Selective extraction, separation, and identification of anthocyanins from Hibiscus sabdariffa L. using solid phase extraction-capillary electrophoresis-mass spectrometry (time-of-flight

Antonio Segura-Carretero1

Miguel A. Puertas-Mejía1, 2

Sonia Cortacero-Ramírez1

Raúl Beltrán3

Carlos Alonso-Villaverde3

Jorge Joven3

Giovanni Dinelli4

Alberto Fernández-Gutiérrez1

1Department of AnalyticalChemistry,Faculty of Sciences,University of Granada,Granada, Spain

2Instituto de Química,Facultad de CienciasExactas y Naturales,Universidad de Antioquia,Medellín, Colombia

3Centre de Recerca Biomèdica,Hospital Universitari de Sant Joan,Reus, Spain

4Department of AgroenvironmentalScience and Technology,University of Bologna,Bologna, Italy

Received April 17, 2007Revised January 29, 2008Accepted January 31, 2008

Research Article

Selective extraction, separation, andidentification of anthocyanins fromHibiscus sabdariffa L. using solid phaseextraction-capillary electrophoresis-massspectrometry (time-of-flight/ion trap)

A method for selective extraction using SPE, electrophoretic separation at basic conditionand the identification by using exact masses and fragmentation patterns has been devel-oped in order to know the anthocyanins in dried calyces of Hibiscus sabdariffa L. A detailedand comparative study of several extraction procedures has been carried out to obtain themaximum number of anthocyanidins from the calyces and then a CE-TOF-MS method inpositive mode using ESI has been developed for the separation and rapid identification ofanthocyanins in H. sabdariffa L. Delphinidin-3-sambubioside, cyanidin-3-sambubiosidehave been detected as main components and cyanidin-3-O-rutinoside, delphinidin-3-O-glucoside and cyanidin-3,5-diglucoside, and chlorogenic acid as minor constituents. Theconfirmation of the anthocyanidins and chlorogenic acid was carried out using fragmenta-tion ions with the IT-mass spectrometer (IT-MS).

Keyworks:

Anthocyanins / CE-MS / CZE / Hibiscus sabdariffa / KarkadeDOI 10.1002/elps.200700819

2852 Electrophoresis 2008, 29, 2852–2861

1 Introduction

Nowadays, the consumer preference for natural productsinstead of artificial ones has acquired an important role inthe food industry, at least in part, due to nutritional supple-mentation and healthy properties associated with them.Among them, karkade, roselle, or byssap (Hibiscus sabdar-iffa L.), an annual herb that belongs to malvacea family, iswidely known as a result of its calyces have been widelyused to prepare cold and hot beverages in many of theworld’s tropical and subtropical countries as well as in phy-totherapeutic applications [1–5]. Many biological activitieshave been extensively reported, such as antihypertensiveand cardioprotective agents [6], hepatoprotector [7], inhibitoragainst porcine pancreatic a-amylase [8], as well as sedative[9], and antioxidant capacity [10]. Moreover, karkade hasgained an important position in the soft drink market andcommercial preparations of H. sabdariffa extract are cur-

rently marketed as supplements due to their apparentpotential health benefits and as a colorant to replace somesynthetic dyes.

The intense red pigments in red calyces of karkade areanthocyanins, which are O-glycosides derivatives of 3,5,7,30-tetrahydroxyflavylium cation and their analysis is usuallycarried out by LC coupled with photodiode array and MSdetection [11, 12]. Differentiation found on anthocyanins arerelated by the number of hydroxyl groups, the nature, posi-tion, and number of sugars (e.g., D-glucose, D-galactose, etc.)attached to the molecule, and the number of aliphatic acids(e.g., acetic, malic, and malonic, etc.) or aromatic acids (e.g.,caffeic, p-coumaric or ferulic acid) linked to sugars in themolecule. Among eighteen known naturally occurringanthocyanidins or aglycones, just six anthocyanidins arecommon in higher plants, they are named as pelargonidin(Pg), peonidin (Pn), cyanidin (Cy), malvidin (Mv), petunidin(Pt), and delphinidin (Dp). The glycosides of the three non-methylated anthocyanidins (Cy, Dp, and Pg) are the mostprevalent in nature [13, 14]. Pale et al. [15] isolated anthocya-nins from calyces of H. sabdariffa and their structures wereelucidated using GC, MS, and NMR methods. From thestems of H. taiwanensis, Pei-Lin et al. [16] isolated new com-pounds, and the structures were determined by spectroscopicand chemical transformation methods. Tian et al. [17] devel-

