Article pubs.acs.org/JAFC
Ionic Liquid-Based One-Step Micellar Extraction of Multiclass Polar Compounds from Hawthorn Fruits by Ultrahigh-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Tandem Mass Spectrometry Shuai-Shuai Hu,† Ling Yi,‡ Xing-Ying Li,† Jun Cao,*,† Li-Hong Ye,§ Wan Cao,† Jian-Hua Da,† Han-Bin Dai,† and Xiao-Juan Liu† †
College of Material Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 310036, China Department of Pharmaceutics, College of Pharmacy, The Ohio State Unversity, Columbus, Ohio 43210, United States § Integrated Chinese and Western Medicine Hospital of Zhejiang Province, Hangzhou 310003, China ‡
ABSTRACT: An ionic liquid (IL)-based one-step micellar extraction procedure was developed for the extraction of multiclass polar analytes (protocatechuic acid, chlorogenic acid, epicatechin, hyperoside, isoquercitrin, quercetin) from hawthorn fruits and their determination using ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF/MS). Compared to conventional organic solvent extractions, this newly proposed method was much easier, more sensitive, environmentally friendly, and effective as well. Several important parameters influencing the micellar extraction efficiency are discussed, such as selection of ILs, surfactant concentration, and extraction time. Under the optimal conditions, good linearity was achieved for each analyte with correlation coefficients (r2) ranging from 0.9934 to 0.9999, and the recovery values ranged from 89.3 to 106% with relative standard deviations lower than 5.5%. Results suggest that the IL-based one-step micellar extraction could be an alternative and promising means in future food analysis. KEYWORDS: hawthorn fruits, ionic liquids, micellar extraction, quadrupole time-of-flight tandem mass spectrometry, ultrahigh-performance liquid chromatography
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INTRODUCTION Ionic liquids (ILs), also known as molten salts, are new solvents resulting from combinations of organic cations and inorganic/ organic anions. These nonmolecular compounds, unlike many traditional molecular solvents, exhibit multiple significant advantages such as low vapor pressure, high thermal stability, nonflammability, good solubility, tunable viscosities, and environmental benignity. Due to their unique properties, ILs have drawn great attention to their applications as novel extraction media in sample preparations. Recent studies have focused on the interactive and retentive behaviors of various small molecules and large biomolecules in liquid−liquid extraction,1 solid−liquid extraction,2 liquid-phase microextraction,3,4 solid-phase microextraction,5,6 and aqueous two-phase extraction employing ILs as extracting agents.7 Due to its high capacity, biocompatible environment, high selectivity, and easy operation, micellar extraction has been recognized as one of the most commonly used techniques for the separation and purification of small molecules and biological macromolecules.8,9 In recent years, some researchers have suggested that ILs composed of long carbon chain radicals and charged hydrophilic heads could form micellar aggregates when dissolved in water above the critical micelle concentration (CMC). At the same time, the dissolved ILs would exhibit characteristics of traditional ionic surfactants.10,11 These ILbased surfactants have been successfully used in one-step extraction and microextraction.10,12,13 Among them, one-step extraction attracted much attention in the extraction of target © XXXX American Chemical Society
ingredients due to its high efficiency and simple procedure. Reviewing the limited reports on one-step extraction, we found that such extraction methods were applied to only one type of analyte, commonly using 1-hexadecyl-3-methylimidazolium bromide ([C16mim]Br) as the extracting solvent.12,13 In recent years, a combination of quadrupole time-of-flight mass spectrometry (Q-TOF/MS) and ultrahigh-performance liquid chromatography (UHPLC) has been proved to be a powerful tool for food analysis, attributed to its rapid separation, high sensitivity, and good selectivity in the analysis of bioactive components and their metabolites in complex matrices.14,15 To our knowledge, a combined technique of ILs based one-step micellar extraction and UHPLC-Q-TOF/MS is novel and promising. As is known to all, foods are usually complex mixtures consisting of a large diversity of molecules, including phenols, polyphenols, amino acids, biogenic amines, peptides, proteins, carbohydrates, etc.16−19 Analysis of foods is, therefore, a topic that demands the development of green, sensitive, and efficient analytical methodologies to guarantee the safety and quality of foods in compliance with legislation. There is no doubt that extraction is a critical and indispensable procedure regarding almost all analytical research on foods. Traditional organic Received: March 11, 2014 Revised: May 20, 2014 Accepted: May 21, 2014
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Table 1. Chemical Structures of Studied ILs
pounds (protocatechuic acid, chlorogenic acid, epicatechin, hyperoside, isoquercitrin, quercetin) in hawthorn fruits. The effects of various experimental parameters, such as selection of ILs and surfactant concentration, as well as extraction time, were investigated systematically. The proposed method is environmentally friendly with good repeatability and is expected to have wider utilization instead of organic solvents in the future.
