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Rapid Fingerprint Analysis of Plant Extracts for Ellagitannins, Gallic Acid, and Quinic Acid Derivatives and Quercetin‑, Kaempferol- and Myricetin-Based Flavonol Glycosides by UPLC-QqQ-MS/MS Marica T. Engström,* Maija Pal̈ ijar̈ vi, and Juha-Pekka Salminen Laboratory of Organic Chemistry and Chemical Biology, Department of Chemistry, University of Turku, FI-20014 Turku, Finland ABSTRACT: This paper describes the development of a rapid method with ultraperformance liquid chromatography−triplequadrupole mass spectrometry that can specifically measure group-specific fingerprints from plant extracts for the following polyphenol groups: (1) ellagitannins, (2) gallic acid derivatives, (3) quinic acid derivatives, (4) quercetin-based flavonol glycosides, (5) kaempferol-based flavonol glycosides, and (6) myricetin-based flavonol glycosides. In addition, the method records simultaneously diode array and full scan mass spectrometry data that can be used to later characterize and quantify the main individual polyphenols if necessary. All of this is achieved within the 10 min period of analysis, which makes the presented method a significant addition to the chemistry tools currently available for the rapid analysis of complex polyphenol mixtures from plant extracts. KEYWORDS: polyphenols, fingerprints, ellagitannin, gallic acid, quinic acid, quercetin, kaempferol, myricetin, LC-MS/MS, MRM

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INTRODUCTION Plants produce a wide variety of polyphenols that may contribute to numerous types of bioactivities. Due to the diverse range of bioactivities and potential health benefits, polyphenols have received more and more attention in recent years.1−3 To achieve a more thorough understanding of the active and inactive polyphenols, one would need to reveal the phenolic fingerprints of active and inactive plants. However, this is typically a very laborious and time-consuming effort and cannot easily be done with hundreds of samples. As a consequence of the growing interest, many efforts have been made to provide sensitive and selective analytical methods for the determination and characterization of polyphenols.4−6 The more traditional ways of analyzing polyphenols have utilized the tendency of their functional groups to undergo characteristic reactions that result in colorful chromophores that can be detected by spectrophotometry. These types of methods include both the general polyphenol methods (e.g., Folin−Ciocalteu assay for total phenolics) and the more selective functional group methods for the detection of proanthocyanidins (e.g., HCl−butanol assay) and hydrolyzable tannins (e.g., NaNO2 assay for ellagitannins and rhodanine method for gallotannins).7−16 Although the spectrophotometric methods are rapid and simple, they lack the specificity for individual compounds. In more sophisticated methods, the analytes are first separated by chromatographic techniques for the accurate quantification and identification of different polyphenols. Reversed-phase liquid chromatography (RP-LC) coupled with a diode array detector (DAD) and/or mass spectrometric detector (LC-MS) are the most widely used analytical tools for the quantification of polyphenols.17,18 The vast development of ultrahigh-performance liquid chromatography combined with a mass spectrometer (UHPLC-MS/MS) has further improved the performance of separation and detection and reduced the time required for qualitative and quantitative analysis.19−21 © XXXX American Chemical Society

The development and increased use of LC-MS/MS instruments has drastically increased the number of studies in which compound-specific fragmentations have been used for the identification and quantitation of single polyphenols by multiple reaction monitoring (MRM) methods.6,18,22−26 These methods are employed with triple-quadrupole mass spectrometers (QqQ-MS) that select compound-specific precursor ions by the first quadrupole and then fragment it into product ions in the collision cell and further select the desired product ions by the last quadrupole for MS detection. The development of these methods is relatively laborious as MS/MS conditions for precursor ion/product ion pairs need to be optimized for each compound separately. However, because compounds in the same polyphenol group typically share similar or the very same building blocks, group-specific methods of polyphenols have been available for MS users via qualitative parent ion and neutral loss scan modes. For instance, all galloyl glucoses and gallotannins produce a gallic acid fragment (170 Da), and all of the parent molecules producing this fragment can be detected by the parent ion scan of m/z 169 (in negative mode). Unfortunately, these types of scanning methods cannot be used for simultaneous detection or semiquantitation of many types of polyphenol groups as a single parent ion scan of, for example, galloyl glucoses and gallotannins between m/z 330 and 2010 (from mono- to dodecagalloyl glucoses, 332−2004 Da) will take approximately 200 ms. This is sufficient to collect 15 data points per any 3 s wide galloyl glucose peak produced by UHPLC, but if a parent ion scan of ellagitannins producing ellagic acid fragment (m/z 301) is measured at the same time by MS/MS, only 7−8 data points can be collected per compound, thus making accurate Received: August 16, 2014 Revised: March 31, 2015 Accepted: April 8, 2015

