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Journal of Chromatography A, 1242 (2012) 43– 58

Contents lists available at SciVerse ScienceDirect

Journal of Chromatography A

jou rn al h om epage: www.elsev ier .com/ locat e/chroma

imultaneous metabolite fingerprinting of hydrophilic and lipophilic compoundsn Echinacea pallida by high-performance liquid chromatography with dioderray and electrospray ionization-mass spectrometry detection

ederica Pellati ∗, Giulia Orlandini, Stefania Benvenutiepartment of Pharmaceutical Sciences, University of Modena and Reggio Emilia, Via G. Campi 183, 41125 Modena, Italy

r t i c l e i n f o

rticle history:eceived 10 February 2012eceived in revised form 6 April 2012ccepted 10 April 2012vailable online 17 April 2012

eywords:chinacea pallidaolyacetylenesolyenesaffeic acid derivativesetabolite fingerprintingPLCass spectrometry

a b s t r a c t

In this study, a detailed phytochemical characterization of Echinacea pallida (Nutt.) Nutt. root extractsand dietary supplements was carried out for the first time by developing advanced chromatographictechniques, based on HPLC with diode array (DAD) and electrospray ionization-mass spectrometry (ESI-MS) detection (with ion trap and triple quadrupole mass analyzers), for the simultaneous analysis ofhydrophilic and lipophilic secondary metabolites. The HPLC analyses were carried out on an Ascentis C18

column (250 mm × 4.6 mm I.D., 5 �m), with a mobile phase composed by H2O and ACN both contain-ing 0.1% formic acid, under gradient elution. The UV spectra, in combination with MS and MS/MS data,allowed the identification of fourteen compounds, including caffeic acid derivatives, polyacetylenes andpolyenes, in the analyzed samples. MS and MS/MS data were discussed in detail and the typical fragmen-tation patterns of each class of secondary metabolites were identified. For the first time, a hydroperoxideintermediate was characterized as an oxidation product of one of E. pallida monocarbonylic acetylenes,providing a confirmation of the mechanism that leads to the generation of hydroxylated derivatives. TheHPLC method was fully validated in agreement with ICH guidelines and then applied to real samples.The quantitative analysis indicated that there was a great variability in the amount of the active com-pounds in the dietary supplements containing E. pallida root extracts: the content of total caffeic acid

derivatives ranged from 2.31 to 11.45 mg/g and the amount of total polyacetylenes and polyenes from6.38 to 30.54 mg/g. In the analyzed samples, the most abundant caffeic acid derivative was found to beechinacoside. Regarding polyacetylenes and polyenes, the most representative compounds were foundto be tetradec-(8Z)-ene-11,13-diyn-2-one, pentedeca-(8Z,11Z)-dien-2-one and pentadec-(8Z)-en-2-one.The developed method can be considered suitable for metabolite fingerprinting and quality control of E.

natu

pallida plant material and

. Introduction

In the ambit of the genus Echinacea (Asteraceae family), thepecies traditionally used in phytotherapy are E. purpurea (L.)oench, E. angustifolia DC. var. angustifolia and E. pallida (Nutt.)utt. [1]. The drug consists of the roots, but, in the case of E.urpurea, aerial parts are also employed. Echinacea extracts areommonly applied in the formulation of dietary supplements anderbal remedies. The main use of these natural products is as

mmunomodulatory in the prevention and treatment of inflamma-ory and viral diseases [1,2].

Echinacea extracts have shown a highly complex chemicalomposition, including polar compounds (caffeic acid derivatives)3,4], non-polar compounds (alkylamides, acetylenic and alkenic

∗ Corresponding author. Tel.: +39 059 205 5144; fax: +39 059 205 5131.E-mail address: federica.pellati@unimore.it (F. Pellati).

021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.chroma.2012.04.025

ral products.© 2012 Elsevier B.V. All rights reserved.

secondary metabolites) [5–7] and high molecular weight con-stituents (polysaccharides and glycoproteins) [8,9].

Regarding caffeic acid derivatives, several compounds havebeen identified in the hydrophilic fractions of Echinacea extracts,including caftaric acid, chlorogenic acid, caffeic acid, cynarin, echi-nacoside and cichoric acid [4,10,11]. In the case of E. pallida roots,the main hydrophilic compound was found to be echinacoside (2)[4,10,11] (Fig. 1A). Small amounts of caftaric acid (1) and cichoricacid (4) have also been described [4,10,11] (Fig. 1A). The main bio-logical activities of these phenolic compounds include antiviral,anti-inflammatory and antioxidant effects [1,2].

Alkamides have been isolated and characterized from E. pur-purea and E. angustifolia [12,13], while E. pallida was found tocontain mainly polyacetylenes and polyenes [6,7]. In the spe-

cific case of E. pallida, ten polyacetylenic and polyenic compoundswith 14 and 15 carbon atoms have been isolated and character-ized from the lipophilic root extract [6,7,14], and their structurehas been determined by means of the application of extensive

44 F. Pellati et al. / J. Chromatogr. A 1242 (2012) 43– 58

HO

HO

O

OO

RhaO

OGlu

OH

O

OH

OH

HO

HO

O

OCH

CH

COOH

O

COOHO

OH

OH

HO

HO

O

O CH

COOH

COOH

OH

O

OH

HO

HO

Caftaric acid(1)

Echinacoside (2)

Cichoric acid(4)

Caffeic acid(3)

(A)

(B)

8-Hydroxy-tetradec-(9 E)-ene -11 ,13-diyn-2-one (5)

O OH O OH O OH

8-Hydroxy-pentad eca-(9 E,13Z)-dien -11 -yn-2-on e (7)

O O O O

Pentade c-(9 E)-ene -11,13 -diyne-2,8-dion e (8)

O O O

Pentade c-(8 Z)-en e-11 ,13-diyn-2-one (11)

Pentade ca-(8Z,13Z)-dien-11-yn-2-one (12)

OO

Pen tad eca-(8Z,11Z)- dien -2-on e (13)

Pen tad ec- (8Z)-en-2-one (14)

8-Hydroxy-pentad ec-(9E)-ene -11 ,13 -diyn-2-on e (6)

Pentadeca-(9E,13Z)-di en-11-yne-2,8-dion e (9)

Tetradec-(8Z)-ene-11,13 -diyn-2-one (10)

F B) Chm

s[miam

pbt2

ig. 1. (A) Chemical structures of caffeic acid derivatives (1–4) in E. pallida roots. (onocarbonylic acetylenes (10–12) and alkenes (13, 14) in E. pallida roots.

pectroscopic (UV, IR, NMR) and spectrometric (MS) techniques6,7,14]. On the basis of the functional groups, these secondary

etabolites can be classified in four chemical classes (Fig. 1B),ncluding three hydroxylated acetylenes (5–7), two dicarbonyliccetylenes (8, 9), three monocarbonylic acetylenes (10–12) and twoonocarbonylic alkenes (13, 14).One of the above mentioned monocarbonyl compounds, namely

entadeca-(8Z,13Z)-dien-11-yn-2-one (12), has been found toe characterized by a good cytotoxic activity, in particular onhe colonic COLO320 cancer cell line, with an IC50 value of.3 ± 0.3 �M after 72 h of exposure, in comparison with the

emical structures of hydroxylated acetylenes (5–7), dicarbonylic acetylenes (8, 9),

anticancer drug 5-fluorouracil (5-FU), currently used in therapy,which presents an IC50 value of 8.7 ± 0.2 �M [15]. This com-pound has also shown an IC50 value of 2.5 ± 0.7 �M on the breastcarcinoma MCF-7 cell line, in comparison with 5-FU with anIC50 value 3.4 ± 0.5 �M [16]. It has also been demonstrated thatthis acetylene is able to arrest the cell cycle in the G1 phase[16] and to induce apoptosis, by increasing the activity of the

caspases 3/7 and the DNA fragmentation [15]. Regarding the poten-tial bioavailability, this secondary metabolite was found to beable to cross the membrane of Caco-2 cells, which representsan optimal in vitro model of intestinal absorption, indicating a

atogr

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afmMealtodnoMiaTTspqafi

2

2

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2

p

F. Pellati et al. / J. Chrom

otential good bioavailability in humans after oral administration15].

In the literature, several HPLC methods have been described forhe determination of polar compounds in Echinacea plant materialnd natural products [4,10,11]. Regarding E. pallida non-polar sec-ndary metabolites, a RP-HPLC-UV/DAD method, based on the usef a monolithic stationary phase, has been developed for the phy-ochemical analysis of polyacetylenes and polyenes in the plant

aterial and dietary supplements [7]. To the best of our knowl-dge, no chromatographic method for the simultaneous analysis ofolar and non-polar compounds in E. pallida root extracts and nat-ral products has been reported so far in the literature. Even if it

s more difficult to optimize the separation of such different con-tituents in one run, a method for the simultaneous determinationf both caffeic acid derivatives and polyacetylenes/polyenes in E.allida is useful because it can reduce both the time and the sampleize required for the analysis. In addition, a technique able to pro-ide a complete fingerprint of the secondary metabolites is of greatnterest in the ambit of phytochemical analysis and quality controlf raw material and derivatives.