solvent extraction is common, popular, and yet with unavoidable disadvantages, for example, contamination to the environment and human health. Until now, IL-based one-step micellar extraction has not been applied for the analysis of multiclass compounds in sample preparation, and no work has been reported on the use of IL-micelles for the extraction of hydrophobic and hydrophilic components from food samples. Hawthorn is well-known as both a fruit and food supplement in Europe and China. Recent studies have demonstrated that hawthorn fruits were mainly used in the treatment of indigestion, hyperlipidemia, hypertension, abdominal pain, and amenorrhea for many years.20 Several analytical methods including high-performance liquid chromatography (HPLC),21 capillary electrophoresis (CE),22 and liquid chromatography− mass spectrometry (LC-MS)23 have been reported to analyze the bioactive constituents, especially phenolic compounds, of this fruit. However, to date, there are no studies related to the utilization of a sub 2 μm particle size column-based LC system for the separation of hawthorn samples, especially coupled with Q-TOF/MS. In the current study, an IL-based one-step micellar extraction followed by UHPLC-Q-TOF/MS was established and further applied for the determination of multiclass phenolic com-
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MATERIALS AND METHODS
Reagents and Samples. The ionic liquids tested in the present study, including 1-ethyl-3-methylimidazolium bromide ([emin]Br), 1hexyl-3-methylimidazolium bromide ([hmin]Br), 1-butyl-3-methylimidazolium methanesulfonate ([bmim]SO3), 1-butyl-3-methylimidazolium tetrafluoroborate ([bmin]BF4), 1-dodecyl-3-methylimidazolium chloride ([C12mim]Cl), 1-dodecyl-3-methylimidazolium bromide ([C12mim]Br), 1-dodecyl-3-methylimidazolium trifluoromethanesulfonate ([C12mim]CF3SO3), 1 -dodecyl-3-methylimidazolium nitrate ([C12mim]NO3), and 1-dodecyl-3-methylimidazolium hydrogen sulfate ([C12mim]HSO3), were purchased from Chengjie Chemical Co. Ltd. (Shanghai, China) and used without further purification (Table 1). HPLC grade methanol and acetonitrile were obtained from SigmaAldrich, Inc. (St. Louis, MO, USA). All other chemicals were of B
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analytical grade (Aladdin Reagent Inc., Shanghai, China), and ultrapure water (Milli-Q system) was used throughout. Analytical standards of protocatechuic acid (1), chlorogenic acid (2), epicatechin (3), hyperoside (4), isoquercitrin (5), and quercetin (6) were provided by Shanghai Winherb Medical Technology Co., Ltd. (Shanghai, China). The hawthorn fruits were obtained from a local drugstore in Hangzhou, China. Preparation of Sample Solution. One gram of accurately weighed hawthorn fruit was extracted with 20 mL of various solvents (ionic liquids, water, or methanol) by ultrasonication for 40 min. The suspension was then filtered through a 0.2 μm disposable membrane filter and stored at room temperature before injection. Instrumental Conditions. Chromatographic analysis was performed on a modular Agilent UHPLC system (Agilent 1290 Infinity LC, Agilent Technologies, Santa Clara, CA, USA) equipped with a vacuum degasser, a binary solvent delivery system, a thermostated autosampler, and a thermostabilized compartment. An Agilent Zorbax Extend-C18 column (2.1 mm × 50 mm, 1.8 μm) was used for separation at a room temperature of 35 °C. The mobile phase was a mixture of acetonitrile (A) and water with 0.1% formic acid (B) using a gradient elution of 10−15% A at 0−2.5 min, 15−15% A at 2.5−3.0 min, 15−40% A at 3.0−6.0 min, and 40−100% A at 6.0−7.0 min, with an equilibrium time of 5 min. The injection volume was 1 μL, and the flow rate was set at 0.4 mL/min. This UHPLC system was connected to a 6530 Q-TOF/MS (Agilent Technologies) equipped with a dual ESI source, operating in the negative ion mode, using the following operation conditions: nebulizer pressure, 45 psig; drying gas, 12 L/min; gas temperature, 350 °C; capillary voltage, 3500 V; fragmentor voltage, 175 V; skimmer voltage, 65 V; octopole RF, 750 V. The data recorded were controlled by MassHunter software (version B 05.00 Qualitative Analysis), and the mass range was recorded in the range m/z 100−2000.
Figure 1. Impact of type of IL on extraction efficiency: (A) [C12mim]Br; (B) [C12mim]CF3SO3; (C) [C12mim]NO3; (D) [C12mim]HSO3; (E) [C12mim]Cl. Extraction conditions: 1.0 g of dried hawthorn samples was mixed with 20 mL of different ionic liquids and then extracted for 40 min at 100 W ultrasonic power. Analytes: protocatechuic acid, chlorogenic acid, epicatechin, hyperoside, isoquercitrin, quercetin.
