Tandem Mass Spectrometry for Sequencing Proanthocyanidins

uted in higher plants and are an integral part of the human diet. A sensitive LC-tandem ... forming (BFF) fission described here for the first time, a...
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Anal. Chem. 2007, 79, 1739-1748

Tandem Mass Spectrometry for Sequencing Proanthocyanidins Hui-Jing Li and Max L. Deinzer*

Department of Chemistry, Oregon State University, Corvallis, Oregon 97331

Proanthocyanidins (PAs) are a group of bioflavonoids consisting of oligomers based on catechin monomeric units. These polyphenolic compounds are widely distributed in higher plants and are an integral part of the human diet. A sensitive LC-tandem mass spectrometric (LC/ESIMSn) method in the positive ion mode for sequencing these ubiquitous and highly beneficial antioxidants is described. The hydroxylation patterns and interflavanoid linkage for A- and B-type PAs were determined by fragment ions derived from a retro-Diels-Alder (RDA) fission, heterocyclic ring fission (HRF), a novel benzofuranforming (BFF) fission described here for the first time, and a quinone methide (QM) fission. The subunit sequence of the PAs was determined by diagnostic ions derived from HRF/RDA fission, HRF/BFF fission, and RDA/HRF fission together with QM fission. A total of 26 PAs were reliably sequenced by the newly established tandem mass spectrometric protocol. It is shown that the protocol based on a combination of these different fragmentation patterns allows for uniquely identifying PA oligomers. Proanthocyanidins (PAs), known as condensed tannins, are flavan-3-ol oligomers and polymers that yield anthocyanidins upon oxidative acid depolymerization reactions. PAs are wieldy distributed throughout the plant kingdom and are present as the second most abundant class of natural phenolic compounds after lignin. PAs exist in a wide range of plant-derived products, and these compounds possess potential health benefits as a result of their antioxidant, anticarcinogenic, and anti-inflammatory actions.1-6 Hops are a rich and convenient source of a broad variety of PAs. The difficulty of extracting and purifying PAs, together with their instability, structural complexity,4 and limited quantities present in hops, makes this source particularly relevant for implementing * To whom correspondence should be addressed. Tel: +1-541-737-1773. Fax: +1-541-737-4371. E-mail: [email protected]. (1) Stevens, J. F.; Miranda, C. L.; Wolthers, K. R.; Schimerlik, M.; Deinzer, M. L.; Buhler, D. R. J. Agric. Food Chem. 2002, 50, 3435-3443. (2) Callemien, D.; Jerkovic, V.; Rozenberg, R.; Collin, S. J. Agric. Food Chem. 2005, 53, 424-429. (3) Jerkovic, V.; Callemien, D.; Collin, S. J. Agric. Food Chem. 2005, 53, 42024206. (4) Merghem, R.; Jay, M.; Brun, N.; Voirin, B. Phytochem. Anal. 2004, 15, 95-99. (5) Maatta-Riihinen, K. R.; Kahkonen, M. P.; Torronen, A. R.; Heinonen, I. M. J. Agric. Food Chem. 2005, 53, 8485-8491. (6) Zhou, Z. H.; Zhang, Y. J.; Xu, M.; Yang, C. R. J. Agric. Food Chem. 2005, 53, 8614-8617. 10.1021/ac061823v CCC: $37.00 Published on Web 01/09/2007

© 2007 American Chemical Society

Chart 1. Flavanol Units of PAs in Hops Conesa

a

n, degree of polymerization; A-F, ring labels.