In this context, this study aims at developing a novel, reliablend fully validated method for the simultaneous analysis of caf-eic acid derivatives and polyacetylenes/polyenes in E. pallida plant

aterial and natural products by HPLC-UV/DAD, HPLC-ESI-MS andS/MS using ion trap (IT) and triple quadrupole (TQ) mass analyz-

rs. The chromatographic conditions were optimized so that caffeiccid derivatives eluted in the first step of the chromatogram, fol-owed by polyacetylenes/polyenes. The sequential elution of thesewo classes of compounds is useful because it allows the parametersf the mass spectrometers to be optimized for these structurallyifferent compounds. A second novel aspect of the proposed tech-ique is that identification of secondary metabolites was carriedut on the basis of UV and MS spectra. The combination of MS andS/MS data with retention times and UV spectra made the peak

dentification very reliable. The fragmentation patterns of caffeiccid derivatives and polyacetylenes/polyenes obtained by IT andQ are discussed in detail for the first time in the present work.he oxidation mechanism of monocarbonylic acetylenes is demon-trated for the first time in this study using MS and MS/MS data. Theractical applicability of the technique was demonstrated by theuantitative analysis of E. pallida root extracts and commerciallyvailable dietary supplements to provide reliable chromatographicngerprints of their bioactive secondary metabolites.

. Experimental

.1. Plant material

Authentic dried roots (600 g) of E. pallida (Nutt.) Nutt. wereindly gifted by Dr. Ing. Gian Stefano Cestarollo, Padua, Italy, inebruary 2011. A voucher specimen of the plant material waseposited at the Botanical Garden of the University of Parma (Italy)accession number EchiPal01). The plant material was protectedrom light and humidity until required for chemical analysis. E. pal-ida roots were ground on an IKA M20 grinder (Staufen, Germany)efore extraction.

Four commercially available dietary supplements containingxtracts of E. pallida roots (capsules and liquid extract) were pur-hased from local pharmacies in Spring 2011. These products arendicated in the text as DS-1/DS-4, respectively.

.2. Chemicals and solvents

Caftaric acid (purity 98%) and cichoric acid (purity 99%) wereurchased from Chromadex (Santa Ana, CA, USA). Echinacoside

. A 1242 (2012) 43– 58 45

(purity ≥ 98%), caffeic acid (purity ≥ 98%), HPLC-grade methanol(MeOH), ethanol (EtOH) acetonitrile (ACN), n-hexane, ethyl acetate(EtOAc), chloroform (CHCl3), formic acid (HCOOH) and sodium sul-fate (Na2SO4) were from Sigma–Aldrich (Milan, Italy). Water waspurified using a Milli-Q PLUS 185 system from Millipore (Milford,MA, USA).

Silica gel column chromatography was performed with Kiesel-gel 60 (40–63 �m, Merck, Darmstadt, Germany). Reversed-phasecolumn chromatography was carried out with LiChroprep RP-18(40–63 �m, Merck).

2.3. Extraction, fractionation and isolation ofpolyacetylenic/polyenic compounds

Powdered dried roots of E. pallida (600 g) were extracted ina Soxhlet apparatus for 16 h using n-hexane (6 L). The extractwas evaporated to dryness under vacuum to give a yellow oil(3.8 g). The extract was then dissolved in a small amount ofn-hexane and EtOAc (2:1, v/v), subjected to silica gel flash col-umn chromatography and eluted with n-hexane/EtOAc (2:1, v/v),affording 140 fractions of 15.5 mL. Each fraction was analyzedby RP-HPLC and combined into 6 fractions (A–E), according tothe chromatographic profile. Fractions A–E were further purifiedby reversed-phase flash column chromatography on LiChroprepRP-18, using H2O/ACN as the mobile phase. After preparative RPchromatography, the eluate containing the compound of inter-est was concentrated under reduced pressure to give an aqueousresidue, which was extracted with CHCl3 (3 × 15 mL). The com-bined organic phases were dried over anhydrous Na2SO4; theresulting anhydrous solution was then filtered and concentratedto dryness under vacuum to give the purified secondary metabo-lite, i.e. compound 5 (6.4 mg from fraction E), 7 (13.8 mg fromfraction D), 10 (92.6 mg from fraction C), 12 (44.5 mg from frac-tion B), 13 (17.0 mg from fraction A) and 14 (105.8 mg fromfraction A).

The degree of purity of the isolated compounds was determinedby HPLC and was found to be higher than 90%. The spectroscopic(UV, IR, NMR) and spectrometric data of the purified secondarymetabolites were in good agreement with the literature [6,7,14].In particular, mono- and bi-dimensional NMR spectra, based on1H, 13C, COSY, HSQC-DEPT, HMBC and NOESY experiments, wereacquired in CDCl3 and compared with data reported by Pellati et al.[6] for compounds 6–9 and 12, Pellati et al. [7] for compounds5, 10 and 14, and Morandi et al. [14] for compound 13. Purifiedcompounds were stored at low temperature (−80 ◦C) under argonatmosphere, protected from light and humidity.

2.4. Sample preparation for HPLC analysis

The pulverized roots (1.0 g) of E. pallida were extracted undermagnetic stirring with 10 mL of MeOH containing 0.1% HCOOHfor 20 min at room temperature. After centrifugation for 5 minat 4000 rpm, the supernatant solution was filtered under vacuumand the residue was re-extracted. The filtrates of the two extrac-tions were combined and concentrated to dryness under vacuumat 35 ◦C. Solvent was then added to the residue to 5 mL of final vol-ume. The extract was filtered through a 0.45-�m PTFE filter into aHPLC vial and capped. All the sample preparations were carried outin duplicate.

Regarding E. pallida dietary supplements (DS-1/DS-3), aweighed amount (1.0 g) of finely powdered material (from the con-

tents of 20 capsules) was extracted according to the procedurepreviously described. One milliliter of liquid formulation (DS-4)was diluted to 5 mL with the solvent, filtered and directly injectedinto the HPLC system.

46 F. Pellati et al. / J. Chromatogr. A 1242 (2012) 43– 58

Table 1Linearity and sensitivity data for E. pallida secondary metabolites.

Compound Linearity range(�g/mL)

Slope (a) Intercept (b) r LOD(�g/mL)

LOQ(�g/mL)

Caftaric acid (1) 3.07–153.40 29.99 (±0.32) −54.36 (±22.94) 0.9991 0.90 3.07Echinacoside (2) 9.06–906.00 7.30 (±0.04) 25.62 (±17.40) 0.9997 2.72 9.06Caffeic acid (3) 2.52–167.82 51.94 (±0.50) −67.28 (±36.56) 0.9991 0.75 2.52Cichoric acid (4) 2.72–136.20 38.65 (±0.17) −28.66 (±10.89) 0.9998 0.82 2.728-Hydroxy-tetradec-(9E)-ene-11,13-diyn-2-one (5) 9.32–466.00 4.88 (±0.01) 4.91 (±2.55) 0.9999 2.80 9.328-Hydroxy-pentadeca-(9E,13Z)-dien-11-yn-2-one (7) 3.58–119.40 18.88 (±0.06) −5.65 (±3.22) 0.9999 1.07 3.58Tetradec-(8Z)-ene-11,13-diyn-2-one (10) 12.52–313.00 6.46 (±0.03) 9.96 (±4.95) 0.9998 3.76 12.52Pentadeca-(8Z,13Z)-dien-11-yn-2-one (12) 2.64–132.20 28.00 (±0.13) 39.11 (±7.79) 0.9998 0.79 2.64Pentadeca-(8Z,11Z)-dien-2-one (13) 13.94–348.50 3.82 (±0.04) 20.79 (±7.18) 0.9992 4.18 13.94

1.6

E heret n in p

2

mamTf

(mH13tflTwcpw

2

(at

Fc

Pentadec-(8Z)-en-2-one (14) 16.38–327.50

xperimental conditions as in Section 2.5. For each curve the equation is y = ax + b, whe intercept and r is the correlation coefficient. Standard error (SE) values are give

.5. HPLC-UV/DAD conditions

Chromatography was performed using an Agilent Technologiesodular model 1100 system (Waldbronn, Germany), consisting of

vacuum degasser, a quaternary pump, an autosampler, a ther-ostatted column compartment and a diode array detector (DAD).

he chromatograms were recorded using an Agilent ChemStationor LC and LC–MS systems (Rev. B.01.03).

The analyses were carried out on an Ascentis C18 column250 mm × 4.6 mm I.D., 5 �m, Supelco, Bellefonte, PA, USA). The

obile phase was composed of (A) 0.1% HCOOH in H2O and (B) 0.1%COOH in ACN. The gradient elution was modified as follows: initial5% B; 0–10 min linear gradient from 15% to 30% B; 10–18 min from0% to 65% B, 18–25 min from 65% to 80% B, 25–30 min from 80%o 90% B, 30–45 min 90% B. The post-running time was 3 min. Theow rate was 1.0 mL/min. The column temperature was set at 30 ◦C.he sample injection volume was 5 �L. The DAD acquisition rangeas 190–450 nm and peak integration was performed at 210 (for

ompounds 10, 11, 13, 14), 226 (for compound 12), 264 (for com-ounds 5–7) and 332 nm (for compounds 1–4). Three injectionsere performed for each sample.

