Article pubs.acs.org/JAFC

High-Performance Liquid Chromatography Separation of cis−trans Anthocyanin Isomers from Wild Lycium ruthenicum Murr. Employing a Mixed-Mode Reversed-Phase/Strong Anion-Exchange Stationary Phase Hongli Jin,†,‡,§ Yanfang Liu,*,‡ Zhimou Guo,‡ Fan Yang,∥ Jixia Wang,‡,§ Xiaolong Li,†,‡,§ Xiaojun Peng,† and Xinmiao Liang‡ †

State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian, Liaoning 116012, People’s Republic of China Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, Liaoning 116023, People’s Republic of China § University of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China ∥ Analytical Chemistry Service Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, Liaoning 116023, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: The cis−trans isomerism is a common phenomenon for acylated anthocyanins. Nevertheless, few studies reported effective methods for the preparation of isomeric anthocyanins from natural products. In this work, a high-performance liquid chromatography (HPLC) method was developed to efficiently purify anthocyanin isomers from Lycium ruthenicum Murr. based on a mixed-mode reversed-phase/strong anion-exchange column (named XCharge C8SAX). Four commercially available columns were evaluated with a pair of isomeric anthocyanins, and the results demonstrated that the XCharge C8SAX column exhibited improved selectivity and column efficiency for the isomers. The chromatographic parameters, including pH, organic content, and ionic strength, were investigated. Optimal separation quality for the anthocyanin isomers was achieved on the XCharge C8SAX column. Six pure anthocyanins, including two pairs of cis−trans isomeric anthocyanins with one new anthocyanin, were purified from L. ruthenicum and identified. All of the results indicated that this method is an effective way to separate anthocyanins, especially for cis−trans isomers. KEYWORDS: anthocyanins, cis−trans anthocyanin isomer, Lycium ruthenicum Murr., mixed-mode stationary phase, HPLC

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INTRODUCTION Anthocyanins, a group of phenolic compounds, confer the characteristic colors to most fruits and vegetables. Increased evidence have proven their possible heath benefits in preventing chronic and degradative diseases, including heart disease and cancer.1 At present, although more than 600 naturally occurring anthocyanins have been characterized, only a limited number of pure anthocyanin standards are commercially available with a steep price. Most bioassays on anthocyanins have to be conducted with crude anthocyanin extracts, likely resulting in the ambiguous explanation of the bioactivity of anthocyanins.2 Therefore, preparation of pure anthocyanins is demanded for their bioactivity investigation. Lycium ruthenicum Murr. is a kind of widespread nutritional food in the salinized desert of the Qinghai−Tibet Plateau. It is also a famous traditional herb recorded in the Tibetan medical classic “Jing Zhu Ben Cao”. L. ruthenicum has been applied to treat many diseases, such as heart disease, abnormal menstruation, and menopause. Besides, the pigments of L. ruthenicum are demonstrated to possess good antioxidant activity,3,4 and thus, anthocyanins in this plant have aroused great interest to many researchers. Recently, Zheng et al.5 characterized anthocyanins in L. ruthenicum by high-performance liquid chromatography (HPLC) with electrospray ionization−mass spectrometry © 2014 American Chemical Society

(ESI−MS), suggesting that most of them are acylated anthocyanins. Chen et al.6 had identified and quantified seven main anthocyanins in L. ruthenicum. The results showed that the anthocyanins were mainly acylated with coumaric acid in both cis and trans configurations. Nevertheless, few anthocyanins in this plant have been isolated and identified by nuclear magnetic resonance (NMR). Information on their chemical structures is still incomplete. The purification of anthocyanins with enough purity and amount for structural elucidation is of great importance. Column chromatography (CC) has been used extensively in the separation of anthocyanins from natural products.7−10 Different columns are able to offer different separation selectivities for anthocyanins, which provide significant contribution to the preparation of anthocyanins.11 Unfortunately, this technique will suffer from laboriousness and low efficiency, owing to consecutive column separation. Poor reproducibility is another drawback that hampers the application of CC in the purification. Alternatively, HPLC, one of the most effective Received: Revised: Accepted: Published: 500

September 23, 2014 December 23, 2014 December 24, 2014 December 24, 2014 DOI: 10.1021/jf504525w J. Agric. Food Chem. 2015, 63, 500−508

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Journal of Agricultural and Food Chemistry

anthocyanins using a RP/strong anion-exchange column as well as to explore the effect of properties of stationary phases on selectivity toward the isomers. The potential of this approach will be demonstrated by the purification of anthocyanin isomers from L. ruthenicum.

techniques to isolate compounds from complex samples, provides many advantages over other techniques, such as high efficiency and good repeatability. It appears to be a practical method for anthocyanin preparation.12,13 Nonetheless, owing to limited separation selectivity, this technique has to be used in combination with CC and taken as a final purification to yield pure anthocyanins.8,11 Preparation of anthocyanins, especially for cis−trans isomers, by HPLC remains to be further improved. Structurally, anthocyanins are heterosides of an aglycone unit (anthocyandin) with the aromatic carboxylates, such as pcoumaric acid, coffeic acid, etc., bonded to the sugar in the molecule. Therefore, the acylated unit of anthocyanin easily generates cis−trans isomers (Figure 1). These stereoisomers