supposed that the two analytes had stronger interactions with the tested IL, giving rise to higher dissolubility in plant cells. With regard to analyte 6, however, these five ILs had no significant effect on the extraction yield with an extraction time of 40 min. These results suggested that the micellar extraction was a complex result of distinct multiple interactions including the viscosity of sample solution and solvent, solubilities of analytes in ILs, the diffusion coefficient, partition coefficient of the solutes, etc. Thus, [C12mim]Cl was selected as extraction solvent in the subsequent experiments. Optimization of Surfactant Concentration and Extraction Time. In previous studies, it was shown that the surfactant concentration was an essential factor which could influence the extraction efficiency of target analytes.25 In this study, the effect of [C12mim]Cl concentration on the extraction of multiclass polar compounds from hawthorn fruit was examined in the range of 50−250 mM, and the results are exhibited in Figure 2. The extraction yields of six analytes were found to increase with the rising surfactant concentration until 150 mM as the maximum and then decrease when the surfactant concentration went higher. The first part of the positive correlation phenomenon was attributed to the higher
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RESULTS AND DISCUSSION Optimization of IL-Based Micellar Extraction. To obtain the highest extraction ratios for the investigated analytes, some parameters of micellar extraction such as type of IL, concentration of IL, and extraction time were explored systematically. Selection of ILs. The structures of ILs have great impacts on their physical and chemical properties, such as density, viscosity, and solubility, which might greatly affect the micellar extraction yield of target compounds.24 To find the optimal IL and evaluate its performance in the micellar extraction of six analytes of hawthorn fruit, 1-dodecyl-3-methylimidazolium type ILs with different anions were tested using protocatechuic acid, chlorogenic acid, epicatechin, hyperoside, isoquercitrin, and quercetin as model compounds. In this study, five long-chain ILs including [C12mim]Cl, [C12mim]Br, [C12mim]CF3SO3, [C 12mim]NO3, and [C 12mim]HSO3 were compared as extraction solvents. These ILs have the same hydrocarbon chain length but different hydrophilic groups, presenting varied properties by different amphiphilic molecular structure. All five IL surfactants were found to be water-soluble and could enhance water miscibility of solutes. Actually, compared with traditional cationic surfactants, the investigated ILs with long chains were demonstrated to have significantly lower CMC values, which means these ILs more easily formed ionic surfactants. Figure 1 indicates that when the other conditions were constant, the anions of ILs strongly affected the extraction yield of the compounds of interest. Moreover, it shows that the extraction yields of hydrophobic and hydrophilic compounds were largely anion-dependent, which was consistent with a previous paper.25 As shown in Figure 1E, [C12mim]Cl exhibited the highest extraction yields for all investigated analytes, especially for analytes 1 and 2 compared with other ILs. It was
Figure 2. Effect of [C12mim]Cl concentrations on the extraction yields of target ingredients. One gram of dried samples was mixed with 50− 250 mM [C12mim]Cl and then extracted for 40 min at 100 W ultrasonic power. Other conditions were as in Figure 1. C
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and methanol had no significant difference in the extraction of target analytes from hawthorn fruit. In other words, the longchain ILs could be an alternative solvent to replace toxic organic solvents. Moreover, IL micelle was a rapid and effective means for simultaneous extraction of hydrophilic and hydrophobic compounds from foods. Performance of UHPLC-Q-TOF/MS. Electrospray ionization (ESI) is a soft ionization technique used widely for the analysis of various analytes ranging from small organic molecules to biomolecules of hundreds of kilodaltons.26 Generally, the sensitivity is often low in the positive or negative ion mode when the analyte of interest is prepared along with the matrix in involatile salts. In this study, it was fortunate to find that the IL matrix provided similar signal intensities compared to commonly used methanol solvent. The results indicated that no significant signal suppression occurred for the six tested analytes and, thus, matrix effect could be neglected in the experiment. To achieve a good separation for multiple ingredient determination, several mobile phase systems including methanol−water, methanol−0.1% formic acid, acetonitrile− water, and acetonitrile−0.1% formic acid were investigated. The result demonstrated that the optimal MS separation of six compounds detected in negative ion mode was achieved by using a gradient elution with 0.1% formic acid−water and acetonitrile at 0.4 mL/min in 6 min (Figure 4). Different columns including an Agilent Zorbax Extend-C18 column (2.1 mm × 50 mm, 1.8 μm), a Zorbax Eclipse XDB-C18 column (2.1
[C12mim]Cl concentration exhibiting greater solvency, resulting in higher rates of analytes transferring to the extraction solvent. After a certain concentration, more [C12mim]Cl would be dissolved into the sample solution, which possibly reduced the molecular interaction of solvent and compounds, giving rising to a decreased solubility of investigated analytes. Thus, the negative correlation phenomenon was observed for analytes 2, 3, 4, and 5 when extracted by higher concentrations of ILs after 150 mM. On the other hand, the extraction efficiency for analytes 1 and 6 rarely changed when the hawthorn sample was extracted. This was probably due to the chemical structures of the two compounds, which made their distribution between the micellar phase and the aqueous phase insensitive to the IL concentration. Considering the extraction efficiency, the optimized concentration of 150 mM [C12mim]Cl was adopted in the following experiments. Generally, extraction time was an important factor usually studied in the conventional extraction process, which could affect the effectiveness of extraction of analytes. In micelleassisted extraction, the effects of extraction time were evaluated in the range of 20−80 min employing 150 mM [C12mim]Cl. Results showed the chromatographic peak areas increased almost linearly when the extraction time went up from 20 to 40 min for all analytes. With longer extraction times, the extraction efficiency decreased on the contrary (data not shown). Therefore, the extraction time was set to 40 min in the following study. Comparison of Different Extraction Methods. To compare the efficiencies of micellar-based and conventional solvent extractions, water, several ILs with the short alkyl chains, and methanol were used to extract six analytes from hawthorn fruit in the present study. It can be observed from Figure 3F that water exhibited much higher extraction yields for
Figure 3. Comparison of different extraction methods: (F) water; (G) [emin]Br; (H) [hmin]Br; (I) [bmim]SO3; (J) [bmin]BF4; (K) methanol. Other conditions were as in Figure 1.