.6. HPLC-ESI-MS and MS/MS conditions

Analyses were performed using two HPLC-ESI-MS/MS systems:a) an Agilent Technologies modular 1200 system, equipped with

vacuum degasser, a binary pump, an autosampler, a thermostat-ed column compartment and a 6310A IT mass analyzer with an

0 5 10 15 20

mAU

0

200

400

600

800

1000

1200

1

2

3 4

5

0 5 10 15 20

mAU

0

200

400

600

800

1000

1200

1

2

3 4

ig. 2. Chromatogram obtained by HPLC-UV/DAD analysis of an E. pallida root extract aonditions as in Section 2.5.

3 (±0.02) 37.11 (±6.97) 0.9994 4.91 16.38

y is the peak area, x is the concentration of the analyte (�g/mL), a is the slope, b isarenthesis. The p value was <0.0001 for all calibration curves.

ESI ion source; (b) an Agilent Technologies modular 1200 system,equipped with a vacuum degasser, a binary pump, an autosampler,a thermostatted column compartment and a 6410B TQ mass ana-lyzer with an ESI ion source. The HPLC column and the appliedchromatographic conditions were the same as reported for theHPLC-UV/DAD system. The flow-rate was split 5:1 before the ESIsource.

For ESI-MS2 (IT), the parameters were set as follows: the capil-lary voltage was 3.5 kV, the nebulizer (N2) pressure was 20 psi, thedrying gas (N2) temperature was 350 ◦C, the drying gas flow was9 L/min and the skimmer voltage was 40 V. Data were acquired byAgilent 6300 Series Ion Trap LC/MS system software (version 6.2).The IT mass spectrometer was operated in the negative ion modeduring the first 17 min of analysis, then switched to the positive ionmode for the remainder analysis time (17–45 min). The full scanacquisition was performed in the m/z range 100–1700. MS2 spec-tra were automatically performed with helium as the collision gasin the m/z range 50–1700 by using the SmartFrag function.

For ESI-MS/MS (TQ), the parameters were set as follows: thecapillary voltage was 4.0 kV, the nebulizer (N2) pressure was 20 psi,the drying gas temperature was 300 ◦C, the drying gas flow was9 L/min and the fragmentor voltage was 90 V. Data were acquiredby Agilent MassHunter Workstation (Rev. B.02.01). TQ was usedin the full-scan negative (0–17 min) and positive (17–45 min) ion

modes in the m/z range 100–1700 and in the product ion scan (PIS)mode using nitrogen as the collision gas (with a collision energy(CE) of 30–35 V for caffeic acid derivatives in the negative ion modeand 5–15 V for polyacetylenes/polyenes in the positive ion mode).

min25 30 35

6

78 9

10

11

12 13 14

min25 30 35

A

B

t (A) 210 and (B) 332 nm. For peak identification, see Fig. 1A and B. Experimental

F. Pellati et al. / J. Chromatogr. A 1242 (2012) 43– 58 47

nm200 250 300 350 400

mAU

0

200

400

600

800

1000

1200

1400

nm200 225 250 275 300 325 350 375

mAU

0

20

40

60

80

100

120

nm200 225 250 275 300 325 350 375

mAU

0

10

20

30

40

50

60

nm200 225 250 275 300 325 350 375

mAU

0

10

20

30

40

50

nm200 225 250 275 300 325 350 375

mAU

0

100

200

300

400

500

600

700

800

nm200 225 250 275 300 325 350 375

mAU

0

50

100

150

200

nm200 225 250 275 300 325 350 375

mAU

0

100

200

300

400

500

A

nm200 225 250 275 300 325 350 375

mAU

0

50

100

150

200

250

300

350 B

D C

F E

H G

F olites:c peak i

2

f1oaotdciaI

ig. 3. Characteristic on-line UV absorption spectra of E. pallida secondary metabompound 9; (F) compounds 10, 11; (G) compound 12; (H) compounds 13, 14. For

.7. HPLC-UV/DAD method validation

The stock standard solution of each commercially available caf-eic acid derivative (1–4) and isolated polyacetylene/polyene (5, 7,0, 12–14) was prepared as follows: an accurately weighed amountf pure compound (0.7–4.5 mg for 1–4; 4.7–6.9 mg for 5, 7, 10nd 12–14) was placed into a 5-mL (for caffeic acid derivatives)r 10-mL (for polyacetylenes/polyenes) volumetric flask, respec-ively; MeOH with 0.1% HCOOH was added and the solution wasiluted to volume with the same solvent. The external standard

alibration curve was generated using five data points, cover-ng the concentration ranges reported in Table 1. Five-microliterliquots of each standard solution were used for HPLC analysis.njections were performed in triplicate for each concentration level.

(A) compounds 1–4; (B) compounds 5, 6; (C) compound 7; (D) compound 8; (E)dentification, see Fig. 1A and B.

The calibration curve was obtained by plotting the peak area ofthe compound at each level versus the concentration of the sam-ple.

The amount of secondary metabolites in E. pallida samples wasdetermined using these calibration curves, when the standard wasavailable. Compounds 6 and 11 were quantified using the calibra-tion curves of compounds with the same chromophore, i.e. 5 and 10,respectively, and their contents were corrected using the molecularweight ratio.

The limit of detection (LOD) of the method represents the ana-

lyte concentration that would yield a signal-to-noise (S/N) ratioof 3; the limit of quantification (LOQ) was evaluated consideringthe analyte concentration that would yield a S/N ratio of 10. TheLOD and LOQ values were experimentally verified by injections of

48 F. Pellati et al. / J. Chromatogr. A 1242 (2012) 43– 58

Table 2Caffeic acid derivatives identified in E. pallida root extracts by HPLC-UV/DAD, HPLC-ESI-MS and MS/MS.

Peak number Retention time(min)

Compound UV �max (nm) MS data (m/z) MS/MS data (m/z)a

Ion trap mass analyzer1 4.6 Caftaric acid 286, 330 623 [2M−H]−

311* [M−H]−179 (11) [C9H7O4]− , 149 (100) [C4H5O6]−

2 6.0 Echinacoside 302, 332 1571 [2M−H]−

785* [M−H]−

392* [M−2H]2−

623 (100) [C29H35O15]− or [C26H39O17]−

179b (23) [C9H7O4]− , 135b (100) [C8H7O2]−

3 7.2 Caffeic acid 298, 320 359 [2M−H]−

179* [M−H]−135 (100) [C8H7O2]−

4 10.3 Cichoric acid 300, 330 947 [2M−H]−

473* [M−H]−311 (14) [C13H11O9]− , 293 (7) [C13H9O8]− , 149 (48) [C4H5O6]− , 135(37) [C8H7O2]−

Triple quadrupole mass analyzer1 4.6 Caftaric acid 286, 330 311* [M−H]− 179 (35) [C9H7O4]− , 135 (100) [C8H7O2]−

2 6.0 Echinacoside 302, 332 1571 [2M−H]−

785* [M−H]−

392* [M−2H]2−

623 (79) [C29H35O15]− or [C26H39O17]− , 461 (10) [C20H29O12]− , 179 (6)[C9H7O4]− , 161 (100) [C9H5O3]−

179c (10) [C9H7O4]− , 161c (17) [C9H5O3]− , 135c (100) [C8H7O2]−

3 7.2 Caffeic acid 298, 320 179* [M−H]− 135 (100) [C8H7O2]−

4 10.3 Cichoric acid 300, 330 947 [2M−H]−

473* [M−H]−179 (32) [C9H7O4]− , 161 (27) [C9H5O3]− , 149 (29) [C4H5O6]− , 135(100) [C8H7O2]−

a The identified product ions were obtained from the fragmentation of the precursor ions marked with an asterisk. Relative intensities of product ions are in parentheses.the prn of th

st

topae

b The product ions at m/z 179 and 135 were obtained from the fragmentation of

c The product ions at m/z 179, 161 and 135 were obtained from the fragmentatio

tandard solutions of the compounds at the LOD and LOQ concen-rations.

The accuracy of the analytical procedure was evaluated usinghe recovery test: this involved the addition of known quantitiesf reference standard compounds to half the sample weight of E.

allida plant material. The fortified samples were then extractednd analyzed using the proposed HPLC method. The results werexpressed as percentage recovery values.

HO

HO

O

O

RhaO

OGl

Echinacoside

[M-H]- m/z 7

[M-2H]2- m/z

HO

HO

O

O

RhaO

OH

m/z 62

HOO

RhaO

OH

OH

O

m/z 4

-

HO

HO

O

OH

m/z 179

HC

m/z 161

HO

m/z 135HO

HO

CC

O

-CO2-H2O

--2Glu-Rha-PhA

CA moiety

-H-

-H-

-H-

HO

Fig. 4. Proposed fragmentation pathways of [M−H

ecursor ion at m/z 392.e precursor ion at m/z 392.

