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Separation, Identification, and Quantitationof Phenolic Acids in Chinese Waxberry (MyricaRubra) Juice by HPLC-PDA-ESI-MSChengjun Wang, Jingbei Zhao, Fan Chen, Yaqian Cheng, and Aihong Guo

Abstract: A high-performance liquid chromatography coupled with photodiode-array spectrophotometry and electro-spray ionization-mass spectrometry (HPLC-PDA-ESI-MS) method for separation and determination of phenolic acids inethyl acetate extracts from Chinese waxberry (Myrica Rubra) juice was developed. Total of 4 phenolic acids (ferulic, caffeic,sinapic, and salicylic acids) were identified by comparing their HPLC retention times, UV-Vis absorption spectra, andsimultaneously recorded mass spectra with authentic standards. Quantitation was carried out by the peak area method.The calibration curves are linear over the concentration range studied with the correlation coefficients, R2, greater than0.99. The contents of ferulic, caffeic, sinapic, and salicylic acids in waxberry juice samples studied were 2.76, 3.58, 2.89,and 1.92 mg/L, respectively, and they occur mainly in bound forms. All relative standard deviations were less than 4%.The recoveries range from 83.6% to 95.8% for the 4 analytes. To the authors’ best knowledge, this is the first report forthe identification of the sinapic and salicylic acids in Chinese waxberry products.

Keywords: HPLC-PDA-ESI-MS, phenolic acids, waxberry (Myrica Rubra) juice

IntroductionEpidemiological studies suggested that consumption of fruits

and vegetables is associated with reduced incidence of mortalityfrom cancer, and cardiovascular and neurological diseases (Seeram2008; Feldman 2001; Middleton and others 2000; Puupponen-Pimia and others 2001; Samman and others 1998). Phenolic com-pounds and other antioxidant phytonutrients contained in plant-derived foods are considered as the active constituents to provideprotection against these diseases. Interest in the role of phenolicantioxidants in human health has prompted research in the separa-tion and characterization of active phenolic compounds in variousfoodstuffs (Amakura and others 2000; Eberhardt and others 2000;Shui and Leong 2002). Chromatographic separation techniquessuch as high-performance liquid chromatography (HPLC) andgas chromatography (GC) were the most popular techniques forthe separation and quantification of phenolic compounds in plantmaterials (Olkowski and others 2003; Chen and others 2001; Zuoand others 2008). Of these techniques, GC coupled with flameionization (FID) and mass spectrometric (MS) detection has pro-vided a high sensitivity and selectivity of phenolics and other polarorganic components in plants. However, GC method is limitedby the volatility or thermal stability of the analytes. Usually, thelaborious and time-consuming derivatization process is requiredin the sample preparation, and it could increase the possibilityof contamination as consequence of undesirable derivative reac-tions (Zuo and Lin 2007). In contrast to GC method, HPLC,especially, coupled with photodiode-array spectrophotometry andmass spectrometry enable direct separation and identification of

MS 20111127 Submitted 9/21/2011, Accepted 11/16/2011. Authors Wang,Zhao, F Chen, and Y Cheng are with College of Chemistry and Materials EngineeringWenzhou Univ., Wenzhou 325035, China. Author Guo is with College of Resourceand Environment, Hebei United Univ., Tangshan 063009, China. Direct inquiriesto author Wang (Email: wang.chengjun@yahoo.com).

many compounds and their derivatives simultaneously in sam-ples without sample pretreatments, and have been widely usedfor analyzing the phenolic compounds (Alonso-Salces and others2004; Yang and others 2011). A combination of HPLC retentiontimes, UV-Vis absorption spectra, and simultaneously recordedmass spectra provides sufficient and reliable data that can be usedfor the identification of the phenolic compounds in samples.

