Artemisia annua L.: Essential oil and acetone extract composition and antioxidant capacity

Indus ial C ops and P oduc s 45 (2013) 170– 181 Con en s lis s a ailable a SciVe se ScienceDi ec Indus ial C ops and P oduc s jou na l h o me pag e: www.else ie .com/loca e/indc op A emisia annua L.: Essen ial oil and ace one ex ac composi ion and an ioxidan capaci y Sand a C. Gou eia∗, Paula C. Cas ilho Cen o de Química da Madei a, CCCEE, Uni e sidade da Madei a, Campus Uni e si á io da Pen eada, piso 0, 9000-390 Funchal, Po ugal a i c l e i n o A icle his o y: Recei ed 26 May 2012 Recei ed in e ised o m 21 Sep embe 2012 Accep ed 17 Decembe 2012 Keywo ds: A emisia annua Ca eoylquinic acids Phenolic Essen ial oil An ioxidan a b s a c Ae ial pa s o A emisia annua g ow h in h ee di e en loca ions o Madei a A chipelago we e s udied. The essen ial oil composi ion was es ablished by GC-MS and he main componen s we e mono- and sesqui e penes; a emisia ke one was no de ec ed. The p esence o phenolic compounds in he ace one ex ac s was in es iga ed by HPLC-DAD-ESI/MSn and a di e sified phenolic p ofile o 40 hyd ocinnamic acid de i a i es and glycosyla ed fla onoids was ound. A ew compounds we e epo ed o he fi s ime in A emisia annua. The an ioxidan capaci y o essen ial oils and ex ac s we e measu ed by h ee di e en in i o assays. Fo he essen ial oils, a e y good an ioxidan esponse was ound and he ex ac s also showed a good an ioxidan capaci y, in pa icula as an i adical sca enge s. © 2012 Else ie B.V. All igh s ese ed. 1. In oduc ion The genus A emisia ( amily o As e aceae) includes mo e han 300 species, mainly small he bs and sh ubs (Yoon e al., 2011). The e a e se e al epo s (Ca alho e al., 2011) desc ibing A emisia plan s as die a y oods and as adi ional he bal medicines agains inflamma o y diseases, in ec ions by ungi, bac e ia and i uses, gas ic ulce , cance among o he s. One o he mos s udied A emisia plan s is A emisia annua L., commonly known as “qinghao” o “annual wo mwood”. I is a plan used o many cen u ies in Chinese olk medicine o he ea men o mala ia and e e and se e al bioac i e me abo- li es ha e been epo ed, he mos impo an o hem being a emisinin, an endope oxide sesqui e pene lac one (Cas ilho e al., 2008). A emisia annua lea es ha e a high con en o essen ial oil (EO) con aining cineole, ␣-pinene, camphene, bo neol, campho , ge mac ene-D and a emisia ke one (Bo a and Sha ma, 2011). The essen ial oil o A emisia annua is e e enced as ha ing an i ungal and an imic obial ac i i y (Ju eau e al., 2002; Ve ma e al., 2011). In addi ion o hese, o he biological p ope ies a e associa ed o mo e pola ex ac s o A emisia annua such ∗Co esponding au ho a : Kemihuse , Linnaeus äg 10, KB6A14, Umeå uni e - si e , SE-901 87 Umeå, Sweden. Tel.: +46 907865182. E-mail add ess: [email p o ec ed] (S.C. Gou eia). as an imala ial, an ibac e ial, an i-inflamma o y, an i umo and an iulce ogenic (Bhakuni e al., 2001; ´ Ca a e al., 2012). The an ioxidan capaci y e e ed in he li e a u e is associa ed o he high fla onoid con en and di e sified ypes o compounds (B isibe e al., 2009; Fe ei a e al., 2010). Also, in i o an i- mala ial and an icance ac i i y o a emisinin and i s de i a i es is enhanced by he p esence ce ain fla onoids (Fe ei a e al., 2010). The phenolic composi ion o A emisia annua has been desc ibed using HPLC-MS/MS echniques by (Lai e al., 2007), (Han e al., 2008) and (Ca bona a e al., 2012); Ca alho and co-wo ke s (Ca alho e al., 2011) epo ed he HPLC-DAD quan ifica ion o se e al phe- nolic compounds. The main phenolic compounds epo ed we e fla onoids and hyd oxycinnama es. Based on li e a u e su ey, he chemical composi ion and biological p ope ies o A emisia annua can ex en- si ely di e acco ding o hei geog aphical o igin and how he plan ma e ial is p ocessed. The in e es in he po en ial applica- ions o his plan is s ill inc easing and in he p esen s udy we analysed A emisia annua plan s g own in Madei a A chipelago, p e iously epo ed as ha ing ca. 1% o a emisin (Cas ilho e al., 2008). The main goals we e o es ablish he phenolic composi ion o c ude ace one and me hanol ex ac s by high-pe o mance liquid ch oma og aphy wi h diode a ay de ec ion coupled wi h mass spec ome y (HPLC-DAD-ESI/MSn); he EO composi ion by GC- MS and he an ioxidan capaci y o he EO and he ex ac s we e e alua ed by h ee di e en assays (DPPH, ABTS and FRAP). 