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DOI: 10.1021/acs.jafc.5b00595 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Example structures of the studied polyphenol subclasses. Compound identities: oenothein B (2), vescalagin (8), pentagalloyl glucose (23), 3-O-caffeoylquinic acid (29), quercetin-3-O-glucoside (31), and kaempferol-3-O-glucoside (41) (HHDP, hexahydroxydiphenoyl; NHTP, nonahydroxytriphenoyl).

based flavonol glycosides, (5) kaempferol-based flavonol glycosides, and (6) myricetin-based flavonol glycosides (Figure 1).

quantitation impossible. If even more parent ion scans for different polyphenol groups are included, then also the measurement of qualitative group-specific fingerprints becomes impossible due to insufficient numbers of data points per single polyphenols. Therefore, more rapid nonscanning MS/MS methods are needed to enable more efficient quantitation and/or fingerprint detection of polyphenol groups. The speed of MRM technology would be especially suitable for this job as one MRM transition can be measured in 5 ms (vs 200 ms of the parent ion scan). However, to our knowledge, MRM-based LC-MS/MS methods for polyphenol subgroups have not been reported to be used for fingerprinting other than proanthocyanidins from plant extracts.27 To screen thousands of plant samples for their bioactive polyphenol content, rapid and specific methods are required. In these cases total concentrations of compounds belonging to different polyphenol subclasses are often of primary interest. If this is done by simultaneously recording the LC-MS/MS traces of many of the major polyphenol groups, overall views of sample-specific polyphenol fingerprints are achieved for every analyzed sample. The present study explored the potential of ultrahigh-performance liquid chromatography combined with a triple-quadrupole mass spectrometer for the rapid fingerprint analysis of different plant polyphenol subclasses from plant crude extracts. Our main interest was in establishing a fast, sensitive, and selective method for the simultaneous analysis of six common plant polyphenol subclasses: (1) ellagitannins, (2) gallic acid derivatives, (3) quinic acid derivatives, (4) quercetin-

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MATERIALS AND METHODS

Chemicals and Reagents. Acetonitrile (LC-MS grade) used in the UPLC analyses was from Sigma-Aldrich (Steinheim, Germany). Formic acid was from VWR (Helsinki, Finland). Water was purified with a Millipore Synergy Water Purification System (Merck KGaA, Darmstadt, Germany). Flavonoids and quinic acids were from ExtraSynthese (Genay, France). Catechin was from Sigma (Sigma Chemical Co., St. Louis, MO, USA). Materials. Pure compounds, simple galloylglucoses, ellagitannins, the less characterized flavonoids, quinic acids, and the gallotannin fractions used were isolated from different plant materials as described in Moilanen and Salminen.28 The identities and purities of these compounds were established by UPLC-DAD-QqQ-MS and HPLCESI-Q-TOF-MS. The simple galloylglucoses and ellagitannins were additionally identified by NMR.28,29 The compounds used are listed in Table 1. Plant materials used for plant extracts (Table 2) were collected from the Botanical Garden of the University of Turku and nearby forests in Turku, Finland. After collection, samples were freezedried and ground into powder. The plant powder was extracted with acetone/water (80:20, v/v) for 2 h, and the extraction was repeated two times. The acetone was evaporated from the extracts, and the extracts were frozen and freeze-dried. Freeze-dried samples were dissolved in 1 mL of water, filtered via 0.20 μm filters, and diluted 5 times with water before UPLC-MS/MS analyses. UPLC-MS/MS Analysis. Sample analysis was carried out with an Acquity UPLC system (Waters Corp., Milford, MA, USA) coupled with a Xevo TQ triple-quadrupole mass spectrometer (Waters Corp.). B


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Journal of Agricultural and Food Chemistry Table 1. Compounds and Fractions Used in the Present Work no.

compound

MW

no.

compound

MW

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

tellimagrandin I oenothein B oenothein A sanguiin H6 lambertianin C agrimoniin gemin A vescalagin castalagin vescavaloninic acid castavaloninic acid cocciferin D2 sanguiin H10 rugosin D pedunculagin chebulagic acid punicalagin geraniin monogalloylglucose digalloylglucose trigalloylglucose tetragalloylglucose pentagalloylglucose gallotannin fraction 1 gallotannin fraction 2 galloylquinic acid digalloylquinic acid

786.6 1569.1 2353.6 1871.3 2805.9 1871.3 1873.3 934.6 934.6 1102.7 1102.7 1869.3 1569.1 1875.3 784.5 954.7 1084.7 952.6 332.3 484.4 636.5 788.6 940.7

28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53

gallocatechin gallate 3-O-caffeoylquinic acid 3,4-O-dicaffeoylquinic acid quercetin-3-O-glucoside quercetin-3-O-galactoside quercetin-4′-O-glucoside quercetin-7-O-glucoside quercetin-3,4′-O-diglucoside quercetin-3-O-rhamnoside quercetin glycoside quercetin glucuronide quercetin hexopentoside quercetin triglycoside kaempferol-3-O-glucoside kaempferol-7-O-glucoside kaempferol-7-O-neohesperoside kaempferol glucuronide kaempferol rhamnoside kaempferol triglycoside kaempferol malonylglucoside kaempferol diglycoside myricetin 3-O-rhamnoside myricetin glucoside myricetin arabinoside myricetin glucuronide myricetin glycoside