Many previous studies have demonstrated high phenolic contentin fruits such as grape, apple, orange, prune, cranberry, blueberry,and their processed products (Sellappan and others 2002; Zhangand Zuo 2004; Ignat and others 2011). However, little informa-tion is available in the literature on the determination of phenolicsin Chinese waxberry products (Bao and others 2005; Fang andothers 2007). The Chinese waxberry is a prominent agriculturalfood crop produced in Zhejiang province, China, with an annualproduction over 300000 tons (data provided by Zhejiang Agricul-tural Office). Besides being consumed fresh, the waxberry fruits,with its special sweet, sour taste and exquisite flavor, are also pro-cessed into juice or sauces. On the other hand, waxberry fruits,leaves, twigs, and bark have been traditionally used in China as afolk remedy to treat gastric intestinal disorders, such as diarrheaand gastroenteritis (Chen 1996). Waxberry extracts have also beenreported to exhibit anticancer, anti-oxidative, antimicrobial, andantiviral effects (Xia and others 2004; Sun and Yao 2009; Yang andothers 2011). The phenolic components of waxberry are believedto be the principal ingredients responsible for these beneficialhealth effects. Although the flavonoids, including myricetin andquercetin, and their glycoside derivatives were identified in thewaxberry fruits (Amakura and others 2000; Bao and others 2005;Fang and others 2007), and the polyphenol tannins were identi-fied in the waxberry kernels (Cheng and others 2008), up to date,data of phenolic acids and other kinds of phenolics from waxberryproducts are lacking. Only Fang and others (2009) and Zhou andothers (2009) have tentatively identified a few phenolic acids in

C© 2012 Institute of Food Technologists R©C272 Journal of Food Science � Vol. 77, Nr. 2, 2012 doi: 10.1111/j.1750-3841.2011.02563.x

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waxberry juice and pomace products. However, previous studiesindicated that phenolic acids, which may be present in plants infree and bound forms, are abundant in berry fruits and constituteabout one-third of the dietary phenols (Robbins, 2003). Thus, itis still necessary to carry out the further study for the identificationand determination of phenolic acids in waxberry products.

The aims of this study were to develop a reliable and effec-tive extraction and HPLC-PDA-ESI-MS method, which couldbe suitable for determination of free and bound phenolic acids inwaxberry products, to separate and characterize the potential phe-nolic antioxidant acids of the waxberry juice using the developedmethod, and to quantify the level of the identified phenolic acidsin waxberry juice samples.

Materials and Methods

Chemicals and standardsStandards of caffeic, sinapic, and salicylic acids were purchased

from Xilong Chemicals. Co. Ltd. (Guangdong, China), and ferulicacid was obtained from Sinopharm Chemical Reagent Co. Ltd.(Shanghai, China). Methanol and acetonitrile (HPLC grade) werepurchased from Tianjin Shield Fine Chemical Co. Ltd. (Tianjin,China). Ethyl acetate and hydrochloric acid were obtained fromJiani Chemistry Co. Ltd. (Wuxi, China). Formic acid was suppliedby TEDIA Co. Inc. (Fairfield, Ohio, U.S.A.). The stock standardsolutions of 4 phenolic acids were first prepared at concentration of100 mg/L in methanol. The working standard solutions containing4 standard compounds, that is, ferulic, caffeic, sinapic, and salicylicacids, were prepared daily in methanol at concentrations of 0.0, 5,10, 15, 20, and 25 mg/L by appropriately combining and dilutingthe individual stock standard solutions. Except where noted, allreagents were of analytical grade and all solution preparations weremade using doubly distilled-deionized water.

Samples preparationWaxberry juice (100% juice, Yumberry brand, Zhejiang

Yangmei Beverage Co. Ltd.) was obtained from a local super-market in Wenzhou, Zhejiang Province, China and stored at 4 ◦Cuntil analysis.

Extraction of free phenolic acids in waxberry juice.Waxberry juice (200 mL) was filtered with a 0.45 μm membraneand a 10-mL aliquot of filtrate was acidified by adding 0.1M HClto maintain pH at 2 and extracted with 5 mL of ethyl acetatetwice. The ethyl acetate phase contained free phenolic acids andwas extracted with 10 mL of 5% NaHCO3 twice. In this way, thephenolic acids were substantially separated from the other phenolicmoieties (Zuo and others 2002). The alkali aqueous solution wasacidified with 0.1M HCl to maintain pH at 2 and extracted againwith 5 mL of ethyl acetate twice. The ethyl acetate extract wasdried under a stream of high purity nitrogen gas. The dry residuewas dissolved in 0.5 mL of methanol prior to HPLC-PDA-ESI-MS analysis.