the hydrophilic components than the hydrophobic ones, indicating itself to be an unsuitable extraction solvent for multiclass polar compounds. When the short-chain ILs (Figure 3G−J) were used as extraction solvents, the yields of hydrophobic compounds were distinctly higher than that of water solvent, and yet lower than that of micellar-based or methanol extraction. In the case of hydrophilic analytes, the change of peak areas resulted from the dissolving of the IL drop in sample solutions. As seen in Figure 3, the analytes extracted by methanol presented much higher yields than those by water and short-chain ILs. In addition, the results shown in Figures 1 and 3 indicated that, as extraction solvents, the micellar solution
Figure 4. TIC of standard solution determined by UHPLC-Q-TOF/ MS. D
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Table 2. Linear Regression Data, Precision, and Limits of Detection (LODs) of the Investigated Compounds precision (RSD%) intraday(n = 6) analyte protocatechuic acid chlorogenic acid epicatechin hyperoside isoquercitrin quercetin
= = = = = =
peak area
retention time
peak area
retention time
LOD (μg/mL)
0.9970 0.9995 0.9984 0.9995 0.9999 0.9934
3.28 1.37 2.15 1.97 2.61 2.63
0.51 0.45 0.42 0.22 0.26 0.10
5.22 3.31 2.36 4.68 4.00 3.03
0.74 0.46 0.45 0.33 0.44 0.26
0.0047 0.0038 0.0054 0.0033 0.0048 0.0030
r
calibration curve y y y y y y
35202x + 17221 64075x + 11624 157405x + 47530 116331x + 16155 150086x + 14966 278146x + 388804
interday(n = 3)
2
Table 3. Retention Time, Detected Mass, Collision Energy, MS/MS Data, and Content of Analytes analyte
retention time (min)
detected mass (m/z)
collision energy (V)
protocatechuic acid chlorogenic acid epicatechin hyperoside isoquercitrin quercetin
0.760 1.144 1.677 3.412 3.629 5.380
154.0178 354.0689 290.0536 464.0874 464.0867 302.0350
10 10 25 25 20 20
mm × 50 mm, 1.8 μm), and a Zorbax StableBond-C18 column (2.1 mm × 50 mm, 1.8 μm) were also investigated. It was found that the Zorbax Extend-C18 column at a temperature of 40 °C was suitable to separate the constituents present in hawthorn fruit. ESI parameters also played major effects in the signal response and peak shape measured for [M − H]− ions of analytes. As a result, higher signal intensity was observed in negative mode at the following conditions: capillary voltage, 3500 V; nebulization gas flow rate, 12 L/min at a temperature of 350 °C; nebulizer, 45 psig; and fragmentor, 175 V. Method Evaluation. To evaluate the proposed micellar extraction method for simultaneous determination of multiclass polar compounds in hawthorn fruits, linearity, precision, limits of detection (LODs), repeatability, and recovery were determined under the optimized conditions. Linearity was investigated at seven different concentration levels ranging from 0.1 to 40 μg/mL for the six analytes. Results shown in Table 2 indicate that good linearity of all target compounds was obtained with correlation coefficients (r2) ranging from 0.9934 to 0.9999. The intraday precision was examined by analyzing six determinations within 1 day, and interday precision was determined within consecutive 3 days among a middle concentration of linear range. As shown in Table 2, the intraand interday variations regarding peak areas and retention time were less than 3.3% (n = 6) and 5.3% (n = 3) for all analytes, respectively, which indicated that the developed method had good precision. The sensitivity of the proposed method was evaluated by determining the LODs at low concentration levels giving a signal-to-noise ratio of 3. The LODs ranged from 0.0030 to 0.0054 μg/mL for all analytes (Table 2). Accredited repeatability was achieved by relative standard deviations (RSDs, n = 5) between 2.1 and 4.3% for the determination of five repeated extractions of hawthorn fruit samples. Recoveries were studied by standard addition method for the accuracy evaluation of the proposed method. Each target compound was added at concentrations ca. 0.5, 1, and 1.5 times the observed concentration in hawthorn fruit samples and then determined by UHPLC-Q-TOF/MS. Results manifested that the recovery values were from 89.3 to 106% for the six analytes with RSDs lower than 5.5%. Considering all validation results,
MS/MS 112.9848 315.7659 247.6703 343.0467 343.0459 229.0457
110.0326 264.9461 144.8024 300.0269 300.0269 151.0038
content (mg/g) 109.0290 191.0568 112.9854 271.0236 151.0035 107.0144
0.0203 0.3727 0.3081 0.1743 0.0870 0.0186
the newly developed method was accurate and reliable for detecting analytes in hawthorn fruit samples. Analysis of Real Samples. The proposed method was applied to the analysis of target compounds in hawthorn samples collected from the local drugstore in China. All analytes 1−6 produced abundant [M − H]− ions as the base peaks in negative ESI-MS spectra because [M − H]− provided better sensitivity compared with the other protonation states. The [M − H]− ions were selected as the precursor ions to produce MS/MS spectra at optimal collision energies (Table 3). In the current research, all of the compounds could be easily identified by their retention time, characteristic accurate masses (error < 5 ppm), and fragment ions (Table 3). Typical LC-MS chromatograms demonstrating the separation of six analytes are shown in Figure 4. The contents of the targeted compounds were found to be different in hawthorn fruit sample (Table 3). The analytical results manifested that the micellar extraction was a precise and reliable method, which could be substituted for the conventional methanol extraction to detect multiclass polar compounds in the complex samples. In conclusion, a new method of IL-based micellar extraction coupled with UHPLC-Q-TOF/MS has been developed for the determination of multiclass polar compounds in hawthorn fruit samples. The optimum micellar extraction conditions were 150 mM [C12mim]Cl, a solid−liquid ratio of 1:20 (g/mL), and an ultrasonication time of 40 min under ultrasonic power of 100 W. Compared to conventional extraction techniques, the developed method provided higher extraction yields and reduced the consumption of volatile organic solvent. More important, the IL-based extraction technique was not only reliable by its good repeatability and precision but also environmentally friendly. This green sample preparation approach is bound to have much wider applications in dealing with food samples.
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AUTHOR INFORMATION
Corresponding Author
*(J.C.) Phone: +86-571-28867909. Fax: +86-571-28867909. Email: [email protected]. E
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Funding
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This study was supported by the General Program of National Natural Science Foundation of China (No. 81274065), Changjiang Scholars and Innovative Research Team in Chinese University (IRT 1231), Scientific Research Foundation of Hangzhou Normal University (2011QDL33), and the newshoot Talents Program of Zhejiang province (2013R421044). Notes
The authors declare no competing financial interest.