The precision of the chromatographic system was tested by per-forming intra- and inter-day multiple injections of an E. pallida rootextract, and then checking the %RSD of retention times and peakareas. Five injections were performed each day for three consecu-tive days.

The precision of the extraction procedure was validated byrepeating the extraction procedure on the same sample of E. pallidaroots. An aliquot of each extract was then injected and quantified.

O

u

OH

O

OH

OH (2)

85

392

O

OH

O

OH

OH3

OH

OH

-CA

61

HOO

RhaO

OGlu

OH

O

OH

OH m/z 623

CA

-H

Glu

PhA moiety-

-H-

-H-

-H-

]− and [M−2H]2− ions of echinacoside (2).

F. Pellati et al. / J. Chromatogr. A 1242 (2012) 43– 58 49

m/z

B

121.0

135.0 144.8

[(M+H)–2H2O]+

x102

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1 159.0

131.0117.0104.8

90.9 143.0

83.0 198.8109.055.1

67.1

171.0

216.7

60 80 100 120 140 160 180 200

A

143.1

145.2

131.1

93.2

20

80 100

121.1

40

60

80

100

Intens. [%]

120

[(M+H)–2H2O]+

159.1

117.1

109.2

0140 160 180 200 m/z

83.2

135.1

199.1

160.1

Fig. 5. Product ion spectra of [(M+H)−H2O]+ of 8-hydroxy-pentadeca-(9E,13Z)-dien-11-yn-2-one (7) isolated from E. pallida roots, using (A) IT and (B) TQ mass analyzers.The identified products ions are written in red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Tcs

ma

s2

3

3

pisoatathhf

his parameter was evaluated by repeating the extraction in dupli-ate on three different days with newly prepared mobile phase andamples.

The specificity of the method was tested by applying the HPLCethod to herbal formulations containing extracts of E. pallida roots

nd excipients.The stability was tested with an E. pallida root extract that was

tored in amber glass flasks at 4 ◦C and at room temperature (about5 ◦C) and analyzed every 12 h.

. Results and discussion

.1. Optimization of extraction and chromatographic conditions

In order to obtain quantitative extraction of both polar and non-olar secondary metabolites from E. pallida roots, several variables

nvolved in the procedure, including the extraction technique, theolvent, the sample-to-solvent ratio, time and repetitions wereptimized. Different extraction solvents, based on MeOH and H2Ot different proportions, were initially evaluated for their extrac-ion efficiency using dynamic maceration (with magnetic stirring)nd sonication. Dynamic maceration with MeOH was found to be

he best extraction method, allowing the highest yields of bothydrophilic and lipophilic compounds from E. pallida roots. MeOHas already been described in the literature as a suitable solvent

or the extraction of polyacetylenes from plant material [17,18]. In

this study, the extraction efficiency of both polar and non-polar sec-ondary metabolites was further increased using MeOH containing0.1% HCOOH as the extraction solvent. The sample-to-solvent ratiooptimization indicated that the highest yield was obtained usinga 1:10 (w/v) ratio. The optimization of extraction time and repe-titions suggested that magnetic stirring for 20 min twice was themost suitable procedure. A final concentration of the two combinedextracts to 5 mL volume was found to be suitable for the analy-sis of both polar and non-polar secondary metabolites in the plantextracts.

Since the applications of the proposed method involve boththe metabolite fingerprinting and the quality control of secondarymetabolites in E. pallida extracts, a conventional C18 column wasselected. Due to the complexity of the matrix, a 25 cm column wasused in order to obtain a satisfactory degree of separation of thecompounds. To determine the optimal chromatographic conditionsfor the separation of the target analytes, various linear gradients of0.1% HCOOH in H2O and 0.1% HCOOH in ACN were tested. The useof HCOOH in the mobile phase allowed to reduce the peak tailing ofphenolic compounds and to enhance the ionization of the analytesin the HPLC-ESI-MS experiments. Under the finally selected gradi-ent conditions described in Section 2.5, peaks were well separated

in a reasonable analysis time, thus allowing the simultaneous deter-mination of both polar and non-polar secondary metabolites in E.pallida extracts. Other tested gradients caused a poor separation ofsome peaks or extended run time.

50 F. Pellati et al. / J. Chromatogr. A 1242 (2012) 43– 58

Table 3Hydroxylated acetylenes identified in E. pallida root extracts by HPLC-UV/DAD, HPLC-ESI-MS and MS/MS.

Peak number Retention time (min) Compound UV �max (nm) MS data (m/z) MS/MS data (m/z)a

Ion trap mass analyzer5 20.0 8-Hydroxy-tetradec-

(9E)-ene-11,13-diyn-2-one

210, 238, 250, 264, 280 419 [M+(M−H2O)+H]+

401 [(M−H2O)2+H]+

241 [M+Na]+

201* [(M+H)−H2O]+

183 [(M+H)−2H2O]+

183 (69) [(M+H)−2H2O]+, 147 (4)[(M+H)−H2O−C4H6]+, 143 (100)[(M+H)−2H2O−C3H4]+, 129 (12)[(M+H)−2H2O−C4H6]+, 105 (3)[(M+H)−H2O−C7H12]+

6 21.0 8-Hydroxy-pentadec-(9E)-ene-11,13-diyn-2-one

212, 240, 252, 266, 282 429 [(M−H2O)2+H]+

255 [M+Na]+

215* [(M+H)−H2O]+

197 [(M+H)−2H2O]+

200 (3) [(M+H)−H2O−CH3]+, 197 (20)[(M+H)−2H2O]+, 171 (15)[(M+H)−2H2O−C2H2]+, 161 (5)[(M+H)−H2O−C4H6]+, 158 (10)[(M+H)−H2O−C3H5O]+, 157 (46)[(M+H)−2H2O−C3H4]+, 147 (19)[(M+H)−H2O−C5H8]+, 143 (6)[(M+H)−2H2O−C4H6]+, 133 (100)[(M+H)−H2O−C6H10]+, 129 (20)[(M+H)−2H2O−C5H8]+, 119 (35)[(M+H)−H2O−C7H12]+

7 21.5 8-Hydroxy-pentadeca-(9E,13Z)-dien-11-yn-2-one

264, 278 451 [M+(M−H2O)+H]+

433 [(M−H2O)2+H]+

257 [M+Na]+

217* [(M+H)−H2O]+

199 [(M+H)−2H2O]+

199 (100) [(M+H)−2H2O]+, 173 (3)[(M+H)−2H2O−C2H2]+, 160 (8)[(M+H)−H2O−C3H5O]+, 159 (68)[(M+H)−2H2O−C3H4]+, 145 (3)[(M+H)−2H2O−C4H6]+, 135 (4)[(M+H)−H2O−C6H10]+, 131 (10)[(M+H)−2H2O−C5H8]+, 121 (8)[(M+H)−H2O−C7H12]+, 117 (10)[(M+H)−2H2O−C6H10]+, 91 (5)[(M+H)−H2O−C8H14O]+ or[(M+H)−2H2O−C8H12]+

Triple quadrupole mass analyzer5 20.0 8-hydroxy-tetradec-

(9E)-ene-11,13-diyn-2-one

210, 238, 250, 264, 280 419 [M+(M−H2O)+H]+

401 [(M−H2O)2+H]+

241 [M+Na]+

233 [(M+H)−H2O+MeOH]+

219 [M+H]+

201* [(M+H)−H2O]+

183 [(M+H)−2H2O]+

186 (11) [(M+H)−H2O−CH3]+, 183 (9)[(M+H)−2H2O]+, 161 (8)[(M+H)−H2O−C3H4]+, 157 (26)[(M+H)−2H2O−C2H2]+, 147 (12)[(M+H)−H2O−C4H6]+, 143 (100)[(M+H)−2H2O−C3H4]+, 130 (8)[(M+H)−H2O−C4H7O]+, 119 (15)[(M+H)−H2O−C6H10]+, 115 (14)[(M+H)−2H2O−C5H8]+, 105 (19)[(M+H)−H2O−C7H12]+, 102 (8)[(M+H)−H2O−C6H11O]+, 75 (8)[(M+H)−H2O−C8H14O]+ or[(M+H)−2H2O−C8H12]+

6 21.0 8-Hydroxy-pentadec-(9E)-ene-11,13-diyn-2-one

212, 240, 252, 266, 282 447 [M+(M−H2O)+H]+

429 [(M−H2O)2+H]+

255 [M+Na]+

237 [(M+Na)−H2O]+

233 [M+H]+

219 [(M+Na)−2H2O]+

215* [(M+H)−H2O]+

197 [(M+H)−2H2O]+

200 (20) [(M+H)−H2O−CH3]+, 197 (33)[(M+H)−2H2O]+, 175 (18)[(M+H)−H2O−C3H4]+, 171 (21)[(M+H)−2H2O−C2H2]+, 158 (17)[(M+H)−H2O−C3H5O]+, 157 (67)[(M+H)−2H2O−C3H4]+, 147 (39)[(M+H)−H2O−C5H8]+, 143 (31)[(M+H)−2H2O−C4H6]+, 133 (40)[(M+H)−H2O−C6H10]+, 129 (47)[(M+H)−2H2O−C5H8]+, 119 (28)[(M+H)−H2O−C7H12]+, 116 (23)[(M+H)−H2O−C6H11O]+