protocols to structurally characterize them. The estimated amount of PAs in hop ranges from 0.5 to 5% on a dry weight basis, depending on variety and geographic origin.6,7 Proanthocyanidins or oligomeric proanthocyanidins, names that are often used interchangeably, consist of sequences of phenolic compounds built from single monomer units called afzelechin, epiafzelechin, catechin, epicatechin, gallocatechin, and epigallocatechin (Chart 1). Although usage of the word “oligomeric” varies somewhat, dimers, trimers, and up to heptamers are generally referred to as oligomeric (n ) 2∼7, Chart 1), whereas larger chains are generally referred to as polymeric or tannins (n ) 8∼24 or more, Chart 1). These flavan-3-ol monomer units are sometimes esterified with gallic acid to form 3-O-gallates. B-Type PAs are flavan-3-ol oligomers and polymers linked mainly through C4f8 or sometimes C4f6 bonds. When an additional ether linkage is formed between C2f7, the compounds are called A-type PAs. This subclass of compounds has two fewer hydrogens than the B-type PAs and can be readily recognized by their [M + H]+ ions that are 2 Da lower in mass. The elucidation of PA structures has generally relied on extensive purification followed by spectral (NMR, MS) and chemical degradation with thiolytic or phloroglucinol adduction. However, NMR resonance multiplicity and broadening associated (7) Moll, M.; Fonknechten, G.; Carnielo, M.; Flayeux, R. MBAA Tech. Q. 1984, 21, 79-87.

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Figure 2. Positive ion ESI tandem mass spectra of PA monomer catechin. (A) MS2 of (M + H)+ ion (m/z 291) and (B) MS3 spectrum of ion with m/z 273.

Scheme 1. Fragmentation Pathways of Monomer Catechin: RDA, HRF, BFF, and Loss of Water Figure 1. (A) HPLC chromatogram of the Oregon-Willamette hop PAs (HPLC separations performed on a 250 mm × 4.6 mm Synergi 4-µm Hydro-RP-80A column with a linear gradient of 5-50% methanol in 1% aqueous formic acid over a 50-min period at 0.8 mL/ min). (B) TIC trace of on-line LC/ESI-MS2 of (E)C-(4,8)-(E)C diastereoisomers (7-10) and the MS2 spectrum (inset) of 9.

with rotational and conformational isomerism often complicates the interpretation of their sequences.8 Chemical degradation requires tedious large-scale separation, multiple purification, and various chemical reactions, and at the end, many structural identities may remain unresolved for lack of authentic samples. Therefore, mass spectrometry coupled with liquid chromatography (LC/MS) has attracted greater attention due to its capacity for reliable analysis and small sample requirements.8-13 Diagnostic ions produced by quinone methide (QM) fission have been utilized exclusively to study the sequence of PAs.8,10 Despite some satisfactory results from these QM fragment ions, background interference, nonexistent signals, and the difficulty to differentiate the ions derived from other mass spectral fragmentation patterns often made it difficult to identify the products, especially in the low-mass region ( 2) spectra, and thus, QM fission was used to sequence them. It is true that RDA, HRF, and BFF fission sequencing is more difficult for the higher PA oligomers, but with higher concentrations of PAs so that MSn (n > 2) spectra could Analytical Chemistry, Vol. 79, No. 4, February 15, 2007

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Figure 5. Positive ion ESI tandem mass spectra of PAs in hop extract mixtures. (A) Full MS of hop extract mixtures, (B) MS2 of tetramer 16 (M + H)+ (m/z 1155), (C) MS2 of pentamer 21 (M + H)+ (m/z 1459), (D) MS2 of A-type dimer 24 (M + H)+ (m/z 593), (E) MS3 of m/z 441 ion (one product ion from m/z 593). (F) MS2 of A-type trimer 26 (M + H)+ (m/z 881) and (G) MS3 of m/z 591 ion (one product ion from m/z 881).

be obtained, the combination of RDA, HRF, BFF, and QM fissions would be more useful for sequencing. Various subunits would thereby be obtained by QM fission, and the combinations of RDA, HRF, and BFF for the subunits provide additional information and most importantly confirmatory evidence. Through systematic QM fission, sequencing of up to eight larger PA tetramers and pentamers in the extracts was successfully performed. Compound 16 ([M + H]+, m/z 1155) detected in the ESI mass spectrum of the PA mixture, observed before chromatographic separation (Figure 5A), was identified as a PA tetramer. Sequencing of this tetramer by QM fissions was straightforward. Diagnostic fragment ions with m/z 867, 865, and 579 were observed (Figure 5B). The ion with m/z 867 ([M + H - QM (288 Da)]+) was produced after QM cleavage of the [M + H]+ ion between the linkage of the upper and second units; thus, the upper unit was identified as (epi)catechin. The ion with m/z 865 ([M + H QM (290 Da)]+) was produced after QM cleavage of the [M + H]+ ion between the linkage of the third and terminal units, identifying the terminal unit as (epi)catechin. The ion with m/z 579 ([M + H - QM (288 + 288 Da)]+ or [867 - QM (288 Da)]+) was produced either after QM cleavage of the [M + H]+ ion or after the QM cleavage of the ion with m/z 867 between the linkage of the second and third units of the [M + H]+ ion. The second unit was, therefore deduced as (epi)catechin. The third unit could 1746 Analytical Chemistry, Vol. 79, No. 4, February 15, 2007