458.4 354.3 516.5 464.4 464.4 464.4 464.4 626.5 448.4 874.7 478.4 596.5 756.6 448.4 448.4 594.5 462.4 431.4 740.6 534.4 594.5 464.4 480.4 450.4 494.4 602.5

344.3 496.4

mixture of different flavonoids (4 μg mL−1) was used to monitor variations in retention time and m/z values. Optimization of Cone Voltages and Collision Energies. When cone voltages and collision energies were optimized for the selected compounds, aqueous solutions of different compounds were directly infused into the ESI source with a syringe pump at a flow rate of 5−40 μL min−1. The concentrations of the aqueous solutions were 40 μg mL−1 for pure compounds and 1 mg mL−1 for the gallotannin fractions. The ranges of cone voltage and collision energy varied between 10 and 170 V and between 5 and 50 eV, respectively. Testing the UPLC-MS/MS Method with Plant Extracts and Polyphenol Mixtures. The method was first tested by three polyphenol mixtures made from pure compounds. This was necessary to avoid coelution of interfering substances with known compounds so that UV chromatograms could be directly compared with MRM chromatograms. The first mixture (1) contained compounds 1, 2, 6, 8, and 29, the second mixture (2) compounds 30, 32, 33, and 41, and the third one (3) compounds 23, 31, 36, 42, 43, and 49 (see Table 1 for compound identities). Mixture 1 was prepared in formic acid/ acetonitrile/water (0.1:5:94.9, v/v/v), and mixtures 2 and 3 were prepared in formic acid/acetonitrile/water (0.1:10:89.9, v/v/v). The dilution range was from 20 μg mL−1 to 0.08 ng mL−1. Three replicates of each sample were run. The mixtures were prepared fresh the same day as the analysis. Plant extracts were prepared as described under Materials, and samples of 1 mg mL−1 were prepared and diluted with water to the final test concentration of 0.2 mg mL−1. The polyphenol mixtures mentioned above were utilized for quantification purposes in the present work. For estimating total concentrations, the responses of compounds belonging to the same polyphenol subgroup were combined and calibration curves prepared by using these values. For the comparison of group-specific and compound-specific methods with single compounds, calibration curves with corresponding compounds were prepared. The dilution range was from 0.08 ng mL−1 to 20 μg mL−1, and only the linear parts of the calibration curves were used for quantification.

Table 2. Plant Species Used in the Present Work no.

plant

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Filipendula ulmaria (meadow sweet), leaves Fumaria of f icinalis (common fumitory), leaves Epilobium hirsutum (great hairy willowherb), leaves Rosa pimpinellifolia (burnet rose), leaves Lysimachia vulgaris (yellow loosestrife), flowers Chamerion angustifolium (fireweed), flowers Vicia cracca (cow vetch), flowers Sorbus hybrida (Swedish service tree), leaves Betula pubescens (white birch), leaves Lythrum salicaria (purple loosestrife), leaves Quercus robur (English oak), leaves Geum urbanum (wood avens), leaves Sorbus aucuparia (rowan), leaves Vincetoxicum hirundinaria (white swallow-wort), leaves Rubus saxatilis (stone bramble), leaves

The UPLC system consisted of a sample manager, a binary solvent manager, a column, and a diode array detector. The column used was a 100 mm × 2.1 mm i.d., 1.7 μm, Acquity UPLC BEH Phenyl column (Waters Corp., Wexford, Ireland). The flow rate of the eluent was 0.5 mL min−1. The elution profile used two solvents, acetonitrile (A) and 0.1% aqueous formic acid (B): 0−0.5 min, 0.1% A in B; 0.5−5.0 min, 0.1−30% A in B (linear gradient); 5.0−6.0 min, 30−35% A in B (linear gradient); 6.0−9.5 min, column wash and stabilization. UV and MS data were collected from 0 to 6 min. Negative ionization mode was used for MS analyzes. ESI conditions were as follows: capillary voltage, 2.4 kV; desolvation temperature, 650 °C; source temperature, 150 °C; desolvation and cone gas (N2), 1000 and 100 L/h, respectively; and collision gas, argon. Catechin (1 μg mL−1) was used to monitor the stability of the ionization efficiency of the mass spectrometer, and a C