7 21.5 8-Hydroxy-pentadeca-(9E,13Z)-dien-11-yn-2-one

264, 278 451 [M+(M−H2O)+H]+

433 [(M−H2O)2+H]+

257 [M+Na]+

239 [(M+Na)−H2O]+

235 [M+H]+

217* [(M+H)−H2O]+

199 [(M+H)−2H2O]+

199 (38) [(M+H)−2H2O]+, 173 (15)[(M+H)−2H2O−C2H2]+, 159 (100)[(M+H)−2H2O−C3H4]+, 149 (21)[(M+H)−H2O−C5H8]+, 145 (39)[(M+H)−2H2O−C4H6]+, 135 (56)[(M+H)−H2O−C6H10]+, 131 (91)[(M+H)−2H2O−C5H8]+, 121 (43)[(M+H)−H2O−C7H12]+, 118 (14)[(M+H)−H2O−C6H11O]+, 117 (75)[(M+H)−2H2O−C6H10]+, 91 (22)[(M+H)−H2O−C8H14O]+ or

+

rsor io

3e

3

a The identified product ions were obtained from the fragmentation of the precu

.2. Identification of the secondary metabolites in E. pallida

xtracts

The HPLC-UV/DAD analysis of an E. pallida root extract at 210 and32 nm indicated a complex composition, as shown in Fig. 2. The

[(M+H)−2H2O−C8H12]

ns marked with an asterisk. Relative intensities of product ions are in parentheses.

corresponding peak identification is shown in Fig. 1A and B. Con-

sidering the complexity of the sample, the overall chromatographicseparation can be considered satisfactory. In the chromatogram ofFig. 2, the first four peaks correspond to caffeic acid derivatives,which, due to their high polarity, eluted first under reversed-phase

F. Pellati et al. / J. Chromatogr

H3C

O OH

H2C

OH OH

H2C

OH

H3C

OH

-H2O

8-Hydroxy-pentadeca-(9E,13 Z)-dien-11-yn-2-one (7)m/z 235

-H2O

m/z 217 m/z 217

H+

H+

H2C

-H2O

m/z 19 9

91

135

121

159

117

145 131

160

91

F1

cp

i(

tu

Fw

ig. 6. Proposed fragmentation pathways of 8-hydroxy-pentadeca-(9E,13Z)-dien-1-yn-2-one (7).

onditions. The other peaks correspond to polyacetylenes andolyenes, which are less polar and, therefore, eluted later.

DAD was employed in the wavelength range 190–450 nm tonvestigate the UV spectra of the target secondary metabolites

Fig. 3).

It was found that 332 nm was the best wavelength for the detec-ion of caffeic acid derivatives (1–4). The UV spectra were alsoseful in the detection of polyacetylenes and polyenes, because

131.4

132.4

0.8

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.9

1 117.1

95.2

89.1

67.3 128.4105.34 14581.2

140 120 100 80 60

x102

131.4

132.4

ig. 7. Product ion spectrum of [M+H]+ of pentadec-(9E)-ene-11,13-diyne-2,8-dione (8)

ritten in red. (For interpretation of the references to color in this figure legend, the read

. A 1242 (2012) 43– 58 51

these compounds have typical chromophoric groups that exhibitedcharacteristic UV profiles [6,7]. Furthermore, the UV spectra of non-polar secondary metabolites allowed to identify the wavelengthswhere these compounds have the highest absorbance values. Poly-acetylenes containing an ene-diyne structure (5, 6) were confirmedby maximal absorption wavelengths at 210, 238, 250, 264 and280 [7,18], while compound 7 with an ene-yne-ene moiety wasobserved to have maximal absorption peaks at 264 and 278 nm[6,18]. The UV absorption maxima of compounds 8 and 9, occurringat 228, 274, 292 and 308 nm and 304 nm, respectively, were con-sistent with extended conjugation of the chromophore [6]. The UVspectra of compounds 10 and 11, characterized by a diyne moiety,had absorption maxima at 194, 228, 240 and 252 nm [7], while com-pound 12, with an ene-yne structure, showed absorption maximaat 226 and 234 nm [6]. The remaining monocarbonylic alkenes (13,14) displayed low UV absorption, with maxima at 194 nm [7,14].

However, retention time data and UV spectra alone could notprovide sufficient information for the correct identification of theconstituents in such a complex matrix. By using HPLC-ESI-MS andMS/MS, it was possible to obtain the quasi-molecular ions andthe product ions, which, in combination with retention times andUV data, made the identification of the target analytes very reli-able. When the reference standards were commercially availableor isolated from the plant material, the identification was furtherconfirmed by comparison of the MS and MS/MS data with those ofthe reference compounds.

The MS and MS/MS spectra of the analyzed samples indicatedthat the negative ion mode provided higher level of sensitivity forcaffeic acid derivatives, while the positive ion mode was more suit-able for polyacetylene and polyene detection. Therefore, the MSdetectors were set in the negative ion mode for the first stage of theanalysis (0–17 min); in the second stage (17–45 min), the detectorwas operated in the positive ion mode to facilitate the detection ofpolyacetylenes and polyenes.

3.2.1. Identification of caffeic acid derivativesThe mass spectra of caffeic acid derivatives in the negative ion

mode consisted of two main peaks, corresponding to the deproto-nated molecular ion [M−H]− and to a proton bound dimer [2M−H]−

of these molecules. In the case of echinacoside (2), a [M−2H]2− ionat m/z 392 was also observed, using both IT and TQ mass analyz-ers. MS/MS experiments from the [M−H]− ion of 2 generated the

predominant product ion at m/z 623 with both IT and TQ, whichcan be attributed either to the loss of a glucose or a caffeic acid(CA) moiety from the deprotonated molecular ion [19]. The furtherproduct ion at m/z 461 observed with TQ can be attributed to a loss

156.3

.3 198.4170.4 231.4

220 200 180 160 m/z

in E. pallida root extracts, using TQ mass analyzer. The identified products ions areer is referred to the web version of the article.)

52 F. Pellati et al. / J. Chromatogr. A 1242 (2012) 43– 58

Table 4Dicarbonylic acetylenes identified in E. pallida root extracts by HPLC-UV/DAD, HPLC-ESI-MS and MS/MS.

Peak number Retention time (min) Compound UV �max (nm) MS data (m/z) MS/MS data (m/z)a

Ion trap mass analyzer8 23.2 Pentadec-(9E)-ene-

11,13-diyne-2,8-dione

228,274, 292,308 253* [M+Na]+

231 [M+H]+

213 [(M+H)−H2O]+

235 (41) [(M+Na)−H2O]+, 195 (62) [(M+Na)−H2O−C3H4]+,153 (54) [(M+Na)−H2O−C6H10]+, 111 (41)[(M+Na)−H2O−C8H12O]+

9 23.6 Pentadeca-(9E,13Z)-dien-11-yne-2,8-dione

304 255 [M+Na]+

233* [M+H]+

215 [(M+H)−H2O]+

215 (52) [(M+H)−H2O]+, 200 (9) [(M+H)−H2O−CH3]+, 119(100) [(M+H)−H2O−C7H12]+

Triple quadrupole mass analyzer8 23.2 Pentadec-(9E)-ene-

11,13-diyne-2,8-dione

228, 274,292, 308 253 [M+Na]+

235 [(M+Na)−H2O]+

231* [M+H]+

213 [(M+H)−H2O]+

198 (6) [(M+H)−H2O−CH3]+, 170 (7)[(M+H)−H2O−C2H3O]+, 156 (8) [(M+H)−H2O−C3H5O]+,132 (20) [(M+H)−C6H11O]+, 128 (21)[(M+H)−H2O−C5H9O]+, 145 (9) [(M+H)−H2O−C5H8]+, 131(5) [(M+H)−H2O−C6H10]+, 117 (100)[(M+H)−H2O−C7H12]+, 89 (19) [(M+H)−H2O−C8H12O]+

9 23.6 Pentadeca-(9E,13Z)-dien-11-

304 255 [M+Na]+

233* [M+H]+

215 +

134 (47) [(M+H)−C6H11O]+, 119 (85)[(M+H)−H2O−C7H12]+, 91 (45) [(M+H)−H2O−C8H12O]+

rsor io

ooo2aCp

cl

t

yne-2,8-dione

a The identified product ions were obtained from the fragmentation of the precu

f a CA moiety from the ion at m/z 623. The product ion at m/z 161bserved with TQ can be assigned to a loss of H2O from a CA residueccurring at m/z 179. The MS/MS spectra of the [M−2H]2− ion of

with both IT and TQ showed two main product ions at m/z 179nd 135, corresponding to a CA residue with subsequent loss of aO2 molecule. The overall proposed scheme for the fragmentationattern of 2 is shown in Fig. 4.

The MS and MS/MS data of caftaric acid (1), caffeic acid (3) and

ichoric acid (4) were found to be in good agreement with theiterature [20,21].