Correspondence: Dr. Antonio Segura Carretero, Research GroupFQM-297, Department of Analytical Chemistry, Faculty ofSciences, University of Granada, C/Fuentenueva s/n, E-18071Granada, SpainE-mail: [email protected]

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2008, 29, 2852–2861 CE and CEC 2853

oped an MS/MS method coupled to HPLC with photodiodearray detection. The methodology showed to be a fast proce-dure for screening anthocyanins in fruits and vegetables.

Over the last years, CE has gained attention as an alter-native tool for analyzing complex matrices such as naturalproducts, food, and food components by providing an effi-cient option to conventional and arduous separation tech-niques [18]. In fact, for anthocyanins, CE can be highlyuseful for efficiently because this separation technique isideal for charged compounds as anthocyanins. Recently,anthocyanins have been isolated from different matricesusing CE [19–21]. Calvo et al. [22] evaluated the migrationtimes of some anthocyanins present in wine using CZEwith photo-DAD, and according to their findings, themigration order depends on the charge and charge/sizeratio. Guadalupe et al. [23] developed a multiple-step ana-lytical method to analyze polymeric phenolics in red wines.This method proved to be valid for analyzing differentfamilies of phenolic compounds such as monomeric phe-nolics and proanthocyanidins. In addition, CE provides acharge-based mode of separation that often is com-plementary to the chromatographic techniques frequentlyused in analytical chemistry. MS detection can considerablyenhance the utility of CE by providing information aboutthe identity of the separated compounds. Therefore, thehyphenation of CE to MS (CE-MS) via an ESI interface is anattractive, cheap and fast analytical tool, and informationobtained is enhanced due to MS detection provides muchmore structural information (MS/MS) [24]. In most of thesecases, CE volatile electrolyte buffer solution is essential toreduce the background noise and not to suppress the ioni-zation efficiency of analytes in ESI. Arráez-Román et al. [25]and Carrasco-Pancorbo et al. [26] have reported the applica-tion of CZE coupled to ESI-MS for the separation andstructural elucidation of different natural products, such aspolyphenols, bitter acids, lignans, and several complexsamples, pointed out of a faster, simple, and direct CZE-ESI-MS methodology. Furthermore, in relation with theanalysis of anthocyanins, Bednar et al. [27] have reported amethod for monitoring anthocyanins in wines using CE-ESI-MS and according to authors, this methodology wassuitable to analyze polyphenolic dyes in wines.

The goal of this work has been the development of amethod for the selective extraction, separation, and identifi-cation using SPE-CE-ESI-MS in order to determine (qualita-tive determination) anthocyanins in hibiscus.

2 Materials and methods

2.1 Chemicals

Delphinidin chloride, cyanin chloride, kuromanin chloride(cyanidin-3-O-glucoside), myrtillin chloride (delphinidin-3-O-glucoside), and chlorogenic acid were purchased fromExtrasynthese, France. Ammonium acetate, ammonium

carbonate, and ammonia (30%) were obtained from Panreac(Barcelona, Spain) and boric acid from Sigma–Aldrich (St.Louis, MO, USA). Buffers were prepared by weighing thequantity indicated in doubly distilled water and adding 2 Mammonia (30%) to adjust the pH. Formic acid and HPLC-grade 2-propanol used in the sheath flow were acquired fromPanreac. All solutions were filtered through 5 mm Millipore(Bedford, MA, USA) membrane filters before being injectedinto the capillary. Distilled water was deionized using a Milli-Q system (Millipore).

2.2 CE-MS

Separations were performed at 25 kV and 257C using barefused-silica capillaries with 50 mm id680 cm (BeckmanInstruments, Fullerton, CA, USA). Before first use theuncoated capillaries were conditioned using a rinse with0.1 M NaOH for 10 min followed by a rinse with water for5 min and finally with running buffer for 30 min. Capillaryconditioning between runs was carried out by flushing thecolumn for 3 min with water and finally for 5 min with theseparation buffer. At the end of the day the capillary wasrinsed with water for 30 min. Injections were made at theanodic end at a pressure of 0.5 psi for 20 s. The selectedrunning buffer was 200 mM boric acid in water and adjust-ment the pH to 9 with ammonia.