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Figure 2. Optimization of cone voltages for the accumulation of precursor ions presenting ellagitannins (A), gallic acid derivatives (B), quinic acid derivatives (C), quercetin-based flavonoids (D, E), kaempferol-based flavonoids (F, G), and myricetin-based flavonoids (H, I). See Table 1 for compound identities. Method Validation. Three mixtures containing different polyphenols (see Testing the UPLC-MS/MS Method with Plant Extracts and Polyphenol Mixtures) were used for the determination of the limit of detection (LOD), limit of quantitation (LOQ), linearity, repeatability, and sensitivity of the developed method. LOD and LOQ were determined as peak to peak values by the signal to noise process function in MassLynx software. Signal to noise ratios (S/N) were > 3 and >10 for LOD and LOQ, respectively. Dilution series were prepared from stock solutions of 40 μg mL−1 and diluted with formic acid/acetonitrile/water (0.1:5:94.9, v/v/v) (mixture 1) or formic acid/acetonitrile/water (0.1:10:89.9, v/v/v) (mixtures 2 and 3). The dilution range was from 0.08 ng mL−1 to 20 μg mL−1. Three replicates of each sample were run for LOD, LOQ, and linearity and five replicates for repeatability.

Negative mode was more sensitive than positive mode (data not shown) and, thus, negative-ion mode was selected to be used in the MRM methods. In addition, negative ion mode fragmented compounds less extensively and thus produced clearer fragmentation patterns. As hypothesized, it was possible to find common precursor ions for fragments of compounds belonging to the same polyphenol subclass. For ellagitannins the selected precursor ion was m/z 301, which is in agreement with the earlier findings where m/z 301 has been reported as the main ellagitannin fragment.32−36 This ion is formed when the hexahydroxydiphenoyl (HHDP) groups characteristic for ellagitannins fragment from the original structure to produce m/z 301.37−39 The optimal cone voltage required for maximal accumulation of m/z 301 in the first quadrupole varied between 90 and 130 V (examples in Figure 2A), depending on the structural differences of the studied molecules: for example, the position of the HHDP group, additional galloyl groups attached to HHDP(s) (such as in valoneoyl and sanguisorboyl groups), size of the molecule, and rigidity of the structure. In general, HHDP groups located at the 2,3-position required higher cone voltages than HHDP groups at the 4,6- or 3,6-position for maximal accumulation of m/z 301. Valoneoyl, sanguisorboyl, and nonahydroxytriphenoyl groups either provided further stability to the ellagitannin structure or needed to be

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RESULTS AND DISCUSSION Optimization of the Cone Voltages. Because compounds that belong to the same polyphenol subgroup share similarities in their structural skeletons,2,30,31 it was hypothesized that it would be possible to develop group-specific MRM methods for different polyphenol subclasses. To optimize MRM methods for ellagitannins, gallic acid derivatives, quinic acid derivatives, quercetin-based flavonol glycosides, kaempferol-based flavonol glycosides, and myricetin-based flavonol glycosides, direct flow injection experiments of the reference compounds (Table 1) were performed in both positive-ion and negative-ion modes. D


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Formation of flavonol aglycone radical ions from flavonols during electrospray ionization mass spectrometry is a wellknown phenomenon.44−48 In the present study, the three flavonoid-based polyphenol groups studied, quercetins, kaempferols, and myricetins, formed two characteristic ions, the aglycone and the aglycone radical ions, in the cone voltage optimizations. These ions were m/z 301 and 300 for quercetinbased flavonoids, m/z 285 and 284 for kaempferol-based flavonoids, and m/z 317 and 316 for myricetin-based flavonoids. The optimal cone voltages for maximal accumulation of those ions varied depending on the structure of the original flavonol glycoside (examples in Figure 2D−I). The cone voltage experiments revealed that for some flavonol glycosides, ions at m/z 316, 284, and 300 derived from the ions at m/z 317, 285, and 301, respectively, by using higher cone voltages, whereas for other flavonol glycosides these ion pairs accumulated with the same cone voltages. Also, the ratio of the two adjacent ions depended on the exact structure of the studied molecule (Figure 3). Being in agreement with previous