The proposed structures of caffeic acid derivative fragments inhe MS and MS/MS spectra obtained by the analysis of E. pallida

-H2O

O O

H2C

OH OH

H2C

O

Pentadec-(9 E)-ene-11,13-di m/z 231

m/z 213

145

131

117

89

1

132

Fig. 8. Proposed fragmentation pathways of pen

[(M+H)−H2O]

ns marked with an asterisk. Relative intensities of product ions are in parentheses.

root extracts were assigned as shown in Table 2; a good matchwith those obtained from the reference standards was found.

3.2.2. Identification of polyacetylenes and polyenesRegarding hydroxylated acetylenes, the [M+H]+ protonated

molecular ions were not detectable or present at low level in thefull scan mass spectra of compounds 5–7, using both IT and TQ.The most abundant monomeric ion species for these analytes were

found to be those attributable to the loss of one H2O molecule fromthe protonated molecular ions [(M+H)−H2O]+ and to the [M+Na]+

adduct ions. Another monomeric ion species commonly observedin the mass spectra of these analytes was attributable to the loss

H+

H+

H3C

O

-H2O

yne-2,8-dione (8)

m/z 213

98

170

156

128

89

tadec-(9E)-ene-11,13-diyne-2,8-dione (8).

F. Pellati et al. / J. Chromatogr. A 1242 (2012) 43– 58 53

[(M+H)–H O]

69.3

97.2

111.2 121.1

135.1

147.1

161.1

191.1

201.1

0

20

40

60

80

100

Intens. [%]

60 80 100 120 140 160 180 200 m/z

149.1

133.1 119.1105.2 123.1 175.1

A

x102

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

169.0

161.0 218.9 109.0 120.9 92.9

80.9 135.255.1 201.0

172.9

60 80 100 120 140 160 180 200 220

[(M+H)–H O]

132.8

118.9 104.979.1

m/z

0

B

Fig. 9. Product ion spectra of [M+H]+ of pentadeca-(8Z,13Z)-dien-11-yn-2-one (12) isolated from E. pallida roots, using (A) IT and (B) TQ mass analyzers. The identifiedp s figur

o[hfoea[m

tfettbs(rTeBeclht

roducts ions are written in red. (For interpretation of the references to color in thi

f a further H2O molecule from the protonated molecular ions, i.e.(M+H)−2H2O]+. The loss of H2O from the protonated molecular ionas already been described in the literature as the primary loss for

alcarinol-type polyacetylenes from Daucus carota [22,23] and forenanthotoxin [17], a toxic polyalkyne present in Oenanthe crocataxtracts, in accordance with the readiness with which hydroxylatedcetylenes dehydrated during ionization. Dimeric ion species, i.e.(M−H2O)2+H]+ and [M+(M−H2O)+H]+, were also identified in the

ass spectra of compounds 5–7.Because of the low abundance of protonated molecular ions,

he MS/MS experiments for hydroxylated acetylenes 5–7 were per-ormed on the [(M+H)−H2O]+ ion species, which can be attributedither to the loss of a H2O molecule from the protonated enolautomer at C-2 of these compounds or to H2O elimination fromhe hydroxyl group located at C-8, which generates a doubleond between carbons C-7 and C-8. Fig. 5 shows the product ionpectra of the [(M+H)−H2O]+ ion of isolated 8-hydroxy-pentadeca-9E,13Z)-dien-11-yn-2-one (7), using IT and TQ mass analyzers,espectively. The proposed fragmentation pattern is shown in Fig. 6.he [(M+H)−H2O]+ precursor ion occurring at m/z 217 for 7 gen-rated a product ion at m/z 199, corresponding to [(M+H)−2H2O]+.oth the precursor and the product ion species of 7 originated sev-ral other product ions by means of the progressive heterolytic

leavage of the C C bonds of the aliphatic chain, which was high-ighted by the subsequent loss of CH2 units (−14 u). The twoomologous series of product ions identified in the MS/MS spec-ra of compound 7 (Fig. 6) are consistent with a fragmentation

e legend, the reader is referred to the web version of the article.)

pattern based on the preferential loss of a first H2O molecule fromthe protonated OH group of the enol form at C-2, followed by theelimination of a further H2O molecule from the OH group locatedat C-8.

The other hydroxylated acetylenes (5, 6) showed a similararray of fragments in their product ion spectra obtained from the[(M+H)−H2O]+ ions at m/z 201 and 215, respectively.

The MS and MS/MS data of compounds 5–7 in E. pallida rootextracts (Table 3) were found to be in agreement with thoseobtained from the analysis of the isolated secondary metabolitesused as reference.

Regarding dicarbonylic acetylenes (8, 9), their fragmentationpatterns were directly evaluated using the MS and MS/MS dataof these compounds in the plant extracts. In fact, due to thevery low content of these constituents in the raw material, itwas not possible to obtain a suitable amount of purified com-pounds for MS and MS/MS characterization. The IT and TQ fullscan MS spectra of compounds 8 and 9 indicated that the [M+H]+

and [M+Na]+ monomeric ion species were the most abundantones. Another additional monomeric ion species, correspondingto [(M+H)−H2O]+, was present in lower amounts. In the caseof pentadec-(9E)-ene-11,13-diyne-2,8-dione (8), the MS/MS spec-trum is discussed in detail for the TQ mass analyzer, which allowed

the fragmentation of the protonated molecular ion. Fig. 7 shows theproduct ion spectrum of the [M+H]+ ion at m/z 231 of compound 8using TQ and Fig. 8 shows the proposed fragmentation pattern. TheMS/MS spectrum of this molecule showed a series of fragments

5 atogr. A 1242 (2012) 43– 58

c[tmCsu

E

([1cai2imssl[li

am[

ea

3

a5mTtmmto

eca

famthps2wiDapstfo

O

-H2O

H2C

H2C

OH

Pentadeca-(8 Z,13Z)-dien-11-yn-2-one (12 )m/z 219

m/z 201

H+

H+

175

161

147

133

119

105

79

4 F. Pellati et al. / J. Chrom

orresponding to the breakdown of the aliphatic chain from the(M+H)−H2O]+ ion species. In the case of dicarbonylic acetylenes,he [(M+H)−H2O]+ ion can be rationalized by the loss of a H2O

olecule from the protonated enol tautomer either at C-2 or at-8. The [M+Na]+ adduct ion of compound 8 at m/z 253 showed theame fragmentation pattern in the product ion spectrum obtainedsing IT.

The complete MS and MS/MS data of dicarbonylic acetylenes in. pallida root extracts are shown in Table 4.

The IT and TQ full scan MS spectra of monocarbonylic acetylenes10–12) and alkenes (13, 14) showed predominant [M+H]+ andM+Na]+ monomeric ion species. With the exception of compound3, the [(M+H)−H2O]+ ions were present in lower amounts. Forompounds 10 and 12, dimeric ion species, such as [(M−H2O)2+H]+

nd [2M+H]+, were also observed. The MS/MS spectra of the [M+H]+

on of purified pentadeca-(8Z,13Z)-dien-11-yn-2-one (12) at m/z19 using IT and TQ showed a product ion at m/z 201, correspond-

ng to [(M+H)−H2O]+, which can be attributed to the loss of a H2Oolecule from the protonated enol tautomer located at C-2. The

ubsequent heterolytic cleavage of the aliphatic chain generatedeveral product ions, which are characterized by the progressiveoss of CH2 units. Fig. 9 shows the product ion spectra of theM+H]+ ion of isolated compound 12 using IT and TQ mass ana-yzers, respectively. The proposed fragmentation pattern is shownn Fig. 10.

Structurally related monocarbonylic acetylenes (10, 11) andlkenes (13, 14) isolated from E. pallida roots showed a similar frag-entation pathway in their product ion spectra obtained from the

M+H]+ ions at m/z 203, 217, 223 and 225, respectively.The MS and MS/MS data of compounds 10–14 in E. pallida root

xtracts (Table 5) were in agreement with those obtained from thenalysis of the purified reference compounds.

.2.3. Identification of the hydroperoxide intermediatePrevious studies have shown that hydroxylated acetylenes 5–7

re not present in E. pallida fresh roots [6,7]. However, compounds–7 have been detected at high level when E. pallida ground plantaterial was stored for several days before the extraction [6,7].

herefore, compounds 5–7 have been considered as “artifacts”, dueo an allylic oxidation reaction of the parent compounds 10–12 with

olecular oxygen [6,7,24]. The dicarbonylic compounds 8 and 9ight result from a further oxidation of the hydroxylated deriva-

ives. Fig. 11 shows the proposed scheme for the oxidation processf monocarbonylic acetylenes.

The allylic oxidation reaction is quite slow in E. pallida crudextracts. On the other hand, the reaction is rapid for the purifiedompounds 10–12, that should be stored under Ar at low temper-ture (−80 ◦C).