CE was coupled using an electrospray interface (ESI)(model G1607A from Agilent Technologies, Palo Alto, CA,USA) to both mass spectrometers. For the connection be-tween CE system and electrospray ion source of the massspectrometers the outlet of the separation capillary was fit-ted into the electrospray needle of the ion source and a flowof conductive sheath liquid made electrical contact betweenthe capillary effluent and water for the electrospray needle.The commercial coaxial-sheath liquid and the electricalcontact at the electrospray needle tip were delivered by a74900-00-05 Cole Palmer syringe pump (Vernon Hills, IL,USA).

2.2.1 IT-MS

An ITmass spectrometer (Bruker Daltonics, Squire 2000TM,Germany) was used in the positive-ion mode and the capil-lary voltage was set at 4050 V. The IT scanned within 200–800 m/z (target mass 400 m/z) range at 13 000 u/s duringseparation and detection in the scan mode. The maximumaccumulation time for the IT was set at 5.00 ms, the targetcount was set at 20.000 and the trap drive level was set at100%. The instrument was controlled by a PC runningEsquire NT software from Bruker Daltonics.

2.2.2 TOF-MS

A TOF spectrometer (microTOF, Bruker Daltonics) wasused. The spectrometer permits whole spectrum acquisitionfollowed by internal calibration, searching against a target


2854 A. Segura-Carretero et al. Electrophoresis 2008, 29, 2852–2861

database of exact monoisotopic masses, and reporting ofpositive findings. TOF-MS acquisition data were processedwith DataAnalysis version 3.3, equipped with a screeningapplication macrocreated together with Bruker Daltonics.The macrosearched for target masses in the database basedon the defined criteria, including mass tolerance(,10 ppm), isotopic pattern match (error less that 5%),retention time window (6 0.3 min), and minimum areacount (.50 000). The isotopic pattern match provides anexact numerical comparison of theoretical and measuredisotopic patterns and provides an additional identificationparameter for accurate mass measurement. The ESI-voltageof the TOF is applied at the end cap of the transfer capillaryto the MS with the spray needle being grounded. The TOF-MS was operated to acquire spectra in the range of 200–800 m/z every 90 ms, 20 000 spectra being averaged for sub-sequent data analysis. Transfer parameters were optimizedfor high sensitivity while keeping the resolution to better10 000 in the mass range of 200–800. The instrument wascontrolled by a PC running Esquire NT software from Bru-ker Daltonics. External calibration was performed usingsodium formate cluster by switching the sheath liquid to asolution containing 5 mM and 0.2% formic acid in water/isopropanol 1:1 v/v at the end of the analysis. An exact cali-bration curve based on numerous cluster masses each dif-fering by 68 Da (NaCHO2) was obtained. Due to the com-pensation of temperature drift in the MicroTOF, this exter-nal calibration provided accurate mass values (better 5 ppm)for a complete run without the need for a dual sprayer setupfor internal mass calibration.

2.3 Extraction procedure of anthocyanidins

Three extraction procedures (extraction procedures A–C)have been proved in this study in order to compare whichshows the high capacity of extraction of mainly anthocya-nins. The extraction procedures A and B are very similar, butwith some modifications, while extraction procedure C isdifferent.

2.3.1 Extraction procedure A

The karkade samples used in this study were collected inGuerlé (Senegal, were it is popularly known as byssap) andthe calyces sun-dried were cleaned. The dried calyces (10 g)were reduced to powder with a mortar and 1.0 g of homoge-nized dry leaves was mixed with 10 mL of MeOH/HCl (99:1v/v) for 4 h at room temperature. During this time the solu-tion was magnetically stirred at 900 rpm. The resultant so-lution, previously filtered, was concentrated to dryness in arotary evaporator under reduced pressure at 357C to get rid ofany residual solvent. During the last step, 2 mL of water wereadded to dissolve the extract, which was then passed througha 5 mm membrane filter and keep to 47C before the analysisby CE-ESI-IT-MS or CE-ESI-TOF-MS.