fragmented before m/z 301 could be formed from the larger fragments. This was confirmed for ellagitannins with valoneoyl groups because these structures first yielded the valonic acid dilactone moiety and then ellagic acid when the cone voltage was further increased. Comparison of oligomeric series of ellagitannins with the same monomeric units suggested that the rigidity brought by the bonds between the monomeric units affected the required cone voltage more than the size of the molecule. For example, for the monomer tellimagrandin I and its dimeric and trimeric macrocyclic oligomers, oenothein B and oenothein A, the optimal cone voltages for the accumulation of m/z 301 were 90, 100, and 130 V, respectively. However, for both dimeric and trimeric sanguiin H-6 and lambertianin C the optimal cone voltage was the same, 120 V. In addition, the difference between the two dimers (oenothein B and sanguiin H-6; 100 vs 120 V) could be due to different types of linkages, DOG and GOD,40 between the monomers. It is thus clear that a single cone voltage is not optimal for all types of ellagitannins and that the use of several cone voltages could produce different types of ellagitannin fingerprints, depending on the ellagitannin composition of the tested plant sample. Similar patterns have been earlier witnessed for other tannin fingerprints as well: large proanthocyanidins required higher cone voltages than their monomers to be witnessed in the crude extracts.27 For gallic acid derivatives the selected precursor ion was m/z 169, which is formed when galloyl groups fragment from the original gallic acid derivative.38,41 Also for gallic acid derivatives, the size of the molecule along with the positions of the galloyl groups affected the required cone voltage for maximal accumulation of m/z 169. The optimal cone voltage varied between 50 and 130 V (examples in Figure 2B). Lowest, 50−70 V, cone voltages were optimal for simple gallic acid derivatives, such as galloyl quinic acids, gallocatechin gallate, and monogalloylglucose. For di-, tri-, tetra-, and pentagalloylglucoses the optimal cone voltages were between 100 and 130 V. Gallotannins have both galloyl groups attached to the core polyol and additional galloyls linked to the core galloyl groups via depside bond(s).41,42 As a consequence, two cone voltages, 50 and 130 V, were obtained for the maximal accumulation of m/z 169. As previously reported,31,41 the results indicated that depside bonds between two galloyls are less stable than ester bonds between the core polyol and galloyl groups. For quinic acid derivatives the cone voltage optimization was straightforward. The tetraol moiety of quinic acid at m/z 191 was selected as the precursor ion.43 The optimal cone voltage for accumulation of the precursor ion m/z 191 varied between 50 and 90 V (Figure 2C) and mainly depended on how easily the other functional groups of the studied compounds were fragmented from the quinic acid moiety. In some cases, these other functional groups could be detected separately as well. For example, galloylquinic acids can be detected by the galloylbased MRM method. On the contrary, caffeoylquinic acids did not fragment into clearly detectable levels of caffeic acid, leaving these compounds to be detected by the quinic acid-based MRM method. Both digalloyl- and dicaffeoyl-substituted quinic acids required higher optimal cone voltages than the monogalloyl (50 → 70 V) or monocaffeoyl (50 → 90 V) derivatives (Figure 2C). This information could be used to screen plant samples for highly substituted quinic acid derivatives, because their quinic acid fingerprint is better visible by high than low cone voltages, whereas the opposite is true for monosubstituted quinic acids.

Figure 3. Ratio of quercetin aglycone and aglycone radical ions in (−)-ESI for quercetin aglycone (A), quercetin-4′-O-glucoside (B), quercetin-3,4′-O-diglucoside (C), quercetin-7-O-glucoside (D), quercetin-3-O-glucoside (E), and quercetin-3-O-galactoside (F).

studies,44,48,49 the results suggested that 3-O-glycosylation favors formation of ions at m/z 300, 284, and 316, whereas 4′- and 7-glycosylations favor formation of ions at m/z 301, 285, and 317. This indicates that the comparison of ratios of different flavonol fingerprints (e.g., m/z 301 vs 300) of a given plant extract may yield information on the types of glycosylation patterns present in the flavonol glycosides (3-Oglycosides vs other types) in that plant. Optimization of Collision Energies. Enhanced selectivity and sensitivity of the method are achieved by collision energies separately optimized for product ions of the specific precursor ions. Such an approach allows the accurate qualitative and quantitative analysis of different polyphenol subgroups directly from plant crude extracts. In contrast to identifying optimal cone voltages, the choice of optimal collision energy was straightforward. For ellagitannins, gallic acid and quinic acid derivatives, and quercetin, kaempferol, and myricetin glycosides, the selected product ions deriving from the selected precursor ions always had the same optimal collision energies. The optimal collision energies are presented in Table 3. Two transitions, one qualifier and one quantifier, were selected for the detection of each group of compounds to avoid detection of false positives. The ratios of the peak areas obtained by the qualitative and quantitative MRM transitions are shown in Table 3. These ratios are constant for true detections and E


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Journal of Agricultural and Food Chemistry Table 3. Group-Specific and Compound-Specific MRM Methods Developed and Used in the Present Study polyphenol class

precursor ion (m/z)

ellagitannins gallic acid derivatives quinic acid derivatives quercetin derivatives kaempferol derivatives myricetin derivatives

301 169 191 301 300 285 284 317 316

compound tellimagrandin I oenothein B agrimoniin vescalagin pentagalloylglucose 3-O-caffeoylquinic acid 3,4-O-dicaffeoylquinic acid quercetin-3-O-glucoside quercetin-3-O-galactoside quercetin-4′-O-glucoside quercetin-3-O-rhamnoside kaempferol-3-O-glucoside kaempferol-7-O-glucoside kaempferol-7-O-neohesperoside myricetin-3-O-rhamnoside a

cone voltage (V)

product ion (quant) (m/z)