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MATERIALS AND METHODS

Plant Material. The fruits of L. ruthenicum were randomly sampled from Dulan (latitude, 36° 26′ N; longitude, 96° 31′ E; altitude, 2774 m). The fruits were ripe, hand-picked, and then preserved in 4 °C for subsequent experiments. Apparatus and Reagents. Purification Factory is a preparative liquid chromatography (LC) system, which consists of two 2525 binary gradient modules (Waters, Milford, MA), an autosampler (Leap Technologies, Carrboro, NC), a 2498 ultraviolet (UV) detector (Waters), and MassLynx software (Waters, version 4.1). Chromatographic separation and analysis were performed on an Alliance HPLC system consisting of a Waters 2695 HPLC pump and a 2489 UV−vis detector. Data acquisition and processing were conducted by Waters Empower software (Milford, MA). Identification of pure compounds was carried out using mass spectrometry (MS) and NMR. MS was performed on a Q-TOF Premier (Waters MS Technologies, Manchester, U.K.). 1H NMR and nuclear Overhauser effect spectrometry (NOESY) were measured on a Bruker DRX-400 spectrometer (1H NMR at 600 MHz), with MeOH-d4/TFA-d (95:5, v/v) as the solvent. ACN was purchased from Merck of HPLC grade (Darmstadt, Germany) and from Yuwang Chemical Reagent Factory of industrial grade (Shandong, China). Methanol was purchased from Yuwang Chemical Reagent Factory of HPLC grade. Trifluoroacetic acid (TFA) and formic acid (FA) were purchased from J&K Chemical of HPLC grade (Hebei, China). Sodium biphosphate (NaH2PO4) was purchased from Sinopharm (Shanghai, China). Phosphoric acid was purchased from Tedia of HPLC grade (Fairfield, OH). Water for the HPLC mobile phase was reverse osmosis Milli-Q water (18.2 MΩ, Millipore, Billerica, MA). The references petunidin 3-O-[6-O-(4-O-(cis-p-coumaroyl)-α-Lrhamnopyranosyl)-β-D-glucopyranoside]-5-O-[β-D-glucopyranoside] (P1) and petunidin 3-O-[6-O-(4-O-(trans-p-coumaroyl)-α-L-rhamnopyranosyl)-β-D-glucopyranoside]-5-O-[β-D-glucopyranoside] (P2) were isolated in our laboratory. Their structures were characterized by MS and NMR. The structures of these two anthocyanins were displayed in Figure 1. The columns used in this work are listed as follows: XTerra MS C18 (4.6 × 150 mm, 5 μm, Waters, Milford, MA), XUnion C18 (4.6 × 150 mm, 5 μm Acchrom, Beijing, China), XUnion C8 (4.6 × 150 mm, 5 μm Acchrom, Beijing, China), and XCharge C8SAX (4.6 × 150 mm, 5 μm Acchrom, Beijing, China). The representative surface chemistry of the XCharge C8SAX stationary phase is shown in Figure 2. The detailed properties of the four columns are listed in Table 1. Phosphate buffers were prepared using NaH2PO4 and adjusted to desired pH with H3PO4 solution at corresponding concentrations before the addition of organic solvent (pH values). The buffers were filtered through 0.22 μm membranes before use.

Figure 1. Structures of the cis−trans anthocyanin isomers from L. ruthenicum.

increase the difficulty in the isolation of anthocyanins from plants. The usual method for separating anthocyanins is almost exclusively performed on conventional C18 columns with aqueous acid/acetonitrile (ACN) as mobile phases.14−16 This classic method shows good repeatability and efficiency for anthocyanin separation, but it has some limitations for isolating anthocyanin isomers. Multiple p-coumaric peaks for a given aglycone, attributed to the isomers, would occur, complicating the isolation and identification of anthocyanins.17 In addition, the nearly co-eluted peaks are observed for some cis−trans anthocyanin isomers on reversed-phase (RP) columns, leading to difficulty in collecting compounds with good purity. Lately, Willemse et al.18 have used hydrophilic interaction chromatography (HILIC) to separate anthocyanins for the first time. Coelution of cis−trans acylated isomers, lower chromatographic efficiency, and higher organic solvent consumption restricted its application in the isomer purification. It is desirable to establish a HPLC method to achieve efficient preparation of isomeric anthocyanins. The mixed-mode columns are capable of bringing interesting selectivity to separate complex mixtures and offer several advantages over conventional RP.19−21 In recent years, Mccallum et al.22 obtained improved separation of anthocyanins in grapes using a mixed-mode RP/anion-exchange column. Superior separation performance for acylated anthocanins was observed. Subsequently, Vergara et al. 23 published that different selectivities of the mixed-mode stationary phase were achieved in the separation of some wine anthocyanins probably based on the complex mechanisms of reverse chromatography and ionexclusion chromatography. In the present work, we aimed to develop an efficient HPLC method to prepare cis−trans isomeric