2.3.2 Extraction procedure B

Samples (10 g) were reduced to powder with a mortar and1.0 g of homogenized dried leaves was extracted with 10 mL ofMeOH/HCl (99:1 v/v) by sonication for 30 min. The resultantsolution was treated according to extraction procedure A.

2.3.3 Extraction procedure C

Extraction was performed according to methods describedpreviously [28, 29] with some modifications. Homogenizeddried calyces (25 g) were mixed with 1 L of acetic acid (15%v/v) for 48 h with magnetic stirring at room temperature.The solution was filtered by Buchner and then passedthrough a 5 mm membrane filter to remove solid particles.The filtrate was mixed with 40 g Amberlite XAD-2 (pore size9 nm, particles size 0.3–1.2 mm) and stirred in a magneticstirrer for 40 min at room temperature, in order to absorbenough anthocyanins quantity. The Amberlite particles werethen packed into a glass column (42 cm63.2 cm) and thecolumn was washed with deionized water until a colorlesssolution was obtained. The anthocyanin remained absorbedon the column was eluted with 1 L of ethanol (70% v/v)–acetic acid (1% v/v). The intense red solution was con-centrated to dryness in a rotary evaporator under reducedpressure at 407C. During the last step, 2 mL of water wereadded to dissolve the extract, which was then passed througha 5 mm membrane filter and keep to 47C before the analysisby CE-ESI-IT-MS and CE-ESI-TOF-MS.

3 Results and discussion

3.1 Development of CE-ESI-MS

The hyphenation of CE and MS represents a powerful tech-nique that was used to aid in the anthocyanin determinationand permits the analysis of vegetables extract without com-plicate pretreatment steps and the combination of MS/MSthat provides characteristic fragmentation patterns thatenable anthocyanins’ characterization and make possible arough determination of the position of the glycoside groups.

Extract C was used to optimize electrophoretic parame-ters according to the following criteria: migration behavior,sensitivity, analysis time, and peak shape.

As we commented in the introduction, CE volatile elec-trolyte buffer solutions are essential to reduce the back-ground noise and not to suppress the ionization efficiency ofanalytes in ESI. On other hand, Bednar et al. [27] usedammonium borate for the separation of several anthocyaninsand these author comment in the article that the use ofborate ammonium as running electrolyte did not cause asignificant contamination of the ion source. For our experi-ence the borate buffers from nonvolatile disodium tetra-borate are noncompatible with CE-MS but the uses of boricacid made possible the use as buffer.



For this reason, two buffers, a volatile buffer commonlyused like ammonium acetate and boric acid/ammonia, havebeen proved. Different concentrations and pH values (8.5–10, in steps of 0.5) were checked, obtaining the best resultswith the buffers containing 60 mM ammonium acetate atpH 10.0 and 200 mM boric acid/ammonia at pH 9.0 (Fig. 1).However, the best separation, in terms of peak efficiency andresolution, baseline stability, and analyte migration time wasobtained with the boric acid/ammonia running buffer, andusing this buffer did not cause a significant contamination ofthe ion source. Voltage applied was in the range of 5–30 kVand finally a voltage of 25 kV was chosen in order to affordthe best resolution together with satisfactory current(around 20 mA) and analysis time. The injection times weremade at the anodic end using N2 pressure of 0.5 psi(1 psi = 6894.76 Pa), and were studied from 5 to 30 s, select-ing 20 s, due to the high sensitivity obtained. These electro-phoretic conditions were established for the subsequentoptimization of the ESI parameters.

The optimization ESI-MS parameters were done con-sidering the height of the MS signal for the main compoundsdetected obtained using the extraction procedure C, becausein general this family of compounds has the same behaviorin the electrospray [30]. Figure 2 shows the ESI-MS parame-ters optimized in order to produce the highest signal inten-sity and adequate MS signals.