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Ionic Liquid-Based One-Step Micellar Extraction of Multiclass Polar Compounds from Hawthorn Fruits by Ultrahigh-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Tandem Mass Spectrometry Shuai-Shuai Hu,† Ling Yi,‡ Xing-Ying Li,† Jun Cao,*,† Li-Hong Ye,§ Wan Cao,† Jian-Hua Da,† Han-Bin Dai,† and Xiao-Juan Liu† †
College of Material Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 310036, China Department of Pharmaceutics, College of Pharmacy, The Ohio State Unversity, Columbus, Ohio 43210, United States § Integrated Chinese and Western Medicine Hospital of Zhejiang Province, Hangzhou 310003, China ‡
ABSTRACT: An ionic liquid (IL)-based one-step micellar extraction procedure was developed for the extraction of multiclass polar analytes (protocatechuic acid, chlorogenic acid, epicatechin, hyperoside, isoquercitrin, quercetin) from hawthorn fruits and their determination using ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF/MS). Compared to conventional organic solvent extractions, this newly proposed method was much easier, more sensitive, environmentally friendly, and effective as well. Several important parameters influencing the micellar extraction efficiency are discussed, such as selection of ILs, surfactant concentration, and extraction time. Under the optimal conditions, good linearity was achieved for each analyte with correlation coefficients (r2) ranging from 0.9934 to 0.9999, and the recovery values ranged from 89.3 to 106% with relative standard deviations lower than 5.5%. Results suggest that the IL-based one-step micellar extraction could be an alternative and promising means in future food analysis. KEYWORDS: hawthorn fruits, ionic liquids, micellar extraction, quadrupole time-of-flight tandem mass spectrometry, ultrahigh-performance liquid chromatography
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INTRODUCTION Ionic liquids (ILs), also known as molten salts, are new solvents resulting from combinations of organic cations and inorganic/ organic anions. These nonmolecular compounds, unlike many traditional molecular solvents, exhibit multiple significant advantages such as low vapor pressure, high thermal stability, nonflammability, good solubility, tunable viscosities, and environmental benignity. Due to their unique properties, ILs have drawn great attention to their applications as novel extraction media in sample preparations. Recent studies have focused on the interactive and retentive behaviors of various small molecules and large biomolecules in liquid−liquid extraction,1 solid−liquid extraction,2 liquid-phase microextraction,3,4 solid-phase microextraction,5,6 and aqueous two-phase extraction employing ILs as extracting agents.7 Due to its high capacity, biocompatible environment, high selectivity, and easy operation, micellar extraction has been recognized as one of the most commonly used techniques for the separation and purification of small molecules and biological macromolecules.8,9 In recent years, some researchers have suggested that ILs composed of long carbon chain radicals and charged hydrophilic heads could form micellar aggregates when dissolved in water above the critical micelle concentration (CMC). At the same time, the dissolved ILs would exhibit characteristics of traditional ionic surfactants.10,11 These ILbased surfactants have been successfully used in one-step extraction and microextraction.10,12,13 Among them, one-step extraction attracted much attention in the extraction of target © XXXX American Chemical Society
ingredients due to its high efficiency and simple procedure. Reviewing the limited reports on one-step extraction, we found that such extraction methods were applied to only one type of analyte, commonly using 1-hexadecyl-3-methylimidazolium bromide ([C16mim]Br) as the extracting solvent.12,13 In recent years, a combination of quadrupole time-of-flight mass spectrometry (Q-TOF/MS) and ultrahigh-performance liquid chromatography (UHPLC) has been proved to be a powerful tool for food analysis, attributed to its rapid separation, high sensitivity, and good selectivity in the analysis of bioactive components and their metabolites in complex matrices.14,15 To our knowledge, a combined technique of ILs based one-step micellar extraction and UHPLC-Q-TOF/MS is novel and promising. As is known to all, foods are usually complex mixtures consisting of a large diversity of molecules, including phenols, polyphenols, amino acids, biogenic amines, peptides, proteins, carbohydrates, etc.16−19 Analysis of foods is, therefore, a topic that demands the development of green, sensitive, and efficient analytical methodologies to guarantee the safety and quality of foods in compliance with legislation. There is no doubt that extraction is a critical and indispensable procedure regarding almost all analytical research on foods. Traditional organic Received: March 11, 2014 Revised: May 20, 2014 Accepted: May 21, 2014
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Table 1. Chemical Structures of Studied ILs
pounds (protocatechuic acid, chlorogenic acid, epicatechin, hyperoside, isoquercitrin, quercetin) in hawthorn fruits. The effects of various experimental parameters, such as selection of ILs and surfactant concentration, as well as extraction time, were investigated systematically. The proposed method is environmentally friendly with good repeatability and is expected to have wider utilization instead of organic solvents in the future.