Figure 2. Representative surface chemistry of the XCharge C8SAX column. 501

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Journal of Agricultural and Food Chemistry Table 1. Physicochemical Properties of the Columns column

ligand

specific surface area (m2/g)

average pore size

total carbon (%)

column efficiency

XTerra MS C18 XUnion C18 XUnion C8 XCharge C8SAX

C18 C18 C8 C8

175 320 320 320

125 100 100 100

15.5% C load 20.3% C load 12.4% C load 9.57% C load

74000 73000 80000 75000

The dead time (to) of the columns was measured by uracil. The column efficiency of all of the columns used in this work was determined by naphthalene with a similar retention factor (k). All results are the average of triplicate determinations. The resolution (R) and selectivity (α) were used for the evaluation of separation quality. These data could be calculated from the system suitability software of the ChemStation. Chromatographic Conditions. HPLC experiments were performed at the flow rate of 1.0 mL/min. The column temperature was maintained at 30 °C throughout using a thermostat. Detection was carried out at 520 nm, unless otherwise specified. For column selection, the mobile phase was composed of NaH2PO4 (10 mM, pH 1.5) and ACN (85:15, v/v). The preparation of fractions 6, 8, and 9 was conducted on an XCharge C8SAX column (4.6 × 150 mm, 5 μm). The mobile phase A was 0.2% TFA (v/v) in water, and the mobile phase B was 0.2% TFA (v/v) in ACN. Different isocratic elution conditions were adopted to separate three fractions. Isocratic elution for fraction 6 was 12% B; isocratic elution for fraction 8 was 14% B; and isocratic elution for fraction 9 was 15% B. HPLC analysis of pure compounds was performed on an XTerra MS C18 column (4.6 × 150 mm, 5 μm). The mobile phase A was 0.2% TFA (v/v) in water, and the mobile phase B was 0.2% TFA (v/v) in methanol. Gradient elution steps were as follows: 0−30 min, 15−50% B; 30−40 min, 90% B. Extraction of Anthocyanins. The fruit of L. ruthenicum Murr. was extracted triply with 20-fold of 70% ethanol for 2 h at pH 2.5 (adjusted with hydrochloric acid). Subsequently, three filtrates were combined and concentrated by rotary evaporation at 50 °C in vacuum. Then, the aqueous extract was prepared on the AB-8 macroporous resin (50 × 520 mm, Chemical Plant of NanKai University, Tianjing, China). The strong polar constituents were removed with aqueous acid (0.5% FA, v/v). Then, the target constituent was eluted with 70% ethanol. It was concentrated by rotary evaporation at 50 °C in vacuum and, finally, lyophilized. Sample Preparation. The anthocyanin sample obtained from extraction described above was dissolved in aqueous acid and subjected to a SCX solid-phase extraction (SPE) cartridge (40 mL, 20 g of sorbent, Acchrom), preconditioned successively with MeOH and distilled water (0.5% FA). Non-anthocyanin compositions were collected with 3 vol of 5% ACN (0.5% FA). Subsequently, anthocyanins were eluted with 3 vol of 30% ACN (1 M NaH2PO4 at pH 2.0). Anthocyanin solution was dried by rotary evaporation at 50 °C in vacuum to remove organic solvent as much as possible and then loaded on the AB-8 macroporous resin. Phosphate was washed out by distilled water (0.5% FA). Anthocyanins were eluted with 70% ethanol. They were concentrated by rotary evaporation at 50 °C in vacuum. Fractionation of anthochanin constitutions was carried out on an XTerra MS C18 column (50 × 150 mm, 5 μm, Waters, Milford, MA) using methanol/water (0.2% TFA) as mobile phases, and in total, 14 fractions were collected according to UV absorption intensity (shown in the Supporting Information). Fractions 6, 8, and 9 were selected in this work to conduct further separation. A total of 100 mg of each fraction (fractions 6, 8, and 9) was dissolved in 2.5 mL of acid water and filtered through 0.22 μm pore size membranes to obtain an anthocyanin sample with a concentration at about 40 mg/ mL.

Figure 3. HPLC chromatograms of the anthocyanin isomers on (A) XTerra MS C18 (4.6 × 150 mm, 5 μm), (B) XUnion C18 (4.6 × 150 mm, 5 μm), (C) XUnion C8 (4.6 × 150 mm, 5 μm), and (D) XCharge C8SAX (4.6 × 150 mm, 5 μm). Isocratic elution with NaH2PO4 (10 mM, pH 1.5) and ACN (85:15, v/v), with a flow rate of 1 mL/min, temperature of 30 °C, and wavelength of 520 nm.

Table 2. Column Parameters for the Separation of Isomeric Anthocyanins on the Four Columns

a

column

a

R

N

XTerra MS C18 XUnion C18 XUnion C8 XCharge C8SAX

1.12 1.12 1.11 1.26

1.18 a 1.43 3.36

25000 a 30000 40000

Could not be determined.

the same particle size, diameter, and column length were tested to separate a pair of cis−trans anthocyanin isomers. The chromatograms for four different columns are shown in Figure 3. The selectivity (a), resolution (R), and column efficiency (N) values for the tested anthocyanins on each column are listed in Table 2. The XTerra MS C18 column, a hybrid-based C18 column, was chosen to separate the anthocyanin isomers for its good hydrophobic selectivity and excellent stability under high acidic conditions. The isomers were properly retained (P1 in Figure 1) with acceptable resolution for analytical purposes (R = 1.18; Table 2). Nonetheless, this analytical resolution seems inadequate for preparative application, because overlapping of the anthocyanin isomers might occur. It is necessary to separate them with satisfactory resolution. Selectivity has the greatest impact upon changing resolution, as compared to efficiency and retention (k). As a result, a column with better separation selectivity toward cis−trans anthocyanin isomers would be the

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RESULTS AND DISCUSSION Column Selection. Column selection is one of the first principal factors for better separation in LC. In this work, four commercially available columns with different solid phases but 502

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Figure 4. HPLC chromatograms of the anthocyanin isomers on the (A) XTerra MS C18 column and (B) XCharge C8SAX column with pH of the mobile phase at (a) 2.32, (b) 2.06, (c) 1.76, and (d) 1.56, with the mobile phase of 85:15 (v/v) phosphate buffer/ACN and ionic strength of 15 mM. Other conditions are the same as those in Figure 3.