Hydrolysis and extraction of (free+bound) phenolicacids in waxberry juice. An 80-mg amount of ascorbic acidwas dissolved in 20 mL of deionized water in a 100-mL round-bottomed flask. A 20-mL aliquot of filtered waxberry juice wasadded. To this solution, 10 mL of 6 N HCl was added slowlywithin 5 min into the flask. Under a nitrogen atmosphere, thesolution was heated at 35 ◦C for 90 min under ultrasonic radia-tions with 40 KHz frequencies in an AS-3120 ultrasonication bath(200 W, Automatic Science instrument Co. Ltd., Tianjin, China).The bound phenolic acids can be hydrolyzed into free form dur-ing this process (Wang and Zuo, 2011). The solution was allowed

to cool and then filtered through 0.45 μm membrane. A 15-mLportion of the filtrate was extracted with ethyl acetate, treatedwith 5% NaHCO3, dried under nitrogen flow, and redissolved inmethanol as described previously in preparation for the extractionof free phenolics.

HPLC-PDA-ESI-MS analysisHPLC-PDA-ESI-MS analysis was carried out on a Finnigan

Surveyor liquid chromatography coupled with a Surveyor pho-todiode array spectrophotometer and a Finnigan LCQ Deca XPPlus electrospray ionization – ion trap mass spectrometer (ThermoFisher Scientific, San Jose, Calif., U.S.A.). Data were collected andprocessed by running Finnigan XCalibur software (version 1.3).Waxberry extracts in 10 μL aliquot were separated on an AgilentEclipse XDB—C18 column (4.6 × 150 mm, 5 μm particle size)by using a isocratic elution of mobile phase consisted of 0.1%formic acid solution and acetonitrile organic modifier (70/30,v/v) with the flow rate 0.5 mL/min. The analytes were detectedby photodiode array detection and electrospray ionization massspectrometry under negative total ion scanning mode. The PDAdetection wavelength was set at 254 nm and UV–Vis absorptionspectra were recorded on-line during HPLC analysis over therange 200 to 600 nm. Mass spectra were simultaneously recordedover the range m/z 50 to 500. The ion transfer tube temperaturewas set at 300 ◦C and the ESI was operated in negative mode atspray voltages of 3000 V. High purity of nitrogen (>99.99%) wasemployed as sheath gas and auxiliary gas at flow rates of 45 and10 arbitrary LCQ units, respectively.

Identification, quantitation, and recovery testThe phenolic acids in the waxberry juice samples were iden-

tified by matching HPLC retention times, UV-Vis absorptionspectra, and mass spectral data with the calibration standards. Allquantification was performed by the external calibration methodbased on peak areas. Calibration curves were constructed by lin-ear regression of the peak area individual standard against theconcentration. All the calibration standards and the extracts ofwaxberry juice samples were run in triplicate. Results are expressedas means ± standard deviation of 3 replicates. To evaluate the ex-traction method for phenolic acids, the recovery tests for bothfree and bound phenolic acids with ethyl acetate were performed.Known amounts of standard mixtures (10 mg/L) were subjectedto the entire analytical procedures with the juice samples intriplicate.

Results and Discussion

HPLC-PDA-ESI-MS separation and identificationof phenolic acids

Prior to employing HPLC-PDA-ESI-MS for the determinationof phenolic acids in waxberry juice extracts, the efficacy of theseparation and detection of phenolics using the HPLC-PDA-ESI-MS technique was tested on a standard mixture of the 4 phenolicacids. The instrument parameters, such as the composition of mo-bile phase and flow rate, were optimized to obtain good selectivityfor phenolic acids during the elution program. The typical chro-matograms of standards and extracts from samples are illustratedin Figure 1, which shows a baseline separation of the 4 phenolicacids and other components was achieved in 20 min. The separa-tion and quantitation of phenolic acids in waxberry juice is difficultbecause of their chemical complexity. A pre-cleanup and fraction-ation process prior to chromatographic analysis is usually necessary

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Phenolic acids in Myrica Rubra juice . . .