110 (90−120) 80 (50−130) 70 (50−90) 85 (60−110) 85 (60−110) 80 (60−110) 80 (60−110) 75 (60−85) 75 (60−85) precursor ion (m/z)

200 125 127 151 271 229 255 271 271

collision energy (eV)

40 15 15 20 25 20 25 20 20 cone voltage (V)

785 783 934 933 939 353 515 515 463 463 463 463 447 447 447 593 463

55 34 38 46 60 24 25 25 40 40 40 40 45 35 40 50 40

product ion (qual) (m/z)

qual/quanta (%)

collision energy (eV)

145 107 109 179 255 257 227 287 287 product ion (m/z)

35 44 ± 3 20 32 ± 8 20 39 ± 3 20 46 ± 2 20 48 ± 3 25 27 ± 4 25 99 ± 4 25 14 ± 1 25 61 ± 2 collision energy (eV)

301 765 301 301 169 191 173 179 300 300 301 301 301 284 285 285 316

40 22 48 64 32 18 25 30 35 25 20 20 20 25 25 30 25

Ratio of the obtained peak areas with qualitative to quantitative MRM transitions (qual/quant × 100%).

detected polyphenol groups are available. Without proper standards, however, MRM fingerprints should not be converted into quantitative results, because this might result in severe over- or underestimation of the polyphenol groups, depending on the standards used. Detection with the presented group-specific MRM method was also compared with compound-specific MRM methods (Figure 4) that were separately optimized for every quantified compound. The results indicated that for all but two compounds the compound-specific methods were more sensitive and yielded better responses than the group-specific methods. The two exceptions were tellimagrandin I and pentagalloyl glucose, which were better detected with the galloyl-specific MRM method than with the compound-specific MRM methods. In fact, tellimagrandin I, which contains one HHDP and two galloyl groups, was better detected by the galloyl than the ellagitannin method (1 in Figure 4B,C). All in all, compound-specific MRM methods cannot be substituted by the developed group-specific methods, but both could be used parallel, depending on the need. For instance, Figure 5 shows how the UPLC-DAD chromatogram at 349 nm was able to reveal nine flavonoids from the crude extract of Epilobium hirsutum (characterization confirmed by UV spectra28). During the same UPLC run, group-specific MRM methods were able to classify three of these compounds as myricetin derivatives, three as quercetin derivatives, and the remaining three as kaempferol derivatives. Simultaneously acquired full scan MS revealed the molecular weights of these compounds, enabling their further characterization. It was found that all three sets of three flavonol glycosides presumably contained the same three

change as a result of false positives. This was demonstrated with the total peak areas of the 15 plant extracts, the individual compounds of each extract, and the pure compounds presented in Table 3. These ratios should be used to verify that the correct polyphenol groups have been detected by the MRM methods. Testing the UPLC-MS/MS Method with Plant Extracts and Polyphenol Mixtures. After optimization of MRMs, the new method was rigorously tested with 14 different plant extracts and 3 mixtures of different polyphenols and their dilution series. All samples were analyzed by using the developed group-specific MRM method and compound-specific MRM methods (Table 3). When the mixtures of pure compounds were analyzed, it was noted that the peak areas obtained with the group-specific MRM method depended both on the cone voltage used and on the number of the units in the studied polyphenol structure that yielded the precursor ions (Figure 4). As suggested already for cone voltage optimization, this indicated differences in the collision-induced dissociation of different molecules belonging to the same polyphenol subgroup. Due to this, the intensities of the peaks obtained, for example, for vescalagin (8) and agrimoniin (6) differed even if both molecules yielded maximum accumulation of m/z 301 when the cone voltage was 110 V (Figure 4B). In contrast, even though the change in applied cone voltage affected absolute peak areas, it did not affect the shape of the concentration versus peak area plots when dilution series were analyzed. This indicates that group-specific fingerprints can be detected even by using nonoptimal cone voltages, and fingerprints can be converted to quantitative data, if suitable standards of each F


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Figure 4. UPLC fingerprints of polyphenol mixtures 1 (A−D) and 3 (E−I) as detected by UV at 280 nm (A, E) and group-specific (black lines) and compound-specific (red lines) MRM methods for ellagitannins (B), gallic acid derivatives (C, I), quinic acid derivatives (D), quercetin-based flavonoids (F), kaempferol-based flavonoids (G), and myricetin-based flavonoids (H). See Table 1 for compound identities.