The oxidation mechanism that leads to hydroxylated acetylenesrom the parent monocarbonylic secondary metabolites has beenttributed to the formation of a very unstable hydroperoxide inter-ediate [24]. In this study, the application of MS and MS/MS

echniques allowed for the first time the identification of aydroperoxide intermediate from a methanolic solution of com-ound 12, stored for several days at room temperature. In the fullcan spectrum recorded on IT, the [(M+H)−H2O]+ ion species at m/z33 of 8-hydroperoxy-pentadeca-(9E,13Z)-dien-11-yn-2-one (15)as found to be the base peak; [M+H]+ and [M+Na]+ monomeric

on species at m/z 251 and 273, respectively, were also detected.imeric ion species were observed and assigned to [(M−H2O)2+H]+

t m/z 465 and [M+(M−H2O)+H]+ at m/z 483, respectively. Theroduct ion spectrum of [(M+H)−H2O]+ ion of 15 (Fig. 12) was con-

istent with the loss of a further H2O molecule, which originatedhe [(M+H)−2H2O]+ base peak at m/z 215. In agreement with theragmentation pathways previously described, the first loss of H2Occurred more likely at the protonated OH group at C-2, while the

Fig. 10. Proposed fragmentation pathway of pentadeca-(8Z,13Z)-dien-11-yn-2-one(12).

second one was speculated to be derived from the OOH group atC-8 (Fig. 13). As shown in Fig. 13, most of the product ions observedin the MS/MS spectrum originated from the heterolytic breakdownof the aliphatic chains of the ion species at m/z 233 and 215.

The results of this study demonstrated for the first time thatthe allylic oxidation of compound 12 proceeds via a hydroperoxideintermediate, which is then reduced to the corresponding alcohol.In this way, the double bond with Z configuration between car-bons C-8 and C-9 is shifted in the position 9 with E configuration inthe artifacts. Fig. 14 shows the proposed mechanism for the allylicoxidation of E. pallida monocarbonylic acetylenes.

3.3. Method validation

HPLC-UV/DAD was selected for quantitative analysis of sec-ondary metabolites in E. pallida samples, in view of the higheravailability and use of this equipment in the phytochemical analysisand quality control of natural products. The method validation wasperformed to show compliance with international requirements

for analytical techniques for the quality control of pharmaceuticals(ICH guidelines) [25].

As shown in Table 1, good linearity was observed for bothhydrophilic and lipophilic secondary metabolites over the tested

F. Pellati et al. / J. Chromatogr. A 1242 (2012) 43– 58 55

Table 5Monocarbonylic acetylenes and alkenes identified in E. pallida root extracts by HPLC-UV/DAD, HPLC-ESI-MS and MS/MS.

Peak number Retention time (min) Compound UV �max (nm) MS data (m/z) MS/MS data (m/z)a

Ion trap mass analyzer10 25.0 Tetradec-(8Z)-ene-

11,13-diyn-2-one194, 228, 240, 252 405 [2M+H]+

369 [(M−H2O)2+H]+

225* [M+Na]+

203 [M+H]+

185 [(M+H)−H2O]+

145 [(M+H)−H2O−C3H4]+

207 (20) [(M+Na)−H2O]+, 182 (29)[(M+Na)−C2H3O]+, 181 (21)[(M+Na)−H2O−C2H2]+, 168 (16)[(M+Na)−C3H5O]+, 167 (26)[(M+Na)−H2O−C3H4]+, 153 (34)[(M+Na)−H2O−C4H6]+, 111 (10)[(M+Na)−H2O−C7H12]+

11 26.5 Pentadec-(8Z)-ene-11,13-diyn-2-one

192, 218, 226, 238, 252 239* [M+Na]+

217 [M+H]+

199 [(M+H)−H2O]+

159 [(M+H)−H2O−C3H4]+

221 (80) [(M+Na)−H2O]+, 195 (3)[(M+Na)−H2O−C2H2]+, 181 (5)[(M+Na)−H2O−C3H4]+, 153 (3)[(M+Na)−H2O−C5H8]+, 125 (58)[(M+Na)−H2O−C7H12]+

12 28.0 Pentadeca-(8Z,13Z)-dien-11-yn-2-one

226,234 437 [2M+H]+

260 [M+H+CH3CN]+

241 [M+Na]+

219* [M+H]+

201 [(M+H)−H2O]+

201 (100) [(M+H)−H2O]+, 175 (9)[(M+H)−H2O−C2H2]+, 161 (39)[(M+H)−H2O−C3H4]+, 147 (7)[(M+H)−H2O−C4H6]+, 133 (13)[(M+H)−H2O−C5H8]+, 119 (13)[(M+H)−H2O−C6H10]+, 105 (16)[(M+H)−H2O−C7H12]+, 79 (4)[(M+H)−H2O−C9H14]+

13 32.8 Pentedeca-(8Z,11Z)-dien-2-one

194 245* [M+Na]+

223 [M+H]+

205 [(M+H)−H2O]+

187 (14) [(M+Na)−H2O−C3H4]+, 91 (5)[(M+Na)−H2O−C10H16]+

14 35.9 Pentadec-(8Z)-en-2-one

194 413 [(M−H2O)2+H]+

247 [M+Na]+

225* [M+H]+

207 [(M+H)−H2O]+

207 (86) [(M+H)−H2O]+, 125 (14)[(M+H)−H2O−C6H10]+, 111 (34)[(M+H)−H2O−C7H12]+

Triple quadrupole mass analyzer10 25.0 Tetradec-(8Z)-ene-

11,13-diyn-2-one194, 228, 240, 252 405 [2M+H]+

225 [M+Na]+

203* [M+H]+

185 [(M+H)−H2O]+

145 [(M+H)−H2O−C3H4]+

185 (19) [(M+H)−H2O]+, 160 (6)[(M+H)−C2H3O]+, 145 (100)[(M+H)−H2O−C3H4]+, 117 (53)[(M+H)−H2O−C5H8]+, 103 (5)[(M+H)−H2O−C6H10]+

11 26.5 Pentadec-(8Z)-ene-11,13-diyn-2-one

192, 218, 226, 238, 252 239 [M+Na]+

221 [(M+Na)−H2O]+

217* [M+H]+

199 [(M+H)−H2O]+

159 [(M+H)−H2O−C3H4]+

199 (30) [(M+H)−H2O]+, 159 (84)[(M+H)−C3H4]+, 131 (86)[(M+H)−H2O−C5H8]+, 117 (56)[(M+H)−H2O−C6H10]+

12 28.0 Pentadeca-(8Z,13Z)-dien-11-yn-2-one

226,234 437 [2M+H]+

241 [M+Na]+

219* [M+H]+

201 [(M+H)−H2O]+

201 (19) [(M+H)−H2O]+, 175 (11)[(M+H)−H2O−C2H2]+, 161 (18)[(M+H)−H2O−C3H4]+, 133 (16)[(M+H)−H2O−C5H8]+, 119 (18)[(M+H)−H2O−C6H10]+, 105 (11)[(M+H)−H2O−C7H12]+, 79 (20)[(M+H)−H2O−C9H14]+

13 32.8 Pentedeca-(8Z,11Z)-dien-2-one

194 245 [M+Na]+

223* [M+H]+

205 [(M+H)−H2O]+

109 (86) [(M+H)−H2O−C7H12]+, 83 (61)[(M+H)−H2O−C9H14]+, 69 (64)[(M+H)−H2O−C10H16]+

14 35.9 Pentadec-(8Z)-en-2-one

194 247 [M+Na]+

225* [M+H]+207 (14) [(M+H)−H2O]+, 71 (8)[(M+H)−H O−C H ]+

rsor io

r0fi2i[sm

a The identified product ions were obtained from the fragmentation of the precu

anges (r > 0.999). The LOD value (Table 1) was in the range.75–2.72 �g/mL for caffeic acid derivatives and 0.79–4.91 �g/mLor polyacetylenes and polyenes. The LOQ value (Table 1) wasn the range 2.52–9.06 �g/mL for caffeic acid derivatives and.64–16.38 �g/mL for polyacetylenes and polyenes. These sensitiv-

ty values were found to be in good agreement with the literature

4,7,11] and indicate that the proposed HPLC-UV/DAD method isufficiently sensitive for the determination of the target secondaryetabolites in E. pallida samples.

O OO

R

Fig. 11. Proposed oxidation process

207 [(M+H)−H2O]+2 10 16

ns marked with an asterisk. Relative intensities of product ions are in parentheses.

With regard to accuracy (Table 6), the percentage recovery val-ues were found to be in the range 97.9–101.7% for hydrophiliccompounds and 95.2–107.0% for lipophilic secondary metabolites.Considering the results of the recovery test, the developed methodcan be considered accurate.

The low intra- and inter-day %RSD values for retention times

(≤0.6), peak areas (≤1.5) and content (SD for echinacoside ≤0.5)indicate the high precision of both the chromatographic systemand the extraction procedure.

H

R

O O

R

of monocarbonylic acetylenes.

56 F. Pellati et al. / J. Chromatogr. A 1242 (2012) 43– 58

83.2

95.1

105.1 119.1

137.0

143.0

157.0

175.0

189.1

197.0

205.1

215.0

0

20

40

60

80

100

Intens. [%]

80 100 120 140 160 180 200 220 m/z

[(M+H)–2H2O]+

133.0

147.0

151.0

161.0165.0

Fig. 12. Product ion spectrum of [(M+H)−H2O]+ of 8-hydroperoxy-pentadeca-(9E,13Z)-written in red. (For interpretation of the references to color in this figure legend, the read

Table 6Recovery data of E. pallida secondary metabolites.