Figure 5. Plot of the log k versus volume fraction of ACN in the mobile phase: (A) XTerra MS C18 column and (B) XCharge C8SAX column, with the mobile phase from 86:14 to 84.5:15.5 (v/v) phosphate buffer at pH 1.5/ACN and ionic strength of 15 mM. Analytes: (■) P1 and (△) P2. Other conditions are the same as those in Figure 3.

Figure 6. Plot of retention times of the anchoyanin isomers versus phosphate concentrations in the mobile phase from 1 to 15 mM: (A) XTerra MS C18 column and (B) XCharge C8SAX column, with the mobile phase of 85:15 (v/v) phosphate buffer at pH 1.5/ACN. Analytes: (■) P1 and (△) P2. Other conditions are the same as those in Figure 3.

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Figure 7. HPLC chromatograms of the anthocyanin isomers on the XCharge C8SAX column with (A) 85:15 phosphate buffer (ionic strength of 15 mM at pH 1.5)/ACN, (B) 86:14 (v/v) 5% (v/v) FA in water (pH 1.6−1.7)/5% (v/v) FA in ACN, and (C) 83:17 (v/v) 0.2% (v/v) TFA in water (pH 1.5−1.6)/0.2% (v/v) TFA in ACN. Other conditions are the same as those in Figure 3.

Figure 9. Purity evaluation of the isolated compounds on the XTerra MS C18 column (4.6 × 150 mm, 5 μm). F6-1 and F6-2 were isolated from fraction 6; F8-1 and F8-2 were isolated from the fraction 8; and P1 and P2 were isolated from fraction 9. Conditions: mobile phase A, 0.2% TFA aqueous solution; mobile phase B, 0.2% TFA in MeOH; gradient, from 15 to 50% B in 0−30 min. Other conditions are the same as those in Figure 3.

the anthocyanin isomers (panels B and C of Figure 3 for XUnion C18 and XUnion C8, respectively). The results showed that the anthocyanins eluted earlier on XUnion C8, indicating that the isomers experienced weaker hydrophobic interaction on XUnion C8. However, selectivity values were similar on these two columns (a = 1.12 and 1.11 for XUnion C18 and XUnion C8, respectively; Table 2). These data revealed that the contribution of the hydrophobic interaction to the selectivity for the cis−trans isomeric anthocyanins was quite limited. The XTerra MS C18 column, despite different bonding techniques, also had a similar selectivity value as either XUnion C18 or XUnion C8. This phenomenon further confirmed the above deduction. The cis− trans isomerism is a form of stereoisomerism. Both of the isomers have exactly the same atoms joined up in exactly the same order. This means that their hydrophobicity might be analogical, and thus, conventional RP columns, which provided a single type of solute−sorbent hydrophobic interaction, would suffer from limited selectivity for the cis−trans anthocyanin isomers. In addition, it was interesting to note that the XUnion C8 column displayed much better resolution than XUnion C18, as shown in Table 2. This could be primarily ascribed to the higher separation efficiency for the isomeric anthocyanins on XUnion C8 than that on XUnion C18 (Table 2). It was reasonable to infer that stationary phases with C8 alkyl ligands were more suitable for separation of the anthocyanin isomers. In a highly acidic mobile phase, anthocyanins mainly exist in the falvylium cation form with a positive charge.24,25 The cis− trans isomers of anthocyanin have a different spatial arrangement, probably resulting in a certain difference in electrostatic

Figure 8. Preparative chromatograms of fractions 6, 8, and 9 under optimized conditions on the XCharge C8SAX column (4.6 × 150 mm, 5 μm). Conditions: mobile phase A, 0.2% TFA aqueous solution; mobile phase B, 0.2% TFA in ACN; and isocratic elution, 12% B for fraction 6, 14% B for fraction 8, and 15% B for fraction 9. Other conditions are the same as those in Figure 3.

optimal choice for their preparation. It appeared interesting that properties of stationary phases would influence the selectivity to the isomers. For RP columns, the hydrophobic interaction was regarded as the most important contribution to retention. Its effect on the selectivity to the anthocyanin isomers was investigated. XUnion C18 and XUnion C8, two conventional RP columns with alkyl C18 and C8 ligands, respectively, were investigated for separation of 504

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Journal of Agricultural and Food Chemistry Table 3. 1H NMR Data for the Isolated Anthocyanins in CD3OD/TFA-d (95:5, v/v) H

F6-1

F6-2

F8-1

F8-2

4-H 6-H 8-H 2′-H 5′-H 6′-H 3′-OCH3 5′-OCH3

8.96 s 7.03 s 7.05 s 7.97 d (2.1)

