope patterns of all these ions were calculatedaccording their formula, and were found to be consistentwith those obtained on the Bio-TOF mass spectrometer. Theabundances of the ions on TOFMS decrease with increasein mass (Fig. 8). The decrease in ionization efficiency ofprocyanidins with increase in DP has been observed by usand other investigators.17,18 We have been able to detect the[M - 2H]2� ions of octamers through 13-mers. No such ionscould be unambiguously distinguished from the backgroundfor proanthocyanidins with higher DP. This demonstratesthat the stability of the deprotonated procyanidins and theirfragments decreases with increase in DP. It is implied thatthe polymers are inclined to cleave into a number of smallfragments rather than a few larger ones, and the smallamount of larger fragments will cleave further into smallerions through a cascade process.

CONCLUSIONS

The detection of various oligomers and polymers is impor-tant for plant physiologists; however, the identification

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of constituent units and the connection sequence of lowoligomers (DP < 5) is crucial for the nutritionist who is inter-ested in studying their bioavailability and possible humanhealth effects. MS/MS coupled with normal-phase HPLCprovides a rapid and sensitive method for proanthocyani-din determination for both purposes. With this method,some minor heterogeneous proanthocyanidins with novelstructures were identified for the first time. The variety ofthe structures of A-type proanthocyanidins was revealed byMS/MS studies. The specificity of MS/MS would be invalu-able for tracing the metabolism of proanthocyanidins in thegut and bloodstream. The dissociation of the polymeric pro-cyanidins in negative ESI has defied previous implicationsthat they would form multiply charged ions. ESI-MS maynot be applicable for polymeric proanthocyanidins.

AcknowledgementsThe authors thank Melody Harrison for her critical review of themanuscript. Mention of a trade name, proprietary product or specificequipment does not constitute a guarantee by the US Departmentof Agriculture and does not imply its approval to the exclusion ofother products that may be suitable.

REFERENCES

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2. Santos-Buelga C, Scalbert A. Proanthocyanidins and tannin-likecompounds—nature, occurance, dietary intake and effects onnutrition and health. J. Sci. Food Agric. 2000; 80: 1094.

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3. Porter LJ. Flavans and proanthocyanidins. In The Flavonoids,Harbone JB (ed). Chapman and Hall: New York, 1988; 21.

4. Cuyckens F, Rozenberg R, De Hoffmann E, Claeys M. Structurecharacterization of flavonoid O-diglycosides by positiveand negative nano-electrospray ionization ion trap massspectrometry. J. Mass Spectrom. 2001; 36: 1203.

5. Ohnishi-Kameyama M, Yanagida A, Kanda T, Nagata T.Identification of catechin oligomers from apple (Malus pumilacv. Fuji) in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and fast-atom bombardmentmass spectrometry. Rapid Commun. Mass Spectrom. 1997; 11:31.

6. Sun W, Miller JM. Tandem mass spectrometry of the B-typeprocyanidins in wine and B-type dehydrodicatechins in anantioxidation mixture of (C)-catechin and (�)-epicatechin. J.Mass Spectrom. 2003; 38: 438.

7. Karchesy JJ, Foo LY, Barofsky B, Arbogast B, Barofsky DF.Negative-ion fast-atom-bombardment mass spectrometry ofprocyanidin oligomers. J. Wood Chem. Technol. 1989; 9: 313.

8. Miketova P, Schram KH, Whitney J, Li M, Huang R, Kerns E,Valcic S, Timmermann BN, Rourick R, Klohr S. Tandem massspectrometry studies of green tea catechins. Identification ofthree minor components in the polyphenolic extract of greentea. J. Mass Spectrom. 2000; 35: 860.

9. Friendrich W, Eberhardt A, Galensa R. Investigation ofproanthocyanidins by HPLC with electrospray ionization massspectrometry. Eur. Food Res. Technol. 2000; 211: 56.

10. Foo LY, Lu Y, Howell AB, Vorsa N. The structure of cranberryproanthocyanidins which inhibit adherence of uropathogenicP-fimbriated Escherichia coli in vitro. Phytochemistry 2000; 54: 173.

11. Lazarus SA, Hammerstone JF, Adamson GE, Schmitz HH.High-performance liquid chromatography/mass spectrometry

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analysis of proanthocyanidins in food and beverages. MethodsEnzymol. 2001; 335: 56.

12. Gu L, Kelm M, Hammerstone JF, Beecher G, Cunningham D,Vannozzi S, Prior RL. Fractionation of polymeric procyanidinsfrom lowbush blueberry and quantification of procyanidinsin selected foods with an optimized normal-phase HPLC–MSfluorescent detection method. J. Agric. Food Chem. 2002; 50: 4852.

13. Prior RL, Lazarus SA, Cao G, Muccitelli H, Hammerstone JF.Identification of procyanidins and anthocyanidins in blueberriesand cranberries (Vaccinium spp.) using high-performance liquidchromatography/mass spectrometry. J. Agric. Food Chem. 2001;49: 1270.

14. Holt RR, Lazarus SA, Sullards MC, Zhu QY, Schramm DD,Hammerstone JF, Fraga CG, Schmitz HH, Keen CL. Procyanidindimer B2 [epicatechin-(4beta-8)-epicatechin] in human plasmaafter the consumption of a flavanol-rich cocoa. Am. J. Clin. Nutr.2002; 76: 798.

15. Karchesy JJ, Hemingway RW, Foo LY, Barofsky E, Barofsky DF.Sequencing procyanidin oligomers by fast atom bombardmentmass spectrometry. Anal. Chem. 1986; 58: 2563.

16. Nonaka G, Morimoto S, Nishioka I. Tannins and relatedcompounds. Part 13. Isolation and structures of timeric,tetrameric, and pentameric proanthocyanidins from cinnamon.J. Chem. Soc., Perkin Trans. 1 1983; 2139.

17. Guyot S, Doco T, Souquet J, Moutounet M, Drilleau J.Characterization of highly polymerized procyanidins in ciderapple (Malus Sylvestris var. Kermerrien) skin and pulp.Phytochemistry 1997; 44: 351.

18. Hayasaka Y, Waters EJ, Cheynier V, Herderich MJ, Vidal S.Characterization of proanthocyanidins in grape seeds usingelectrospray mass spectrometry. Rapid Commun. Mass Spectrom.2003; 17: 9.

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