carbohydrates as building blocks: the first compound in the set of three peaks always gave the mass of 180 Da for the carbohydrate (possibly glucose or galactose), the second compound gave the mass of 132 Da (xylose/lyxose, but more presumably arabinose), and the third compound gave the mass of 146 Da (rhamnose) for the carbohydrate part of the glycoside. This could not have been achieved by full scan only as aglycone fragments are not always unequivocally witnessed. Furthermore, these findings were confirmed by compoundspecific MRMs (see examples in Figure 5F−I). This illustrates the logic of how the created group-specific methods should be used to facilitate compound characterization and the measurement of group-specific fingerprints. For every species and polyphenol group analyzed, one should first reveal the percentage of the fingerprint produced by the qualifier MRM transition of that produced by the quantifier MRM transition. If the ratio deviates from values shown in Table 3, then further examination of the sample by UV and full scan MS is needed either to verify the results obtained by the fingerprint method

or to reveal the causes for false positives. For instance, some flavonols and flavones share the same mass for their aglycone part (e.g., 286 Da for luteolin and kaempferol), but these two types of flavonoids are easily separated by both UV spectra and MS/MS spectra recorded for the aglycone part. The latter difference in MS/MS spectra explains why flavones produce variable ratios with the qualifier/quantifier transitions from those of flavonols. In the present work, quantification was established by preparing calibration curves with pure compounds (polyphenol mixtures 1, 2, and 3). The results obtained by group- and compound-specific MRM methods (Table 3) for the 15 plant extracts are presented in Table 4. Results were expressed as milligrams per gram of plant dry weight. The developed groupspecific MRM method was able to produce quantitative data comparable to the compound-specific MRM methods for all of the studied polyphenols. However, in some extracts detection of tellimagrandin I (1) with the compound- and group-specific methods yielded divergent results (see Table 4). Due to the G


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Figure 5. UPLC fingerprints of water-soluble flavonoids of Epilobium hirsutum (great hairy willowherb) leaves as detected by UV at 349 nm (A), full scan (E), and MRM methods for myricetin-based flavonoids (B), quercetin-based flavonoids (C), and kaempferol-based flavonoids (D). Panels F−I show detection with compound-specific MRM methods for myricetin-3-O-rhamnoside (F), quercetin-3-O-galactoside (G), kaempferol-3-Oglucoside (H), and quercetin-3-O-rhamnoside (I). Insets in (A) and (E) present UV and mass spectra for selected peaks. FL, flavonoid; MyrGlyc, myricetin glycoside; QueGlyc, quercetin glycoside; KaeGlyc, kaempferol glycoside.

coelution of compounds from the same polyphenol subgroup, tellimagrandin I could not be detected separately anymore by the developed group-specific MRM method, but instead a combined signal of several ellagitannins was detected. This resulted in overestimated quantification when compared to the results obtained by the compound-specific methods. This further demonstrated the purpose for which the method was developed: for fingerprint analysis and quantification of total concentrations of different polyphenol subgroups, not for quantification of individual compounds. The results indicated also that in the present work the matrix effect of the plant extracts was not remarkable, and calibration curves produced

with pure compounds were sufficient for obtaining quantitative data. This highlights the importance of correct sample concentration: to minimize matrix effect, sample concentration should be preferably adjusted to such a level where no detection rises above the 1 × 106 limit. Also, compounds used for quantification must be carefully selected. In present work, compounds used in the standard mixtures were selected so that they would as well as possible resemble the range of polyphenols in the studied plant extracts. If this is not possible, the quantitation results should be treated as relative ones, not absolute, and expressed as equivalents of the compounds used as standards. H


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Table 4. Quantitative Results Obtained by the Developed Group-Specific MRM Method for the Different Polyphenol Subclasses in the Studied Plant Extracts and Comparison of Quantification of Single Compounds in the Studied Plant Extracts with the Developed Group-Specific MRM Method and Compound-Specific MRM Methodsa plant extract quantitative results

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

total galloyl totalellagitannin total quinic acid total kaempferol total quercetin total myricetin

6.7 41.3 6.2 0.8 9.3

4.3 147.4 1.8 1.8 14.8

1.4 69.7

0.4

0.5

0.4

0.4

1.0 127.3 2.5 0.4 0.3

0.1

4.0 55.3

6.4 0.4 1.4 0.4

2.2 142.7 0.1 0.1 0.1

0.4

0.7 0.9

1.4 2.7 6.1 5.3 4.3 0.2

3.2 151.9

1.2 2.4 7.8

3.3 30.9 4.1 14.7 12.6 1.6 quantitative

31.8 1.4 3.1

7.1 0.7 1.2

a

14.9 18.6 2.3 1.4 1.7 0.5 results (mg g−1)