8.95 s 7.02 s 7.03 s 7.97 d (2.1)

Anthocyandin 8.97 s 7.03 s 7.04 s 8.0 d (1.9)

7.80 d (2.1) 4.01 s

7.80 d (2.1) 4.01 s

7.85 d (2.0) 4.01 s

1″ 2″ 3″ 4″ 5″ 6a 6b

5.53 d (7.7) 3.50−3.80

5.54 d (7.8) 3.2−4.1

5.19 d (7.8) 3.2−4.1

1‴ 2‴ 3‴ 4‴ 5‴ 6a 6b

3.96 4.07 5.22 d (7.8) 3.50−3.80

P1

P2

8.92 s 7.03 s 7.03 s 7.82 s

8.90 s 7.01 s 7.01 s 7.93 s

8.96 s 7.03 s 7.06 s 7.99 d (1.5)

7.82 s

7.77 s 3.96 s

7.82 d (1.8) 4.01 s

3-O-Glucopyranoside 5.50 d (7.7) 3.45−3.86

5.49 d (7.8) 3.46−4.07

5.52 d (7.8) 3.43−3.92

3.97 4.06 5-O-Glucopyranoside 5.20 d (7.8) 3.36−4.06

5.53 d (7.8) 3.84 3.78 3.70 3.75 3.98 4.06

5.19 d (7.9) 3.46−4.07

5.20 d (7.8) 3.43−3.92

5.22 d (7.8) 3.82 3.76 3.72 3.73 3.87 3.96

4.72 s

4.66 s 3.43−3.92

4.92 t (9.8) 1.01 d (6.2)

4.81 s 3.43−3.92 0.86 d (6.2)

4.74 s 3.61 3.59 3.92 t (9.7) 3.58 1.02 d (6.2)

7.43 d (8.6) 6.82 d (8.6) 6.82 d (8.6) 7.43 d (8.6) 6.26 d (15.9) 7.59 d (15.9)

7.58 d (7.0) 6.69 d (6.9) 6.69 d (6.9) 7.58 d (7.0) 5.56 d (12.7) 6.77 d (12.7)

7.44 d (8.5) 6.83 d (8.4) 6.83 d (8.4) 7.44 d (8.5) 6.26 d (15.9) 7.58 d (15.9)

3.85 3.86 3-Glucosyl

1′′′′′ 2′′′′′ 3′′′′′ 4′′′′′ 5′′′′′ 6a 6b

5.00 d (7.2) 3.50−3.80

5.05 d (7.3) 3.2−4.1

3.84 3.94

1′′′′ 2′′′′ 3′′′′ 4′′′′ 5′′′′ −CH3

4.75 s 3.50−3.80

4.68 s 3.50−3.80

4.93 t (9.7) 3.50−3.80 1.0 d (6.2)

4.83 m 3.50−3.80 0.86 d (6.2)

2 3 5 6 α β

7.50 d (8.6) 7.13 d (8.6) 7.13 d (8.6) 7.50 d (8.6) 6.29 d (16.0) 7.58 d (16.0)

7.62 d (8.0) 7.10 d (8.0) 7.10 d (8.0) 7.62 d (8.0) 5.69 d (12.5) 6.86 d (12.5)

6″-O-Rhamnopyranosyl 4.72 s 3.50−3.80 4.92 t (9.7) 1.03 d (6.3) Hydroxycinnamic Acid 7.08 s 6.79 d (8.2) 6.93 d (8.2) 6.22 d (15.9) 7.52 d (15.9)

contribution to the improved selectivity. Accordingly, the resolution of the isomers on this mixed-mode column was superior to that on RP columns (Table 2). Moreover, it is worth mentioning that, in previous papers, the mixed-mode column performed lower column efficiency and relatively longer analysis time for separation of anthocyanins in comparison to RP columns.23 The XCharge C8SAX column exhibited high column efficiency to the anthocyanins, which was nearly twice as much as that on the XTerra MS C18 column (N = 40 000 and 25 000 for XCharge C8SAX and XTerra MS C18, respectively; Table 2). Hypothetically, the strength of ionic

interaction. Hence, the mixed-mode RP/ion-exchange stationary phase might be useful to provide improved selectivity. On the basis of the polar-copolymerized approach, the XCharge C8SAX column has both alkyl C8 chains and quaternary ammonium groups on the silica surface, providing a hydrophobic interaction along with strong anion-exchange ability. As shown in Figure 3D, the anthocyanin isomers were completely separated on this mixed-mode column. Furthermore, XCharge C8SAX had better selectivity than the three other RP columns. It is possible that the presence of positively charged groups in the XCharge C8SAX column resulted in different extents of ionic repulsion with the isomeric anthocyanins and then provided a significant 505