The choice of sheath liquid has a significant effect on thesensitivity and electrical contact between CE and ESI. Initi-ally we have tested different types of sheath-flow liquids:2-propanol/water (60:40 v/v); 2-propanol/water (60:40 v/v)containing 0.1% formic acid; and methanol/water (80:20 v/v)with 0.25% acetic acid. Using 2-propanol/water (60:40 v/v)and methanol/water (80:20 v/v) sheath liquid with 0.25%

acetic acid, the current broke down after 2 min, possibly dueto poor electrical contact between the CE and ESI, which mayhave been a result of the nature of modifier in the solution.However, the use of a sheath liquid of 60:40 v/v 2-propanol/water containing 0.1% v/v formic acid provided higher cur-rent stability and MS signal. Therefore, 60:40 v/v 2-propanol/water with 0.1% v/v formic acid was selected as sheathliquid. The sheath liquid was expected to dilute the CZEsample zone as it passed concentrically around the CZE col-umn effluent and mixed with it, but no significant differ-ences were observed in the range from 0.22 to 0.34 mL/hprobably due to incomplete mixing in the Taylor cone[31].While higher flow rates result in a lower S/N, due to thedilution of the separated compounds, lower flow rates pro-duce an unstable spray. Finally, an intermediate flow rate of0.24 mL/min was selected as optimum in terms of signalresponse and stability. The ESI-MS operating conditionswere optimized by adjusting the needle-counter electrodedistance and applied electrospray potentials, while a samplesolution was injected and separated in the CE-ESI-MSinstrument. The optimum nebulizer/drying gas conditionswere 6 psi nitrogen and 5 L/min nitrogen (heater tempera-ture 3007C).

It was also observed in Fig. 2 that compound stability(applied electrospray potentials) played an important role forkarkade compounds. Therefore, at higher percentages of thisvariable the MS signal decreases as the number of moleculesintroduced into MS is low, while with lower percentages themajority of the compounds becomes more stable rising theMS signal. Thus, we have chosen 100% of compound stability.

As a result, the following CE-ESI-MS conditions wereused: running buffer 200 mM borate-ammonium, pH 9,voltage 25 kV, 20 s injection time. A sheath liquid was com-

Figure 1. Running buffer: ammonium acetate/ammonia 60 mM, pH 10 (A), boric acid/ammonia200 mM, pH 9 (B). Initial conditions: voltage,25 kV; injection time, 10 s at 0.5 psi. Sheathliquid: 2-propanol/water 60:40 v/v and formicacid (0.1%) at a flow rate of 0.24 mL/h. Dryinggas: 5 L/min. Temperature: 3007C. Nebulizer gaspressure: 6 psi. MS analyses were carried outusing positive polarity. Compound stability:100%. MS scan 100–800 m/z (target mass400 m/z). Sample: dried calyces of karkade(extract C).



Figure 2. Optimization of ESI-MS parameters. Conditions: buffer, 200 mM boric acid/ammonia pH 9; voltage, 25 kV; injection time, 10 s at0.5 psi and sheath liquid, 2-propanol/water 60:40 v/v, and formic acid (0.1%). MS analyses were carried out using positive polarity. MS scan100–800 m/z (target mass 400 m/z). Sample: dried calyces of karkade (extract procedure C).

posed of 2-propanol/water (60:40 v/v) and 0.1% formic aciddelivered at a flow rate of 0.24 mL/h. The optimum nebulizerwas 6 psi, dry gas 5 L/min, and dry temperature 3007C. MSanalyses were carried out using a compound stability of 25%.

3.2 Identification of karkade compounds and

structure information using MS/MS

The analysis by CE-ESI-MS using IT and TOF analyzersshowed that anthocyanins glycosides derivatives were pres-ent in the methanolic extract of dried calyces of karkade.

According to previous results [27], we expected that antho-cyanins migrate as anions due to the equilibrium betweenborate complexes of phenolics groups in ortho position onthe anthocyanin framework, afterwards when they havereached the electrospray, they were positively charged.

3.2.1 IT and TOF-MS

Figure 3 shows the base peak electropherogram and theextracted ion electropherogram for the identified compoundsof the extract C under CE-ESI-MS conditions, and revealed



Figure 3. Base peak electropherogram andextracted ion electropherogram. CE-microTOF-MS conditions: 50 mm id fused-silica capillary,80 cm total length. Buffer: 200 mM boric acid/ammonia, pH 9. Voltage: 25 kV. Injection time:20 s at 0.5 psi. Sheath liquid: 2-propanol/water60:40 v/v containing 0.1% v/v formic acid flowrate 0.24 mL/h. MS scan 100–800 m/z. Sample:dried calyces of karkade (extract C). For otherconditions see text.