10.3 4.0 0.2

compound

group-specific MRM/compound-specific MRM

1 2 6 8 23 29 31 + 32 36 41 49

7.6/8.1; 11.0/7.8; 5.5/2.5; 8.8/4.8; 0.9/0.2; 9.9/0.2; 25.2/0.8; 2.1/0.1 28.0/29.2; 26.2/27.4 27.9/21.2 2.2/3.4; 3.0/4.3 3.8/4.6; 1.4/0.9; 2.7/3.0 1.4/4.2; 0.5/1.8 2.8/2.6; 0.5/0.7; 0.3/0.3; 0.8/0.9; 0.2/0.1; 0.4/0.4; 1.0/1.3; 1.2/1.5 0.3/0.3; 0.1/0.1; 0.2/0.8; 4.6/5.3; 0.8/0.9; 0.4/0.3; 0.2/0.1 0.1/0.2; 0.3/0.3; 0.1/0.1; 0.1/0.1; 0.1/0.2; 0.4/0.6; 0.1/0.1; 0.3/0.4; 1.5/2.0; 0.1/0.2 3.9/3.6; 1.2/1.5; 0.5/0.6; 0.0/0.1; 0.0/0.1

5.9 7.9

Results are expressed as mg g−1 of dry material. See Tables 1 and 2 for compound and plant extract identities, respectively.

and 10−0.3 μg mL−1, respectively. For both quercetin and kaempferol glycosides LOD was 0.3 ng mL−1 and LOQ, 1.2 ng mL−1. For myricetin-based flavonol glycosides LOD and LOQ were 40 and 80 ng mL−1, respectively. As witnessed in a previous section when the method was tested with polyphenol mixtures, these results further indicated that, in general, the developed UPLC-MS/MS method was more sensitive for the quercetin and kaempferol glycosides than for the other studied polyphenol subclasses. Also, the LOD, LOQ, and linear ranges varied slightly within each polyphenol subclass, depending on the exact structure of the studied molecule. The presented values were obtained with molecules for which the present method was most insensitive; that is, with other types of molecules the method would have performed better. The repeatability of the method was determined using Student’s t test at the 95% confidence interval. Five replicates in two different concentrations (20 and 5 μg mL−1) were analyzed, and the results were presented as confidence intervals of the sums of peak areas obtained for ellagitannins, gallic acid and quinic acid derivatives, and quercetin-, kaempferol-, and myricetin-based flavonol glycosides. The relative standard deviation (RSD) for all six samples with five replicates was determined from these results. The RSD for peak areas was 2.4%, which demonstrates the pronounced reliability of the developed UPLC-MS/MS method. The results suggested that the quantitative determination of ellagitannins and quinic acid derivatives was slightly less accurate than for the other polyphenol classes (RSD, 3.1, 2.7, and 1.8−2.4%, respectively). Naturally, more accurate results were obtained when operating at the linear range than above the linear range. In this study we created novel methods for the analysis of six common classes of plant polyphenols. UPLC enabled rapid chromatography of different kinds of polyphenols, whereas TQMS fragmented compounds into group-specific precursor and product ions for detection with MRM methods. With the

One of the most common problems for the developed methods is related to both ellagitannins and quercetin-based flavonoids producing precursor ions with the very same m/z value (m/z 301), and therefore LC-MS/MS methods have been used in previous studies to distinguish their conjugates.32,33 In the present study, it was noted that even if the ellagitannin and quercetin MRM transitions had different product ions (301 → 200/301 → 145 and 301 → 151/301 → 179 respectively), a small false-positive ellagitannin response (0.1−1%) was obtained from quercetin derivatives when their concentrations were >1 μg mL−1. For qualitative analysis this will not cause any problems because the false-positive response of ellagitannins is easily distinguished directly from the MRM chromatograms: Figure 6C shows a false-positive detection of ellagitannins that is due to high levels of quercetin glycosides (panel B), and panel F shows the true ellagitannin detection that is not disturbed by the 0.1−1% false positive caused by the quercetin glycosides (panel E). As noted above, these findings can be confirmed by comparing peak areas obtained by quantifier versus qualifier MRM transitions as well. All in all, although the new MRM methods significantly aid in the mission of fingerprinting plant extracts for various polyphenol groups, they cannot be used as such without careful inspection of the data produced. Once the findings are confirmed for each studied species, the methods can be used to more routinely screen the differences of polyphenol fingerprints between individual plants of the selected species. Method Validation. LOD, LOQ, and linearity were determined with the presented method for polyphenols present in the three mixtures containing different polyphenols. For both ellagitannins and gallic acid derivatives LOD and LOQ were 150 and 310 ng mL−1, respectively. For quinic acid derivatives LOD was 20 ng mL−1 and LOQ, 80 ng mL−1. The linear range was the same, 15−0.7 μg mL−1 for ellagitannins, gallic acid, and quinic acid derivatives. For the quercetin, kaempferol, and myricetin glycosides the linear ranges were 7.5−0.02, 7.5−0.08, I


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Figure 6. UPLC traces at 280 nm for polyphenol extracts of Fumaria of f icinalis flowers (A) and Rubus saxatilis leaves (D). Panels B and E show MRM fingerprints detected for quercetin-based flavonoids, and panels C and F show MRM fingerprints detected for ellagitannins. Quercetins produce

Rapid Fingerprint Analysis of Plant Extracts for ... - ACS Publications

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