easily be identified as (epi)catechin after establishing the identies of the first, second, and terminal units, and 16 was thus identified as (epi)catechin-(epi)catechin-(epi)catechin-(epi)catechin. Compound 21 ([M + H]+, m/z 1459) (Figure 5A), is a PA pentamer. Diagnostic fragment ions with m/z 1171, 1169, 867, and 579 were observed (Figure 5C). The ion with m/z 1171 ([M + H - QM (288 Da)]+) was likely produced after QM cleavage of the [M + H]+ ion between the linkage of the upper and second units allowing the upper unit to be identified as (epi)catechin. The ion with m/z 1169 ([M + H - QM (290 Da)]+) was produced after QM cleavage of the [M + H]+ between the linkage of the fourth and terminal units, which identified the terminal unit as (epi)catechin. The ion with m/z 867 ([M + H - QM (288 + 304 Da)]+ or [1171 - QM (304 Da)]+) was produced either after QM cleavage of [M + H]+ or after QM cleavage of the ion with m/z 1171 between the linkage of the second and third units of the [M + H]+ ion, which allows the second unit to be deduced as (epi)gallocatechin. The ion with m/z 579 ([M + H - QM (288 + 304 + 288 Da)]+, [1171 - QM (304 + 288 Da)]+, or [867 - QM (288 Da)]+) was produced after QM cleavage of the [M + H]+ ion, the ion with m/z 1171, or m/z 867 between the linkage of the third and fourth units of the [M + H]+ ion, and the third unit is thus (epi)catechin, the fourth unit is then (epi)catechin. This process was used to identify the sequences of 16-23 (Table 4).

Table 4. Positive Ion ESI Tandem Mass Diagnostic Ions (m/z) and Sequences of B-Type Hop PA Oligomers 16-23a compd

subunit sequences

[M + H+

diagnostic ions

16 17 18 19 20 21 22 23

(E)C-(E)C-(E)C-(E)C (E)C-(E)C-(E)GC-(E)C (E)C-(E)GC-(E)GC-(E)C (E)GC-(E)GC-(E)GC-(E)C (E)C-(E)C-(E)C-(E)C-(E)C (E)C-(E)GC-(E)C-(E)C-(E)C (E)C-(E)GC-(E)GC-(E)C-(E)C (E)C-(E)C-(E)C-(E)C-(E)C-(E)C

1155 1171 1187 1203 1443 1459 1475 1731

867, 865, 579 883, 881, 595 899, 897, 595 913, 899, 595 1155,1153,867, 579 1171,1169,867, 579 1187,1185,883, 579 1441,1143, 1155, 867, 579

a Neutral losses are shown in parentheses. m/z was omitted. (E)C and (E)GC are abbreviations for (epi)catechin and (epi)gallocatechin. The symbol (E) indicates there are two possibilities: “catechin or epicatechin or gallocatechin or epigallocatechin”.