two main anthocyanins with retention times at 11.8 min (Cy-3-sambubioside) and 11.9 (Dp-3-sambubioside). In order toobtain an unequivocal identification of compounds, we usedthe compound stability parameters, which gave informationabout fragmentation patterns. Consequently, compound sta-bility played an important role for calyces compounds. InFig. 4 the MS and MS/MS spectra of the most importantcompounds in dried flower of H. sabdariffa L. are shown, andresulted in clear and characteristic fragmentation patterns.Different voltages were used to evaluate the fragmentationpattern with MS/MS. Under the conditions used for thisapplication, high reproducibility was obtained and the frag-mentations patterns obtained with anthocyanins was alwaysthe same. In fact, the glycosidic bonds between the flavyliumring and sugars directly linked to it were cleaved. MS/MSspectra showed the fragments corresponding to the agly-cones. The fragmentation pattern of C-3 substituted antho-cyanins produced only one fragment, corresponding to them/z of the aglycon. The only exception to this pattern was theCy-3,5-diglucoside. MS/MS of this anthocyanin producedfragments that corresponded to the aglycon, the C-3, and theC-5 substituted anthocyanin.

On the basis of the agreement between the scanned mo-lecular masses using the MS detectors and known anthocya-nins molecular weight we confirmed in all karkade extracts

the presence of Dp-3-sambubioside at m/z 597 and Cy-3-sambubioside at m/z 581. Additionally, we detected thepresence of Cy-3-O-rutinoside (m/z 595), Dp-3-O-glucoside(m/z 465), and Cy-3,5-O-diglucoside (m/z 611) as minorconstituents (Fig. 5). The identity of the anthocyanins wasconfirmed by fragmentation ions (MS2) and using the accu-rate mass data obtained from positive ion mode, which gaveinformation about their aglyconic moiety of the anthocyanin.Additionally, isotopic patterns were used to confirm the mo-lecular mass of the major anthocyanins detected in karkadeextracts, and the experimentally isotopic patterns show thatm/z values for the three isotopologues obtained from differ-ent anthocyanins are in accordance with the calculatedvalues (Fig. 6). Thus, it is possible to prove that the com-pounds under study correspond with the assignment pro-posed. Table 1 shows that the exact mass measurementswere within acceptable limits for present purpose. Thedeviations from calculated masses were below 2 mDa inmost measurements. The same product ions were observedfor each mass spectrum and the ion signal intensities dif-fered moderately. Therefore, despite the different analyzersused to detect target molecules, the CE-ESI-IT and CE-ESI-TOF methodologies yield almost identical product ion massspectra not only for the anthocyanins examined but also forchlorogenic acid examined.



Figure 4. MS (A1, B1, C1, D1) and MS2 (A2, B2, C2, D2) spectra of the most important dried calyces of karkade compounds. MS/MS ofcompounds in extract C. (A) Dp-3-sambubioside, (B) Cy-3-sambubioside, (C) Dp-3-glucoside, and (D) Cy-3,5-diglucoside. All conditions asin Fig. 2.



Figure 5. Structures of antho-cyanidins detected in H. sabdar-iffa L., by CE-ESI-MS. (1) Cy-3-O-rutinoside, (2) Cy-3,5-digluco-side, (3) Cy-3-sambubioside, (4)Dp-3-sambubioside, (5) Dp-3-O-glucoside, and (6) chlorogenicacid.

Figure 6. Isotopic patterns of the main peaksdetected in dried calyces of karkade (extract C).(A) Cy-3-sambubioside, (B) Dp-3-sambubioside,and (C) Cy-3-rutinoside. All conditions as Fig. 2.



Table 1. Mass exact measurements by CE-ESI-microTOF

m/z Experimental m/z Calculated Error (ppm) Sigma Analytes

355.1006a) 355.1024 5.011 0.0194 Chlorogenic acid465.1037b) 465.1028 2.110 0.0286 Delphinidin-3-O-glucoside581.1507b) 581.1501 0.978 0.0377 Cyanidin-3-sambubioside595.1664b) 595.1657 1.064 0.0361 Cyanidin-3-O-rutinoside597.1462b) 597.1450 1.948 0.0374 Delphinidin-3-sambubioside611.16282 611.1607 3.548 0.0232 Cy-3,5-O-diglucoside