Compound Recovery (%,mean ± RSD, n = 3)

Caftaric acid (1) 97.9 ± 1.1Echinacoside (2) 101.7 ± 0.1Caffeic acid (3) 100.4 ± 0.3Cichoric acid (4) 101.3 ± 0.58-Hydroxy-tetradec-(9E)-ene-11,13-diyn-2-one (5) 98.6 ± 1.28-Hydroxy-pentadeca-(9E,13Z)-dien-11-yn-2-one (7) 100.3 ± 0.6Tetradec-(8Z)-ene-11,13-diyn-2-one (10) 95.2 ± 0.5Pentadeca-(8Z,13Z)-dien-11-yn-2-one (12) 105.8 ± 0.4Pentadeca-(8Z,11Z)-dien-2-one (13) 107.0 ± 2.3Pentadec-(8Z)-en-2-one (14) 99.1 ± 0.6

ER

piFap

ad

TCm

11111

o

xperimental condition as in Section 2.5.SD (%) = (SD/mean) × 100.

The chromatograms obtained by the analysis of commercialroducts indicated that the HPLC method can discriminate E. pall-

da components from the other constituents of the formulations.urthermore, peak purity tests were performed using the dioderray detector to demonstrate that the analyte chromatographic

eak was pure and not attributable to more than one component.

Regarding stability, the analytes in solution did not show anyppreciable change in the chromatographic profile over 72 h andegradation products were not detected.

able 7ontent of caffeic acid derivatives and polyacetylenes/polyenes in E. pallida plant materg/g).a

Peak number Compound name Plant ma

1 Caftaric acid 0.06 ± 0.02 Echinacoside 4.53 ± 0.43 Caffeic acid 0.07 ± 0.04 Cichoric acid 0.09 ± 0.05 8-Hydroxy-tetradec-(9E)-ene-11,13-diyn-2-one 0.58 ± 0.06 8-Hydroxy-pentadec-(9E)-ene-11,13-diyn-2-one 0.28 ± 0.07 8-Hydroxy-pentadeca-(9E,13Z)-dien-11-yn-2-one 0.20c

0 Tetradec-(8Z)-ene-11,13-diyn-2-one 0.69 ± 0.01 Pentadec-(8Z)-ene-11,13-diyn-2-one 0.11 ± 0.02 Pentadeca-(8Z,13Z)-dien-11-yn-2-one 0.30c

3 Pentedeca-(8Z,11Z)-dien-2-one 1.45 ± 0.04 Pentadec-(8Z)-en-2-one 1.85 ± 0.1

Total caffeic acid derivatives 4.75 ± 0.4Total polyacetylenes/polyenes 5.46 ± 0.2

a Data are expressed as mean (n = 6) ± standard deviation (SD). In the case of the plantf the capsule content.b Data are expressed as mg/mL.c SD < 0.005.

dien-11-yn-2-one (15), using IT mass analyzer. The identified products ions areer is referred to the web version of the article.)

The validation data highlighted the suitability of the proposedmethod for the quali- and quantitative analysis of E. pallida sec-ondary metabolites.

3.4. Analysis of E. pallida plant material and natural products

The validated method was applied for the quantitative analy-sis of hydrophilic and lipophilic secondary metabolites in E. pallidaroots and dietary supplements. Data are shown in Table 7 andexpressed as mg/g of dry weight.

The total amount of caffeic acid derivatives in E. pallida rootswas 4.75 ± 0.44 mg/g, while that of total polyacetylenes/polyeneswas 5.46 ± 0.23 mg/g. Echinacoside (4) was found to be the mainconstituent among caffeic acid derivatives; its content in theplant material (4.53 ± 0.49 mg/g) was lower in comparison withprevious reports [4,10,11]. 8-Hydroxy-tetradec-(9E)-ene-11,13-diyn-2-one (5) was the main compound (0.58 ± 0.04 mg/g) inthe group of hydroxylated acetylenes (5–7). Tetradec-(8Z)-ene-11,13-diyn-2-one (10), pentedeca-(8Z,11Z)-dien-2-one (13) andpentadec-(8Z)-en-2-one (14) were the most abundant constituents

in the class of monocarbonylic acetylenes (10–12) and alkenes (13,14). The content of individual polyacetylenes and polyenes in E.pallida roots was of the same order of magnitude of that reportedin a previous study [7].

ial and dietary supplements (DS-1/DS-4) by HPLC-UV/DAD (data are expressed as

terial DS-1 DS-2 DS-3 DS-4b

2 0.25 ± 0.02 0.25 ± 0.04 0.28 ± 0.04 0.09c

9 10.91 ± 1.60 1.99 ± 0.04 4.61 ± 0.15 1.73 ± 0.072 0.15 ± 0.01 0.03c 0.08c 0.02c

1 0.14 ± 0.01 0.04c 0.08c 0.03c

4 0.73 ± 0.04 0.27c 0.74 ± 0.01 0.17c

2 1.23 ± 0.02 0.31 ± 0.01 – 0.08c

0.89 ± 0.03 0.19c – 0.07c

3 5.59 ± 0.18 0.65 ± 0.01 – 0.60 ± 0.013 1.53 ± 0.05 0.11c – 0.12c

4.07 ± 0.12 0.28 ± 0.01 1.41 ± 0.04 0.26c

9 10.11 ± 0.60 2.91 ± 0.15 2.75 ± 0.02 0.31 ± 0.010 6.39 ± 0.44 4.31 ± 0.16 1.48 ± 0.12 0.15 ± 0.01

4 11.45 ± 1.64 2.31 ± 0.06 5.05 ± 0.17 1.87 ± 0.083 30.54 ± 0.87 9.03 ± 0.24 6.38 ± 0.18 1.76 ± 0.04

material, n = 18. Regarding dietary supplements, the results are expressed as mg/g

F. Pellati et al. / J. Chromatogr

O OO H

H2C

OH OO H

H2C

OOH

H+

H+

-H2O

8-Hydroperoxy-pentadeca-(9E,13Z)- dien-11- yn-2 -one (15) m/z 25 1

m/z 233

-H2O

H2C

O

m/z 21 5

165

151

137

175

161

147

133

119

189

Fd

tc1fasoao

F

[

[

ig. 13. Proposed fragmentation pathway of 8-hydroperoxy-pentadeca-(9E,13Z)-ien-11-yn-2-one (15).

The analysis of commercial products showed differences inhe quantitative composition of the compounds of interest: theontent of total caffeic acid derivatives ranged from 2.31 to1.45 mg/g and the level of total polyacetylenes and polyenesrom 6.38 to 30.54 mg/g. In particular, the preparation indicateds DS-1 contained high amounts of both hydrophilic and lipophilicecondary metabolites, whereas DS-4 displayed low level. The

ther samples (DS-2 and DS-3) contained an intermediate level ofctive compounds. It is well-known that the chemical compositionf natural products can differ significantly: even the same plant

R'

H

H

RH

HO

O

R'

OOH

H

R

H

R'

OH

H

R

H

ig. 14. Proposed mechanism for the allylic oxidation of monocarbonylic acetylenes.

[[[

[

[

[

[

[

. A 1242 (2012) 43– 58 57

material may vary depending on genetic variation and envi-ronmental factors; furthermore, drying temperature, extractionmethods, formulations and storage conditions may be the causesof the variability in the composition.

4. Conclusion

A HPLC method for the simultaneous analysis of caffeic acidderivatives, polyacetylenes and polyenes in E. pallida natural prod-ucts has been developed for the first time. Detailed MS and MS/MSdata of hydrophilic and lipophilic constituents in the plant extractsare provided. Under the applied conditions, both IT and TQ massanalyzers allowed a good fragmentation degree of the target ana-lytes and, therefore, produced useful structural information. UsingMS detection, a hydroperoxide intermediate was found to beinvolved in the oxidation process of monocarbonylic acetylenes.

This method represents an improvement on existing ones,because the MS/MS fragmentation patterns of the secondarymetabolites were used for peak identification. Through the on-line spectroscopic and spectrometric data of both hydrophilic andlipophilic compounds, this method allows a complete identificationand standardization of the plant material. The validation proce-dure and the application to real samples indicated that this methodaffords reliable analysis and is appropriate for metabolite finger-printing and quality control of complex matrices, such as E. pallidaroots, extracts and dietary supplements.

Acknowledgments

The authors acknowledge Banca Popolare dell’Emilia Romagna(BPER) and CPL Concordia for the financial support. The authors areparticularly grateful to Prof. Fabio Prati (Department of Chemistry,University of Modena and Reggio Emilia) for valuable discussionsduring this work and Dr. Diego Pinetti (Centro InterdipartimentaleGrandi Strumenti, CIGS, University of Modena and Reggio Emilia)for his technical support. The authors are also grateful to Dr. Ing.Gian Stefano Cestarollo (Padua, Italy) for kindly providing E. pallidaraw plant material.

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