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column. The resolution of the isomers was effectively improved on this mix-mode column with decreasing pH in the mobile phase (Figure 4B). At pH of 2.32, only one broad peak was observed (panel a of Figure 4B). When pH decreased to 2.06, a slight resolution of the isomers was achieved (panel b of Figure 4B). Baseline separation appeared at a pH value of 1.76 (panel c of Figure 4B), and better resolution as well as sharp peak shapes were obtained with pH of 1.56 (panel d of Figure 4B). This was due in part to the alleviation of the secondary equilibria effect by the decreased pH. More importantly, the electrostatic repulsion between the anthocyanin isomers and quaternary ammonium in the XCharge C8SAX column might provide remarkable contribution to the improvement of separation selectivity. The ionization of quaternary ammonium groups is barely changed within the studied pH values, whereas the charge of anthocyanins is pH-dependent. The percentage of flavylium cation species would increase with the decrease of pH. Meanwhile, the discrepancy in electrostatic force between the cis−trans isomeric anthocyanins became increasingly evident, leading to quite different strengths of ionic repulsion with positive charge in the XCharge C8SAX column. Thus, separation selectivity was improved constantly, as shown in Figure 4B. Effect of the Organic Content. The XCharge C8SAX column has hydrophobic alkyl C8 chains, and the anthocyanin isomers have some hydrophobic portions in the molecules. Two compounds could be retained through hydrophobic interaction. The linear solvent strength theory is often used to evaluate retention of a compound in reverse-phase liquid chromatography (RPLC)28

Figure 10. Chemical structures of the isolated anthocyanins.

repulsion between the positive charge on the stationary phase and falvylium cations could affect the separation efficiency.21 To summarize, benefiting from the presence of an appropriate level of positive charge on the surface of the solid phase, the XCharge C8SAX column displayed improved selectivity and better column efficiency for separation of the cis−trans anthocyanin isomers, which would be favorable for their preparation. Mobile Phase Optimization. To obtain optimal separation performance on XCharge C8SAX, various mobile phase experimental factors, such as pH, organic content, and buffer concentration, were studied. The same experiments were also performed on the XTerra MS C18 column to further understand the retention mechanism of anthocyanins. Effect of the pH. The pH value of elution has profound influence on the ionization of anthocyanins25 and should be taken into consideration in the HPLC method development. As presented in Figure 4, separation of the anthocyanin isomers within the pH values of 2.32−1.56 was implemented on C18 and mixed-mode columns. The ionic strength and organic content were maintained constant at 15 mM and 15%, respectively. On the XTerra MS C18 column (Figure 4A), the isomeric anthocyanins were co-eluted as one broad peak at pH ranging from 2.32 to 1.76 (panels a, b and c of Figure 4A). When pH decreased to 1.56 (panel d of Figure 4A), partial separation of the isomers was acquired. The secondary equilibria of anthocyanins, which would cause on-column band broadening,26,27 was alleviated with a decreasing pH value. As a result, a highly acidic condition was of significance to ensure prevalence of the falvylium cations and improved chromatographic performance. However, the resolution for the isomers is still limited for preparative separation. In contrast, the XCharge C8SAX column exhibited much better separation quality for the isomers than the XTerra C18

log k = log k w −

ϕo 2

(PH2O − Po)

(1)

where PH2O is the polarity of water, Po is the polarity of the organic solvent, Φo is the volume fraction of the organic solvent in percentage, and kw is the retention factor of an analyte in water. In this section, the influence of the organic modifier content in the mobile phase on the separation of the isomers was investigated under various ACN concentrations (from 14 to 15.5%). The ionic strength was kept constant at 15 mM, and the pH was fixed at 1.5. The log k versus the volume fraction of ACN was plotted in Figure 5. Obviously, the retention factor decreased linearly with the increase of the ACN content on these two columns. The correlation coefficients for the isomers were 0.9997/0.9998 on the XTerra MS C18 column and 0.9999/ 0.9999 on the XCharge C8SAX column. These results demonstrated that the anthocyanin isomers experienced RPLC retention behavior on both columns. The retention of the anthocyanins would be greatly susceptible to the change of the organic content on the XCharge C8SAX column. Effect of the Buffer Ionic Strength. Given that the electrostatic repulsive interaction might be involved in the retention of anthocyanins on the XCharge C8SAX column, the influence of the ionic strength in the mobile phase on the separation of the isomers was further assessed under different buffer concentrations (from 1 to 15 mM). The organic content was kept at 15%, and the pH was fixed at 1.5. The plots of the retention time versus the ionic strength are presented in Figure 6. On the XTerra C18 column (Figure 6A), retention of the two isomers remained almost constant with the increase of the buffer concentration. Therefore, no obvious ion-exchange interaction existed. In comparison, the retention enhanced with an increasing buffer concentration on the XCharge C8SAX column 506

DOI: 10.1021/jf504525w J. Agric. Food Chem. 2015, 63, 500−508

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Journal of Agricultural and Food Chemistry