solvent extraction is common, popular, and yet with unavoidable disadvantages, for example, contamination to the environment and human health. Until now, IL-based one-step micellar extraction has not been applied for the analysis of multiclass compounds in sample preparation, and no work has been reported on the use of IL-micelles for the extraction of hydrophobic and hydrophilic components from food samples. Hawthorn is well-known as both a fruit and food supplement in Europe and China. Recent studies have demonstrated that hawthorn fruits were mainly used in the treatment of indigestion, hyperlipidemia, hypertension, abdominal pain, and amenorrhea for many years.20 Several analytical methods including high-performance liquid chromatography (HPLC),21 capillary electrophoresis (CE),22 and liquid chromatography− mass spectrometry (LC-MS)23 have been reported to analyze the bioactive constituents, especially phenolic compounds, of this fruit. However, to date, there are no studies related to the utilization of a sub 2 μm particle size column-based LC system for the separation of hawthorn samples, especially coupled with Q-TOF/MS. In the current study, an IL-based one-step micellar extraction followed by UHPLC-Q-TOF/MS was established and further applied for the determination of multiclass phenolic com-
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MATERIALS AND METHODS
Reagents and Samples. The ionic liquids tested in the present study, including 1-ethyl-3-methylimidazolium bromide ([emin]Br), 1hexyl-3-methylimidazolium bromide ([hmin]Br), 1-butyl-3-methylimidazolium methanesulfonate ([bmim]SO3), 1-butyl-3-methylimidazolium tetrafluoroborate ([bmin]BF4), 1-dodecyl-3-methylimidazolium chloride ([C12mim]Cl), 1-dodecyl-3-methylimidazolium bromide ([C12mim]Br), 1-dodecyl-3-methylimidazolium trifluoromethanesulfonate ([C12mim]CF3SO3), 1 -dodecyl-3-methylimidazolium nitrate ([C12mim]NO3), and 1-dodecyl-3-methylimidazolium hydrogen sulfate ([C12mim]HSO3), were purchased from Chengjie Chemical Co. Ltd. (Shanghai, China) and used without further purification (Table 1). HPLC grade methanol and acetonitrile were obtained from SigmaAldrich, Inc. (St. Louis, MO, USA). All other chemicals were of B
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analytical grade (Aladdin Reagent Inc., Shanghai, China), and ultrapure water (Milli-Q system) was used throughout. Analytical standards of protocatechuic acid (1), chlorogenic acid (2), epicatechin (3), hyperoside (4), isoquercitrin (5), and quercetin (6) were provided by Shanghai Winherb Medical Technology Co., Ltd. (Shanghai, China). The hawthorn fruits were obtained from a local drugstore in Hangzhou, China. Preparation of Sample Solution. One gram of accurately weighed hawthorn fruit was extracted with 20 mL of various solvents (ionic liquids, water, or methanol) by ultrasonication for 40 min. The suspension was then filtered through a 0.2 μm disposable membrane filter and stored at room temperature before injection. Instrumental Conditions. Chromatographic analysis was performed on a modular Agilent UHPLC system (Agilent 1290 Infinity LC, Agilent Technologies, Santa Clara, CA, USA) equipped with a vacuum degasser, a binary solvent delivery system, a thermostated autosampler, and a thermostabilized compartment. An Agilent Zorbax Extend-C18 column (2.1 mm × 50 mm, 1.8 μm) was used for separation at a room temperature of 35 °C. The mobile phase was a mixture of acetonitrile (A) and water with 0.1% formic acid (B) using a gradient elution of 10−15% A at 0−2.5 min, 15−15% A at 2.5−3.0 min, 15−40% A at 3.0−6.0 min, and 40−100% A at 6.0−7.0 min, with an equilibrium time of 5 min. The injection volume was 1 μL, and the flow rate was set at 0.4 mL/min. This UHPLC system was connected to a 6530 Q-TOF/MS (Agilent Technologies) equipped with a dual ESI source, operating in the negative ion mode, using the following operation conditions: nebulizer pressure, 45 psig; drying gas, 12 L/min; gas temperature, 350 °C; capillary voltage, 3500 V; fragmentor voltage, 175 V; skimmer voltage, 65 V; octopole RF, 750 V. The data recorded were controlled by MassHunter software (version B 05.00 Qualitative Analysis), and the mass range was recorded in the range m/z 100−2000.
Figure 1. Impact of type of IL on extraction efficiency: (A) [C12mim]Br; (B) [C12mim]CF3SO3; (C) [C12mim]NO3; (D) [C12mim]HSO3; (E) [C12mim]Cl. Extraction conditions: 1.0 g of dried hawthorn samples was mixed with 20 mL of different ionic liquids and then extracted for 40 min at 100 W ultrasonic power. Analytes: protocatechuic acid, chlorogenic acid, epicatechin, hyperoside, isoquercitrin, quercetin.