for reliable identification and quantification. In this study, free phe-nolic acids in waxberry juice were extracted and separated fromthe sample matrix with ethyl acetate and 5% NaHCO3. Figure 1Billustrates the separation of the free phenolic acids extracted fromwaxberry juice. The identities of the phenolic acids were estab-lished and confirmed by comparison of HPLC retention times,UV-Vis absorption spectra, and recorded mass spectra with refer-ence standards under the same chromatographic conditions. The

ESI-MS− spectrums of identified phenolic acids, including caf-feic, ferulic, sinapic, and salicylic acids, in this investigation arepresented in Figure 2. The overall identification characteristics ofthe phenolic acids studied are listed in Table 1. Total of 3 freephenolic acids corresponding to peaks 1, 2, and 4 were identi-fied as ferulic, caffeic, and salicylic acids, respectively, in the ethylacetate extracts of waxberry juice samples. In details, the peak1 presented spectral characteristics of the caffeic acid with UV

Caffeic acid

Ferulic acid

Sinapic acid

Salicylic acid

1

1

2

2

3

4

4

A

B

C

Figure 1–HPLC chromatograms of (A) 4 phenolicacids standards (20 mg/L); (B) ethyl acetateextracts of waxberry juice; (C) ethyl acetateextracts of hydrolyzed waxberry juice.

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λmax at (230, 325) nm and ESI-MS pseudomolecular and frag-ment ions at m/z (179.1 [M-H]−, 135.3[M-COOH]−), and itsretention time was at 4.86 min. Compared with the standard, thiscompound was unambiguously identified as caffeic acid. Peaks 2and 4 were eluted out at 7.75 min and 16.02 min, and showedthe UV absorption spectral characteristics with λmax at (240, 295)nm and (240, 300) nm, respectively. The pseudomolecular ionsm/z 193.1 [M-H]− and 137.1 [M-H]−, and fragment ions m/z93.3 [M-COOH]− (peak 4) were observed in ESI-MS− spectrafor those 2 peaks, and they were identified as ferulic and salicylicacids, respectively, by comparison with the authentic standards.Many previous studies have shown that phenolic acids are presentin fruits mainly in combined forms such as glycosides and esters

Table 1– HPLC retention timesa, UV-Vis absorption spectralcharacteristics, and identified ions present in the mass spectraof phenolic acids in standards and ethyl acetate extracts fromwaxberry juice samples by HPLC-PDA-ESI-MS.

Peak nr HPLC tR UV λmax ESI-MS− Identified phenolic(Figure 2) (min) (nm) (m/z) acids (MWb)

1 4.86 ± 0.11 230, 325 179, 135 Caffeic acid (180)2 7.75 ± 0.08 240, 295 193 Ferulic acid (194)3 9.81 ± 0.07 235, 320 223 Sinapic acid (224)4 16.02 ± 0.05 240, 300 137, 93 Salicylic acid (138)aReported as mean ± standard deviation of triplicate analysis;bMW = molecular weight.

(Chen and others 2001; Chen and Zuo 2007). To analyze the to-tal phenolic acids, waxberry juice was hydrolyzed with ultrasonic-assisted acidic hydrolysis (Wang and Zuo, 2011). Ascorbic acid wasadded to prevent the oxidation of the phenolic acids. After hydrol-ysis, the released phenolic acids were extracted and analyzed byHPLC-PDA-ESI-MS. The corresponding chromatogram is pre-sented in Figure 1C. Except for 3 identified free phenolic acids,sinapic acid was identified with the UV λmax at (235, 320) nmand ESI-MS pseudomolecular ions at m/z 223.1 [M-H]−, and itsretention time was at 9.81 min. Other peaks in chromatogramsFigure 1B and 1C were not able to be clearly resolved due to theabsence of standards and insufficient characteristic data, and furtherresearch is needed to elucidate the structure of those peaks. To thebest of our knowledge, only free phenolic acids, including gallic,p-hydroxybenzoic, vanillic, caffeic, p-coumaric acid, and ferulicacids have been reported previously in waxberry fruit, juice, orpomace products (Fang and others 2007, 2009; Zhou and oth-ers 2009). The salicylic and sinapic acids documented here wereidentified in waxberry products for the first time.