acid was cis configuration. In comparison of the 1H NMR and NOESY data to F6-2 in the literature,8 F6-2 was identified as petunidin 3-O-[6-O-(4-O-(4-O-cis-(β-D-glucopyranoside)-pcoumaroyl)-α-L-rhamnopyranosyl)-β-D-glucopyranoside]-5-O[β-D-glucopyranoside]. To the best of our knowledge, it is a new anthocyanin. F8-1, red powder, [M + H]+: m/z 949.2598, calculated for C43H49O24, m/z 949.2608. 1H NMR data are shown in Table 3. In comparison of the 1H NMR and NOESY data to the literature,8 F8-1 was identified as petunidin 3-O-[6-O-(4-O(trans-p-caffeoyl)-α-L-rhamnopyranosyl)-β-D-glucopyranoside]5-O-[β-D-glucopyranoside]. F8-2, red powder, [M + H]+: m/z 919.2469, calculated for C42H47O23, m/z 919.2505. 1H NMR data are shown in Table 3. In comparison of the 1H NMR and NOESY data to the literature,8 F8-2 was identified as delphinidin 3-O-[6-O-(4-O(trans-p-coumaroyl)-α-L-rhamnopyranosyl)-β-D-glucopyranoside]-5-O-[β-D-glucopyranoside]. P1, red powder, [M + H]+: m/z 933.2647, calculated for C43H49O23, m/z 933.2659. 1H NMR data are shown in Table 3. In comparison of the 1H NMR and NOESY data to the literature,8 P1 was identified as petunidin 3-O-[6-O-(4-O-(cis-pcoumaroyl)-α-L-rhamnopyranosyl)-β-D-glucopyranoside]-5-O[β-D-glucopyranoside]. P2, red powder, [M + H]+: m/z 933.2650, calculated for C43H49O23, m/z 933.2659. 1H NMR data are shown in Table 3. In comparison of the 1H NMR and NOESY data to the literature,8 P2 was identified as petunidin 3-O-[6-O-(4-O-(transp-coumaroyl)-α-L-rhamnopyranosyl)-β-D-glucopyranoside]-5O-[β-D-glucopyranoside]. The chemical structures of P1 and P2 are shown in Figure 1, while others are shown in Figure 10. To the best of our knowledge, F6-2 is a new anthocyanin. Except for P2, all other anthocyanins were isolated from L. ruthenicum for the first time. In summary, a HPLC method had been successfully developed to realize effective preparation of cis−trans isomeric anthocyanins from L. ruthenicum using the XCharge C8SAX column. Four commercially available columns were evaluated to separate a pair of anthocyanin isomers. The results indicated that the XCharge C8SAX column performed improved selectivity and better column efficiency in terms of separation of the isomers. Accordingly, discrimination of the isomers on this mix-mode column was superior to that on RP columns. Moreover, the mobile phases, including pH, organic content, and ionic strength, were investigated. The data explicitly demonstrated that combined mechanisms of the hydrophobic interaction and ionic repulsion were involved in anthocyanin retention on the mixed-mode column, which was a possible explanation for the improved selectivity to the isomers. On the basis of the established approach, six pure anthocyanins, containing two pairs of cis−trans anthocyanin isomers with one new anthocyanin, were isolated from L. ruthenicum and identified by MS and NMR. Five of them were purified from this plant for the first time. The overall results indicated that this method was feasible to efficiently separate anthocyanins, especially for cis− trans isomeric anthocyanins, benefiting from the improved separation selectivity.

(Figure 6B), which is due to the reduction of Donnan exclusion between flavylium cations and positive quaternary ammonium groups in the stationary phase as a result of the electrostatic screen effect.29 From the above discussion, the XCharge C8SAX column provided a hydrophobic interaction along with an electrostatic repulsion for the separation of the anthocyanin isomers under the highly acidic conditions, which could be a reasonable explanation for its superior selectivity. Effect of the Additive Types. Phosphate buffer has poor volatility and needs to be removed through the desalting process. Substitution of phosphate with other common acidic additives, including FA and TFA, was tested to separate the isomers on the XCharge C8SAX column. To maintain a pH value of the mobile phase around 1.5, 5% FA (pH 1.6−1.7) or 0.2% TFA (pH 1.5− 1.6) was adopted. The organic content in the mobile phase was optimized to acquire a similar retention on the XCharge C8SAX column with different additives. As shown in Figure 7, FA and TFA could provide good chromatographic performance and, thus, were able to replace phosphate buffer. Moreover, as for preparative application, a satisfactory result was observed with TFA as the additive (as shown in the Supporting Information). The pH changed slightly for solutions containing TFA, even with large loading amounts, which could improve anthocyanin preparation. Hence, 0.2% TFA was recommended as mobile phase additives for separation of the isomers on the XCharge C8SAX column. Purification of Anthocyanin Isomers from L. ruthenicum on the XCharge C8SAX Column. On the basis of the above results, a HPLC method was developed to separate the anthocyanin isomers using the XCharge C8SAX column. Three XTerra MS C18 purified fractions (fractions 6, 8, and 9) from L. ruthenicum were taken as examples for target purification. Chromatographic conditions for each fraction were optimized. The preparation chromatograms of fractions 6, 8, and 9 on the XCharge C8SAX column were presented in Figure 8. Six compounds were yielded with sufficient amounts and dried by lyophilization. The purity of these compounds was checked by HPLC, which is shown in Figure 10. F6-2 is 85% pure, and other compounds are more than 90% pure. It was noticed that compounds F6-1 and F6-2 had close retention times on the XTerra MS C18 column (Figure 9). They were difficult to isolate and collect with favorable purity. However, these two compounds could be easily obtained with high purity on the XCharge C8SAX column. Similar results were observed for compounds F8-1 and F8-2 as well as compounds P1 and P2. These data demonstrated that this mixed-mode column could be used as the second-dimensional separation in anthocyanin preparation, because of its complementary separation selectivity. Structure Elucidation. Identification of the pure compounds was performed by mass spectrum and NMR. The results of specific structural identification were as follows: F6-1, red powder, [M + H]+: m/z 1095.3169, calculated for C49H59O28, m/z 1095.3187. 1H NMR data are shown in Table 3. In comparison of the 1H NMR and NOESY data to the literature,8 F6-1 was identified as petunidin 3-O-[6-O-(4-O-(4O-trans-(β-D-glucopyranoside)-p-coumaroyl)-α-L-rhamnopyranosyl)-β-D-glucopyranoside]-5-O-[β-D-glucopyranoside]. F6-2, red powder, [M + H]+: m/z 1095.3169, calculated for C49H59O28, m/z 1095.3187. 1H NMR data are shown in Table 3. It was similar to F6-1. However, the olefinic proton signals of pcoumaric acids of F6-2 had smaller coupling constants (J = 12.5 Hz) than that of F6-1 (J = 16.0 Hz), indicating that its p-coumaric