Off-Line LC/ESI-MSn Analyses of A-type PAs from Hop Extract Oligomeric Mixtures. Two A-type hop PAs (24, 25) were also initially identified as being present in the oligomeric mixtures (Figure 5A). These were ultimately separated as pure compounds (Figure 1). Compound 24 (Figure 1) ([M + H]+, m/z 593; TR, 5.55 min) is an A-type PA dimer (Figure 5D) (Supporting Information part 4) and has a mass that is 2 Da less than that of the corresponding B-type PA dimer. The fragment ion with m/z 441 (593 - 152 Da) arises from RDAF fission of the [M + H]+ ion and identifies this unit as gallocatechin. Subsequent HRFC fission took place to give the ion with m/z 315 (441 - 126 Da) (Figure 5E). Using the rules established above, 24 was sequenced as (epi)gallocatechin-A-(epi)catechin. Compound 25 ([M + H]+, m/z 593; TR, 6.67 min) showed exactly the same tandem mass spectra as 24, which means the sequence of 25 was also (epi)gallocatechin-A-(epi)catechin (“-A-” represents an A-type C2f7 interflavanoid linkage). Compounds 24 (Figure 1) and 25 eluted at nearly the same time in the reversed-phase column, which indicated they are diastereoisomers. Moreover, they should possess C4f8 interflavanoid linkages due to the lower collision energy (35%) required to get the most abundant ions in the tandem spectrum. QM fission of A-type PAs requires breaking two bonds (Scheme 4), in contrast to the B-type PAs that requires only one bond to cleave. In these studies, the signals of QM fission product ions resulting from cleavage of the A-type bonds were too weak to be observed. In this case, RDAF/HRFC fission achieved the sequencing objective (Figure 5E, Scheme 4), and other fragmentations (HRFC/RDAF, HRFC/ H2O/BFFF) confirmed the result (Figure 5D, Scheme 4). Compound 26 ([M + H]+, m/z 881, TR, 10.79 min) (Figure 1, Figure 5A), is an A-type PA trimer. The fragment ions can easily be interpreted from the above reasoning. The diagnostic fragment ion with m/z 591 ([M + H - QM (290 Da)]+) (Figure 5F) was produced after QM cleavage of [M + H]+ between the linkage of the central and terminal units. Thus, the terminal unit was identified as (epi)catechin and the upper-central unit (ion at m/z 591) as an A-type dimer (Figure 5F). The MS3 spectrum of the m/z 591 ion (Figure 5G) yielded an ion with m/z 465 ([591 HRF (126 Da)]+) resulting from HRF fission (rule 4). The diagnostic fragment ion with m/z 343 ([591 - HRF (126 Da) BFF (122)]+) could result from BFF fission at the terminal unit of the m/z 591 ion, which would confirm its identity as (epi)catechin; hence, the upper unit has to be (epi)gallocatechin. Thus, 26 was sequenced as (epi)gallocatechin-A-(epi)catechin-(epi)-

catechin. In this case, QM fission could only have provided parts of the sequence. QM fission combined with HRF/BFF fission was the strategy that helped sequence this trimer. This case illustrates that sequencing PAs can require various combinations of fragmentations (indirect MS protocol) and that just one type of fission (direct MS protocol) may not be sufficient for unambiguous identification. On-Line LC/ESI-MS2. Off-line HPLC isolation of 1-15 and 24-26 was used in order to structural elucidate each of these compounds by MSn. On-line LC-ESI-MS2 experiment was performed to illustrate the utility of the approach. The ion with m/z 579 was scanned to record the total ion current (TIC) of dimers 7-10 (Figure 1B). The MS/MS spectra of the collisionally activated m/z 579 peak revealed the QMCDV (m/z 291), RDAc or RDAF (m/z 427), and HRFC/RDAF (m/z 301) fragmentations (Table 2) as illustrated for PA 9 (Figure 1B inset). The PA oligomeric mixtures were also introduced into the instrument online and MS/MS performed on 1, 2, 5, 6, and 14 (Figure 1A), thus proving that these compounds can be structurally analyzed by on-line LC/ESI-MS.2 It is important to note that oligomers, with the same molecular weights, but different retention times result from diastereoisomerism. PAs with the same molecular weights but with a different order of monomer units would also show differences in retention times, but such compounds were not encountered in these studies. However, isomeric PAs differing in their sequences do exist as reported, for example, for dimers of prodelphinidins, C-GC and GC-C from grapes.19 If such compounds or larger isomeric oligomers were present in a PA mixture, they could, in principle, be sequenced by LC/MS3 using hybrid triple-quadrupole linear ion trap or similar instrumentation that can trap a product ion sufficiently long to perform multiple fragmentations. CONCLUSION The newly established direct and indirect analytical protocols based on tandem mass spectrometry have been systematically exploited in this contribution to sequence 26 PAs in hops ranging from monomers to hexamers. The most important value of this procedure is that when one fragmentation protocol fails to distinguish between oligomers with the same mass, for example, one of the other fragmentation protocols will always provide a unique pattern that allows a distinction between these isomers. (20) Ito, S.; Joslyn, M. A. Nature 1964, 204, 475-476. (21) Dykes, L.; Rooney, L. W.; Waniska, R. D.; Rooney, W. L. J. Agric. Food Chem. 2005, 53, 6813-6818.