supposed that the two analytes had stronger interactions with the tested IL, giving rise to higher dissolubility in plant cells. With regard to analyte 6, however, these five ILs had no significant effect on the extraction yield with an extraction time of 40 min. These results suggested that the micellar extraction was a complex result of distinct multiple interactions including the viscosity of sample solution and solvent, solubilities of analytes in ILs, the diffusion coefficient, partition coefficient of the solutes, etc. Thus, [C12mim]Cl was selected as extraction solvent in the subsequent experiments. Optimization of Surfactant Concentration and Extraction Time. In previous studies, it was shown that the surfactant concentration was an essential factor which could influence the extraction efficiency of target analytes.25 In this study, the effect of [C12mim]Cl concentration on the extraction of multiclass polar compounds from hawthorn fruit was examined in the range of 50−250 mM, and the results are exhibited in Figure 2. The extraction yields of six analytes were found to increase with the rising surfactant concentration until 150 mM as the maximum and then decrease when the surfactant concentration went higher. The first part of the positive correlation phenomenon was attributed to the higher
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RESULTS AND DISCUSSION Optimization of IL-Based Micellar Extraction. To obtain the highest extraction ratios for the investigated analytes, some parameters of micellar extraction such as type of IL, concentration of IL, and extraction time were explored systematically. Selection of ILs. The structures of ILs have great impacts on their physical and chemical properties, such as density, viscosity, and solubility, which might greatly affect the micellar extraction yield of target compounds.24 To find the optimal IL and evaluate its performance in the micellar extraction of six analytes of hawthorn fruit, 1-dodecyl-3-methylimidazolium type ILs with different anions were tested using protocatechuic acid, chlorogenic acid, epicatechin, hyperoside, isoquercitrin, and quercetin as model compounds. In this study, five long-chain ILs including [C12mim]Cl, [C12mim]Br, [C12mim]CF3SO3, [C 12mim]NO3, and [C 12mim]HSO3 were compared as extraction solvents. These ILs have the same hydrocarbon chain length but different hydrophilic groups, presenting varied properties by different amphiphilic molecular structure. All five IL surfactants were found to be water-soluble and could enhance water miscibility of solutes. Actually, compared with traditional cationic surfactants, the investigated ILs with long chains were demonstrated to have significantly lower CMC values, which means these ILs more easily formed ionic surfactants. Figure 1 indicates that when the other conditions were constant, the anions of ILs strongly affected the extraction yield of the compounds of interest. Moreover, it shows that the extraction yields of hydrophobic and hydrophilic compounds were largely anion-dependent, which was consistent with a previous paper.25 As shown in Figure 1E, [C12mim]Cl exhibited the highest extraction yields for all investigated analytes, especially for analytes 1 and 2 compared with other ILs. It was
Figure 2. Effect of [C12mim]Cl concentrations on the extraction yields of target ingredients. One gram of dried samples was mixed with 50− 250 mM [C12mim]Cl and then extracted for 40 min at 100 W ultrasonic power. Other conditions were as in Figure 1. C
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and methanol had no significant difference in the extraction of target analytes from hawthorn fruit. In other words, the longchain ILs could be an alternative solvent to replace toxic organic solvents. Moreover, IL micelle was a rapid and effective means for simultaneous extraction of hydrophilic and hydrophobic compounds from foods. Performance of UHPLC-Q-TOF/MS. Electrospray ionization (ESI) is a soft ionization technique used widely for the analysis of various analytes ranging from small organic molecules to biomolecules of hundreds of kilodaltons.26 Generally, the sensitivity is often low in the positive or negative ion mode when the analyte of interest is prepared along with the matrix in involatile salts. In this study, it was fortunate to find that the IL matrix provided similar signal intensities compared to commonly used methanol solvent. The results indicated that no significant signal suppression occurred for the six tested analytes and, thus, matrix effect could be neglected in the experiment. To achieve a good separation for multiple ingredient determination, several mobile phase systems including methanol−water, methanol−0.1% formic acid, acetonitrile− water, and acetonitrile−0.1% formic acid were investigated. The result demonstrated that the optimal MS separation of six compounds detected in negative ion mode was achieved by using a gradient elution with 0.1% formic acid−water and acetonitrile at 0.4 mL/min in 6 min (Figure 4). Different columns including an Agilent Zorbax Extend-C18 column (2.1 mm × 50 mm, 1.8 μm), a Zorbax Eclipse XDB-C18 column (2.1
[C12mim]Cl concentration exhibiting greater solvency, resulting in higher rates of analytes transferring to the extraction solvent. After a certain concentration, more [C12mim]Cl would be dissolved into the sample solution, which possibly reduced the molecular interaction of solvent and compounds, giving rising to a decreased solubility of investigated analytes. Thus, the negative correlation phenomenon was observed for analytes 2, 3, 4, and 5 when extracted by higher concentrations of ILs after 150 mM. On the other hand, the extraction efficiency for analytes 1 and 6 rarely changed when the hawthorn sample was extracted. This was probably due to the chemical structures of the two compounds, which made their distribution between the micellar phase and the aqueous phase insensitive to the IL concentration. Considering the extraction efficiency, the optimized concentration of 150 mM [C12mim]Cl was adopted in the following experiments. Generally, extraction time was an important factor usually studied in the conventional extraction process, which could affect the effectiveness of extraction of analytes. In micelleassisted extraction, the effects of extraction time were evaluated in the range of 20−80 min employing 150 mM [C12mim]Cl. Results showed the chromatographic peak areas increased almost linearly when the extraction time went up from 20 to 40 min for all analytes. With longer extraction times, the extraction efficiency decreased on the contrary (data not shown). Therefore, the extraction time was set to 40 min in the following study. Comparison of Different Extraction Methods. To compare the efficiencies of micellar-based and conventional solvent extractions, water, several ILs with the short alkyl chains, and methanol were used to extract six analytes from hawthorn fruit in the present study. It can be observed from Figure 3F that water exhibited much higher extraction yields for
Figure 3. Comparison of different extraction methods: (F) water; (G) [emin]Br; (H) [hmin]Br; (I) [bmim]SO3; (J) [bmin]BF4; (K) methanol. Other conditions were as in Figure 1.