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ASSOCIATED CONTENT

S Supporting Information *

Fractionation of anthocyanin constitutions on a Prep XTerra MS C18 column (Figure S1) and preparation of fraction 9 on the XCharge C8SAX column with (A) 5% (v/v) FA and (B) 0.2% 507

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analysis, and selected reaction monitoring. J. Chromatogr. A 2005, 1091, 72−82. (15) Wu, X. L.; Prior, R. L. Systematic identification and characterization of anthocyanins by HPLC−ESI−MS/MS in common foods in the United States: Fruits and berries. J. Agric. Food Chem. 2005, 53, 2589−2599. (16) Nayak, C. A.; Srinivas, P.; Rastogi, N. K. Characterisation of anthocyanins from Garcinia indica Choisy. Food Chem. 2010, 118, 719− 724. (17) Valls, J.; Millan, S.; Pilar Marti, M.; Borras, E.; Arola, L. Advanced separation methods of food anthocyanins, isoflavones and flavanols. J. Chromatogr. A 2009, 1216, 7143−7172. (18) Willemse, C. M.; Stander, M. A.; de Villiers, A. Hydrophilic interaction chromatographic analysis of anthocyanins. J. Chromatogr. A 2013, 1319, 127−140. (19) Nogueira, R.; Lammerhofer, M.; Lindner, W. Alternative highperformance liquid chromatographic peptide separation and purification concept using a new mixed-mode reversed-phase/weak anionexchange type stationary phase. J. Chromatogr. A 2005, 1089, 158−169. (20) Li, J.; Shao, S.; Jaworsky, M. S.; Kurtulik, P. T. Simultaneous determination of cations, zwitterions and neutral compounds using mixed-mode reversed-phase and cation-exchange high-performance liquid chromatography. J. Chromatogr. A 2008, 1185, 185−193. (21) Wei, J.; Guo, Z.; Zhang, P.; Zhang, F.; Yang, B.; Liang, X. A new reversed-phase/strong anion-exchange mixed-mode stationary phase based on polar-copolymerized approach and its application in the enrichment of aristolochic acids. J. Chromatogr. A 2012, 1246, 129−136. (22) McCallum, J. L.; Yang, R.; Young, J. C.; Strommer, J. N.; Tsao, R. Improved high performance liquid chromatographic separation of anthocyanin compounds from grapes using a novel mixed-mode ionexchange reversed-phase column. J. Chromatogr. A 2007, 1148, 38−45. (23) Vergara, C.; Mardones, C.; Hermosin-Gutierrez, I.; von Baer, D. Comparison of high-performance liquid chromatography separation of red wine anthocyanins on a mixed-mode ion-exchange reversed-phase and on a reversed-phase column. J. Chromatogr. A 2010, 1217, 5710− 5717. (24) Fleschhut, J.; Kratzer, F.; Rechkemmer, G.; Kulling, S. E. Stability and biotransformation of various dietary anthocyanins in vitro. Eur. J. Nutr. 2006, 45, 7−18. (25) Kennedy, J. A.; Waterhouse, A. L. Analysis of pigmented highmolecular-mass grape phenolics using ion-pair, normal-phase highperformance liquid chromatography. J. Chromatogr. A 2000, 866, 25− 34. (26) De Villiers, A.; Cabooter, D.; Lynen, F.; Desmet, G.; Sandra, P. High performance liquid chromatography analysis of wine anthocyanins revisited: Effect of particle size and temperature. J. Chromatogr. A 2009, 1216, 3270−3279. (27) de Villiers, A.; Cabooter, D.; Lynen, F.; Desmet, G.; Sandra, P. High-efficiency high performance liquid chromatographic analysis of red wine anthocyanins. J. Chromatogr. A 2011, 1218, 4660−4670. (28) Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography, 2nd ed.; Wiley: New York, 1979; p 260. (29) Fallas, M. M.; Buckenmaier, S. M. C.; McCalley, D. V. A comparison of overload behaviour for some sub 2 μm totally porous and sub 3 μm shell particle columns with ionised solutes. J. Chromatogr. A 2012, 1235, 49−59.

(v/v) TFA (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +86-411-84379541. Fax: +86-411-84379539. Email: [email protected]. Funding

This work was supported by the Project of the National Science Foundation of China (21305138) and the External Cooperation Program of the Bureau of International Cooperation (BIC), Chinese Academy of Science (Grant 121421KYSB20130013). Notes

The authors declare no competing financial interest.

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