a) [M 1 H]1

b) [M]1

3.3 Comparison of different extraction systems

With the aim to demonstrate the capability of the CE-ESI-MSmethod for the analysis of complex samples, the method wasapplied to the analysis of different karkade extracts preparedusing the three extraction procedures described above. Underthe best CE-ESI-MS parameters established it is possible toanalyze the different compounds present in the different typesof extracts and to carry out a comparative study of the extrac-tion capacity of the procedures. Table 2 lists the compoundsfound in the different extraction procedures. In extracts A andB, besides of Cy-3-samb and Dy-3-samb also were detected theprotonated parent ion [M 1 H]1 at m/z 355.1, correspondingto chlorogenic acid, and finally six compounds in extract C.The peaks at m/z 355.1, 465.1, and 611.2 were easily identifiedand confirmed by co-injection using standards and MS/MSstudies. The others compounds, for which no standard exists,the use of MS/MS spectra recorded, the accurate mass of theTOFand the literature on karkade showed that it is possible toachieve the tentative identification.

Table 2. Relation m/z found in different extracts

Extract A Extract B Extract C Analytes

355.1 355.1 355.1a) Chlorogenic acid– – 465.1b) Delphinidin-3-O-glucoside581.2 581.2 581.2b) Cyanidin-3-sambubioside– – 595.1b) Cyanidin-3-O-rutinoside597.2 597.2 597.2b) Delphinidin-3-sambubioside– – 611.2b) Cy-3,5-O-diglucoside

a) [M 1 H]1

b) [M]1

Finally, in order to characterize a major number of com-pounds using the proposed method, we selected the extractobtained from extraction procedure C and analyzed by CE-ESI-MS in positive mode. Dp-3-sambubioside and Cy-3-sambubioside have been isolated in previous works as themain compounds present in dried flower of H. sabdariffa L.using a wide variety of CE, HPLC, and HPLC-MS methodol-ogies [32, 33] so far, the present work is a new contribution asanalytical tool to detect and identify anthocyanins in karkadeextract using CE-ESI-MS.

4 Concluding remarks

The identification of anthocyanins from dried calyces of kar-kade sample was carried out using CE-ESI-MS with IT andTOF analyzers. The major anthocyanins present in theextracts of karkade were identified by comparing bothmigration time and MS data obtained from karkade samplesand bearing in mind all the data reported in the literature.We also checked that the fragmentation patterns for all theanthocyanins present in the karkade and their correspondingstandards were the same. We can point out that, in this study,CE-MS coupling was a rapid and sensitive method thatallowed us to identify the main pigments present in karkadeextracts. In conclusion, CE-ESI-MS with ionization in posi-tive mode provides a suitable rapid and efficient technique todetermine anthocyanins from methanolic extracts of naturalproducts. Moreover, to our knowledge, this is the first timethat the application of CE-MS for the determination of pig-ments from H. sabdariffa L. and the possibilities of CE-ESI-MS for monitoring flavonoids in preparations of cold and hotbeverages has been described.

The authors acknowledge financial support from the Minis-terio de Educación y Ciencia, Spain (project CTQ2005-01914/BQU). M. A. P.-M., was recipient of a short-term fellowshipthrough Coimbra Group Scholarships Programme for youngProfessors and Researchers from Latin American Universities. Wethank J. A. Smith for proofreading this manuscript.

The authors have declared no conflict of interest.

5 References

[1] Carvajal-Zarrabal, O.,Waliszewski, S. M., Barradas-Dermitz,D. M. A., Orta-Flores, Z. et al., Plant Food Hum. Nutr. 2005, 60,153–159.

[2] Herrera-Arellano, A., Flores-Romero, S., Chávez-Soto, M. A.,Tortoriello, J., Phytomedicine 2004, 11, 375–382.

[3] Chen, C. C., Hsu, J. D., Wang, S. F., Chiang, H. C. et al., J. Agri.Food Chem. 2003, 51, 5472–5477.



[4] Lin, H. H., Chen, J. H., Kuo, W. H., Wang, C. J., Chem. Biol.Interact. 2007, 165, 59–75.

[5] Ali, B. H., Wabel, A. L., Blunden, N., Phytother. Res. 2005, 19,369–375.

[6] Odigie, I. P., Ettarh, R. R., Adigun, S. A., J. Ethnopharmacol.2003, 86, 181–185.

[7] Amin, A., Hamza, A. A., Life Sci. 2005, 77, 266–278.