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This is the first time to our knowledge that such an extensive and diverse group of PAs has been observed from this plant. All of the fragmentation mechanisms were proposed based on data obtained by ESI-MSn. These indirect analytical protocols have enriched and expanded the collection of analytical approaches available for sequencing PAs and should be useful for identifying other natural products. The new approach was found to provide for the reliable determination of PAs extracted from a source that contains only trace levels of these compounds. The identification of both A-and B-type linkages through the use of LC/MSn alone eliminates a number of tedious separation steps. PAs are ubiquitous and are found in fruits,20 cereals/beans,21 nuts,22 beverages,23 spices,24 vegetables,25 herbal medicines,26 and a number of other plant sources. The efficient sequencing protocols for A- and B-type PAs described here will benefit researchers in food sciences, agencies and monitoring groups, health science researchers, pharmacists, and chemists, who must quickly screen, analyze, or confirm the presence of PAs in natural sources and amended, or adulterated consumer products. Abbreviations: C, catechin; EC, epicatechin; (E)C, (epi)catechin; GC, gallocatechin; (E)GC, (epi)gallocatechin; PAs, (22) Stich, H. F.; Ohshima, H.; Pignatelli, B.; Michelon, J.; Bartsch, H. J. Natl. Cancer Inst. 1983, 70, 1047-1050. (23) Brown, A. G.; Eyton, W. B.; Holmes, A.; Ollis, W. D. Nature 1969, 221, 742-744. (24) Schulz, J. M.; Herrmann, K. Z. Lebensm.-Unters. Forsch. 1980, 171, 278280. (25) Gu, L. w.; Kelm, M. A.; Hammerstone, J. F.; Beecher, G.; Holden, J.; Haytowitz, D.; Gebhardt, S.; Prior, R. L. J. Nutr. 2004, 134, 613-617. (26) Shao, Z. H.; Vanden, H. T. L.; Li, Ch. Q.; Schumacker, P. T.; Becker, L. B.; Chan, K. Ch.; Qin, Y. M.; Yin, J. J.; Yuan, Ch. S. Am. J. Chin. Med. 2004, 32, 89-95.

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proanthocyanidins; LC/ESI-MSn, liquid chromatography/electrospray ionization tandem mass spectrometry; BFF, benzofuranforming fission; RDA, retro-Diels-Alder fission; HRF, heterocyclic ring fission; QM, quinone methide fission; APCI, atmospheric pressure chemical ionization; TLC, thin-layer chromatography; QMCDV the ion derived from the QM fisson of ring-C/ring-D linkage bond by the loss of upper unit; QMCDv, the ion derived from the QM fisson of ring-C/ring-D linkage bond by the loss of lower unit; QMFGV, the ion derived from the QM fisson of ringF/ring-G linkage bond by the loss of upper unit; QMFGv, the ion derived from the QM fisson of ring-F/ring-G linkage bond by the loss of lower unit. ACKNOWLEDGMENT Financial support from the Anheuser-Busch Companies and the Hop Research Council is gratefully acknowledged. This work was made possible in part by grant P30 ES00210 from the National Institute of Environmental Health Sciences, NIH. We thank the Mass Spectrometry Facility of the Environmental Health Sciences Center at Oregon State University. SUPPORTING INFORMATION AVAILABLE Assignment of the fragment ions (MS2, MS3 spectra) of the representative compounds 1, 4, 11 and 24. This material is available free of charge via the Internet at http://pubs.acs.org

Received for review September 27, 2006. Accepted November 30, 2006. AC061823V