Quantitative analysis free and bound phenolic acidsin waxberry juice samples

The quantitative

(Continued )

evaluations of the identified phenolic acidswere performed on the basis of the HPLC-PDA data. As presentedin Table 2, all calibration graphs were linear over the concentration

Figure 2–ESI-MS− spectrums of caffeic, ferulic,sinapic, and salicylic acids in standards andsamples.

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ranges tested with correlation coefficients, R2, greater than 0.99.The detection limits were lower than 0.60 mg/L for all phenolicacids. The recovery tests were performed to evaluate the ethylacetate extraction method for phenolic acids. Different knownamounts of standard compounds were subjected to the entire an-alytical procedures with the samples in triplicate. As evidencedby the data given in Table 3, the percentage recoveries of 83.6%to 95.8% were obtained for studied phenolic acids and the rela-tive standard deviations were less than 4%. The present procedureoffers good precision and reproducibility, and can be applied inphenolic acid analysis in juice samples. Results from the quantifi-cation of both free and bound phenolic acids in waxberry juice are

Table 2–Calibration equationsa and detection limitsb for quanti-tative analysis of phenolic acids by HPLC-PDA.

Linear DetectionPhenolic Calibration Coefficient dynamic limitsacids equations (R2) ranges (mg/L) (mg/L)

Caffeic acid y = 1.1×105 x+2418 0.9985 0.96 to 100 0.29Ferulic acid y = 1.1×105 x−8731 0.9969 1.10 to 100 0.33Sinapic acid y = 9.9×104 x−6327 0.9989 0.70 to 100 0.21Salicylic acid y = 3.3×104 x−4702 0.9994 1.93 to 100 0.58ay is the peak area; x is the concentration of standards.bDetection limits were based on 3 times of signal to noise.

presented in Table 3. The concentrations of total caffeic, ferulic,sinapic, and salicylic acids were 2.76, 3.58, 2.89, and 1.92 mg/L,respectively. As expected, only minor amounts of phenolic acidsoccur in the free state in waxberry juice. All phenolic acids arepresent predominantly as conjugated forms (over 44.6% to 100%),which can be liberated by hydrolysis. The results presented hereindicate that the content of phenolic acids in waxberry juice isslightly different from those previously studied. The gallic acidwas reported as the dominant phenolic acid in fresh waxberryjuice (Fang and others 2009). However, it was not found at all incurrent commercial juice product. This may be due to differencesin fruit source, ripeness, manufacture process of juice products,

Table 3– Free and total contenta, extraction recoveriesb, andRSDs of identified phenolic acids in Chinese waxberry juice.

PercentageFree form (Free+bound) of free Recovery RSD

Phenolic acids (mg/L) form (mg/L) form (%) (%) (%)

Caffeic acid 1.53 ± 0.09 2.76 ± 0.12 55.4 92.7 3.2Ferulic acid 1.16 ± 0.16 3.58 ± 0.14 32.4 83.6 2.1Sinapic acid NDc 2.89 ± 0.08 – 88.9 3.8Salicylic acid 0.72 ± 0.06 1.82 ± 0.07 38.2 95.8 1.9aReported as mean ± standard deviation of triplicate analysis;bRecovery was reported as the average value of triplicate analysis;cNot detected.

Figure 2–Continued

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and length of storage time, as well as differences in the proceduresused for obtaining samples.

ConclusionsThe developed HPLC-PDA-ESI-MS method has been proved

an accurate and reliable technique for the simultaneous separa-tion, identification, and quantification of major phenolic acids inethyl acetate extracts from plant-derived foods. It was success-fully employed in the determination of phenolic acids in Chinesewaxberry juice samples, and 4 phenolic acids, including caffeic,ferulic, sinapic, and sylicylic acids were identified. Of these, toauthors’ best knowledge, sinapic and sylicylic acids are first re-ported in waxberry (Myrica rubra) products. Quantitative analysisindicated that waxberry juice contains abundant phenolic acidsand they occur mainly in bound forms.

AcknowledgementsThe project was supported by Scientific Research Foundation

for the Returned Overseas Chinese Scholars, State EducationMinistry.

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