[8] Hansawasdi, C., Kawabata, J., Kasai, T., Biosci. Biotechnol.Biochem. 2000, 64, 1041–1043.

[9] Amos, S., Binda, L., Chindo, B. A., Tseja, A. et al., Pharm.Biol. 2003, 41, 325–329.

[10] Chen, C. C., Chou, F. P., Ho, Y. C., Lin, W. L. et al., J. Sci. FoodAgric. 2004, 84, 1989–1996.

[11] Zhang, Z., Kou, X., Fugal, K., Mclaughlin, J., J. Agric. FoodChem. 2004, 52, 688–691.

[12] Chirinos, R., Campos, D., Betalleluz, I., Giusti, M. M. et al., J.Agric. Food Chem. 2006, 54, 7089–7097.

[13] Giusti, M. M., Wrolstad, R. E., Biochem. Eng. J. 2003, 14,217–225.

[14] Stintzing, F. C., Carle, R., Trends Food Sci. Technol. 2004, 15,19–38.

[15] Pale, E., Koiuda-Bonafos, M., Nacro, M., Comptes RendusChimie 2004, 7, 973–980.

[16] Pei-Lin, W. U., Tian-Shung, W. U., Cai-Xia, H. E., Chia-Hao, S.U., Kuo-Hsiung, L. E. E., Chem. Pharm. Bull. 2005, 53, 56–59.

[17] Tian, Q., Giusti, M. M., Stoner, G. D., Schwartz, S. J., J.Chromatogr. A 2005, 1091, 72–82.

[18] Cifuentes, A., Electrophoresis 2006, 27, 283–303.

[19] Saenz-Lopez, R., Fernandez-Zurbano, P., Tena, M. T., J.Chromatogr. A 2004, 1052, 191–197.

[20] Bednar, P., Tomassi, A. V., Presutti, C., Pavlikova, M. et al.,Chromatographia 2003, 58, 283–287.

[21] Ichiyanagi, T., Kashiwada, Y., Ikeshiro, Y., Hatano, Y. et al.,Chem. Pharm. Bull. 2004, 52, 226–229.

[22] Calvo, D., Saenz-Lopez, R., Fernandez-Zurbano, P., Tena, M.T., Anal. Chim. Acta. 2004, 524, 207–213.

[23] Guadalupe, Z., Soldevilla, A., Saenz-Navajas, M. P., Ayes-taran, B., J. Chromatogr. A 2006, 1112, 112–120.

[24] Simó, C., Barbas, C., Cifuentes, A., Electrophoresis 2005, 26,1306–1318.

[25] Arráez-Román, D., Cortacero-Ramírez, S., Segura-Carretero,A., Martín-Lagos Contreras, J.-A., Fernández-Gutiérrez, A.,Electrophoresis 2006, 27, 2197–2207.

[26] Carrasco-Pancorbo, A., Arráez-Román, D., Segura-Carre-tero, A., Fernández-Gutiérrez, A., Electrophoresis 2006, 27,2182–2196.

[27] Bednar, P., Papouskova, B., Muller, L., Bartak, P. et al., J. Sep.Sci. 2005, 28, 1291–1299.

[28] Matsui, T., Ueda, T., Oki, T., Sugita, K. et al., J. Agric. FoodChem. 2001, 49, 1948–1951.

[29] Hou, D.-X., Tong, X., Terahara, N., Luo, D., Fujii, M., Arch.Biochem. Biophys. 2005, 440, 101–109.

[30] Gómez-Romero, M., Arráez-Román, D., Moreno-Torres, R.,García-Salas, P. et al., J. Sep. Sci. 2007, 30, 595–603.

[31] Hernandez-Borges, J., Neusüss, C., Cifuentes, A., Pelzing,M., Electrophoresis 2004, 25, 2257–2281.

[32] Giusti, M. M., Rodríguez-Saona, L. E., Griffin, D., Wrolstad,R. E., J. Agric. Food Chem. 1999, 47, 4657–4664.

[33] Gradinaru, G., Biliaderis, C. G., Kallithraka, S., Kefalas, P.,García-Viguera, C., Food Chem. 2003, 83, 423–436.


Documents

Selective extraction, separation, and identification of anthocyanins from Hibiscus sabdariffa L. using solid phase extraction-capillary electrophoresis-mass spectrometry (time-of-flight