the hydrophilic components than the hydrophobic ones, indicating itself to be an unsuitable extraction solvent for multiclass polar compounds. When the short-chain ILs (Figure 3G−J) were used as extraction solvents, the yields of hydrophobic compounds were distinctly higher than that of water solvent, and yet lower than that of micellar-based or methanol extraction. In the case of hydrophilic analytes, the change of peak areas resulted from the dissolving of the IL drop in sample solutions. As seen in Figure 3, the analytes extracted by methanol presented much higher yields than those by water and short-chain ILs. In addition, the results shown in Figures 1 and 3 indicated that, as extraction solvents, the micellar solution
Figure 4. TIC of standard solution determined by UHPLC-Q-TOF/ MS. D
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Table 2. Linear Regression Data, Precision, and Limits of Detection (LODs) of the Investigated Compounds precision (RSD%) intraday(n = 6) analyte protocatechuic acid chlorogenic acid epicatechin hyperoside isoquercitrin quercetin
= = = = = =
peak area
retention time
peak area
retention time
LOD (μg/mL)
0.9970 0.9995 0.9984 0.9995 0.9999 0.9934
3.28 1.37 2.15 1.97 2.61 2.63
0.51 0.45 0.42 0.22 0.26 0.10
5.22 3.31 2.36 4.68 4.00 3.03
0.74 0.46 0.45 0.33 0.44 0.26
0.0047 0.0038 0.0054 0.0033 0.0048 0.0030
r
calibration curve y y y y y y
35202x + 17221 64075x + 11624 157405x + 47530 116331x + 16155 150086x + 14966 278146x + 388804
interday(n = 3)
2
Table 3. Retention Time, Detected Mass, Collision Energy, MS/MS Data, and Content of Analytes analyte
retention time (min)
detected mass (m/z)
collision energy (V)
protocatechuic acid chlorogenic acid epicatechin hyperoside isoquercitrin quercetin
0.760 1.144 1.677 3.412 3.629 5.380
154.0178 354.0689 290.0536 464.0874 464.0867 302.0350
10 10 25 25 20 20
mm × 50 mm, 1.8 μm), and a Zorbax StableBond-C18 column (2.1 mm × 50 mm, 1.8 μm) were also investigated. It was found that the Zorbax Extend-C18 column at a temperature of 40 °C was suitable to separate the constituents present in hawthorn fruit. ESI parameters also played major effects in the signal response and peak shape measured for [M − H]− ions of analytes. As a result, higher signal intensity was observed in negative mode at the following conditions: capillary voltage, 3500 V; nebulization gas flow rate, 12 L/min at a temperature of 350 °C; nebulizer, 45 psig; and fragmentor, 175 V. Method Evaluation. To evaluate the proposed micellar extraction method for simultaneous determination of multiclass polar compounds in hawthorn fruits, linearity, precision, limits of detection (LODs), repeatability, and recovery were determined under the optimized conditions. Linearity was investigated at seven different concentration levels ranging from 0.1 to 40 μg/mL for the six analytes. Results shown in Table 2 indicate that good linearity of all target compounds was obtained with correlation coefficients (r2) ranging from 0.9934 to 0.9999. The intraday precision was examined by analyzing six determinations within 1 day, and interday precision was determined within consecutive 3 days among a middle concentration of linear range. As shown in Table 2, the intraand interday variations regarding peak areas and retention time were less than 3.3% (n = 6) and 5.3% (n = 3) for all analytes, respectively, which indicated that the developed method had good precision. The sensitivity of the proposed method was evaluated by determining the LODs at low concentration levels giving a signal-to-noise ratio of 3. The LODs ranged from 0.0030 to 0.0054 μg/mL for all analytes (Table 2). Accredited repeatability was achieved by relative standard deviations (RSDs, n = 5) between 2.1 and 4.3% for the determination of five repeated extractions of hawthorn fruit samples. Recoveries were studied by standard addition method for the accuracy evaluation of the proposed method. Each target compound was added at concentrations ca. 0.5, 1, and 1.5 times the observed concentration in hawthorn fruit samples and then determined by UHPLC-Q-TOF/MS. Results manifested that the recovery values were from 89.3 to 106% for the six analytes with RSDs lower than 5.5%. Considering all validation results,
MS/MS 112.9848 315.7659 247.6703 343.0467 343.0459 229.0457
110.0326 264.9461 144.8024 300.0269 300.0269 151.0038
content (mg/g) 109.0290 191.0568 112.9854 271.0236 151.0035 107.0144
0.0203 0.3727 0.3081 0.1743 0.0870 0.0186
the newly developed method was accurate and reliable for detecting analytes in hawthorn fruit samples. Analysis of Real Samples. The proposed method was applied to the analysis of target compounds in hawthorn samples collected from the local drugstore in China. All analytes 1−6 produced abundant [M − H]− ions as the base peaks in negative ESI-MS spectra because [M − H]− provided better sensitivity compared with the other protonation states. The [M − H]− ions were selected as the precursor ions to produce MS/MS spectra at optimal collision energies (Table 3). In the current research, all of the compounds could be easily identified by their retention time, characteristic accurate masses (error < 5 ppm), and fragment ions (Table 3). Typical LC-MS chromatograms demonstrating the separation of six analytes are shown in Figure 4. The contents of the targeted compounds were found to be different in hawthorn fruit sample (Table 3). The analytical results manifested that the micellar extraction was a precise and reliable method, which could be substituted for the conventional methanol extraction to detect multiclass polar compounds in the complex samples. In conclusion, a new method of IL-based micellar extraction coupled with UHPLC-Q-TOF/MS has been developed for the determination of multiclass polar compounds in hawthorn fruit samples. The optimum micellar extraction conditions were 150 mM [C12mim]Cl, a solid−liquid ratio of 1:20 (g/mL), and an ultrasonication time of 40 min under ultrasonic power of 100 W. Compared to conventional extraction techniques, the developed method provided higher extraction yields and reduced the consumption of volatile organic solvent. More important, the IL-based extraction technique was not only reliable by its good repeatability and precision but also environmentally friendly. This green sample preparation approach is bound to have much wider applications in dealing with food samples.
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AUTHOR INFORMATION
Corresponding Author
*(J.C.) Phone: +86-571-28867909. Fax: +86-571-28867909. Email: [email protected]. E
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Funding
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This study was supported by the General Program of National Natural Science Foundation of China (No. 81274065), Changjiang Scholars and Innovative Research Team in Chinese University (IRT 1231), Scientific Research Foundation of Hangzhou Normal University (2011QDL33), and the newshoot Talents Program of Zhejiang province (2013R421044). Notes
The authors declare no competing financial interest.
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