0926-6690/$ – see on ma e © 2012 Else ie B.V. All igh s ese ed. h p://dx.doi.o g/10.1016/j.indc op.2012.12.022 S.C. Gou eia, P.C. Cas ilho / Indus ial C ops and P oduc s 45 (2013) 170– 181 171 2. Ma e ials and me hods 2.1. Chemicals and s anda ds The ollowing eagen s we e pu chased om Me ck (Da ms ad , Ge many): disodium phospha e dodecahyd a ed (99%), po assium pe sul a e (99%), e ous sul a e hep ahyd a e (99%), glacial ace ic acid (100%), sodium ca bona e (p.a.) and sodium chlo ide (99.5%). 2,2-diphenyl-1-pic ylhyd azyl (DPPH) (>95%), T olox (≥99.8%, HPLC), 2,2azinobis-(3-e hylbenz hiazoline-6-sul onic acid) (ABTS) (≥99%, HPLC), 2,4,6-T i(2-Py idyl)-s- iazine (TPTZ) (≥99.0%, TLC) and Folin-Ciocal eu’s phenol eagen we e pu chased om Fluka (Lisbon, Po ugal). Po assium chlo ide (>99.5%), gallic acid (99%, HPLC), po assium ace a e (p.a.), u in (≥98%, HPLC) and e ic chlo ide hexahyd a e (97–100%) we e pu chased om Pan eac (Ba celona, Spain); po assium dihyd ogen phospha e (99.5%), alu- minium chlo ide (98%) and sodium ace a e ihyd a e (pu e) we e pu chased om Riedel-de Haën (Hano e , Ge many). All sol en s used o plan ex ac ion we e AR g ade, pu chased om Fishe (Lisbon, Po ugal). HPLC-MS g ade ace- oni ile (99.9%, LabScan, Gliwice, Poland) and ul a-pu e wa e (Milli-Q Wa e s pu ifica ion sys em, EUA) we e used o HPLC analysis. S anda ds p epa ed in e hanol (100 ␮g/mL): apigenin (>99%), lu eolin (>99%), que ce in (>99%), p-couma ic acid (>99%) and ca - eic acid (>99%) om Ex asyn hese (Lyon, F ance), kaemp e ol (>99%) and 5-O-ca eoylquinic acid (99%) om Ac os O ganics (Geel, Belgium). 1,3-O-dica eoylquinic acid, 1,5-O-dica eoylquinic acid, 3,4-O-dica eoylquinic acid, 3,5-O-dica eoylquinic acid, 4,5- O-dica eoylquinic acid and 3,4,5-O- ica eoylquinic acid (>98% by HPLC o all) we e ob ained om Chengdo Biopu i y Phy ochemi- cals, L d China (Sichuan, China). 2.2. Plan ma e ial and sample p epa a ion Samples o A emisia annua we e ob ained om seeds o a emisinin ich hyb ids (CPQBA × POP) kindly o e ed by UNI- CAMP, B azil and cul i a ed in h ee di e en loca ions o Madei a A chipelago (P eces, Ja dim Bo ânico and Po o San o). Be o e flowe ing, he whole plan s we e cu and d ied. Plan s g own in Madei a Bo anical Ga den (Ja dim Bo ânico) expe imen- al field eached abou 3.5 m high. These plan s we e di ided in o wo lo s and one o hem was d ied unde di ec sunligh in o de o maximize a emisinin p oduc ion (Cas ilho e al., 2008). Lea es om he op hi d o plan s we e p ocessed sepa a ely om hose o he es o he plan . Vouche s we e deposi ed in he Madei a Bo anical Ga den He ba ium collec ion. The d ied plan ma e ial was g ound o fine powde in a mechanic g inde . Po ions o his powde ed plan we e sepa a ely ex ac ed by solid liquid ex ac ion, using wo sol en s (ace one and me hanol): The plan ma e ial (50 g) was mace a ed wi h 500 mL o sol en , a oom empe a u e, o 24 h. In all cases, he solu ions we e fil e ed and concen a ed unde educed p essu e in a o a y e apo a o (40 ◦C) and kep in he da k a −20 ◦C un il es ed. Essen ial oils we e ob ained by hyd odis illa ion in a Cle enge ype appa a us. 2.3. Essen ial oil analysis by GC-MS analysis Quali a i e and quan i a i e composi ion o essen ial oils was pe o med by GC and GC-MS analysis, using a HP 5889 se ies II and a Va ian Sa u n 3 ion ap sys em, espec i ely in bo h cases, columns DB-5 (30 m × 0.15 mm × 0.25 ␮m; J. W. Scien ific) we e used; helium N60 was used as ca ie gas. The analysis condi ions we e: ini ial empe a u e 40 ◦C o 14 min, g adien o 1.5 ◦C/min up o 250 ◦C, iso he m o 10 min. Injec o (and ans e line) empe a u e was 270 ◦C. 1 ␮L was injec ed in spli less mode. GC-MS analysis was pe o med by ESI in a mass ange 24 o 400 m/z wi h a delay ime o 2 min. Iden ifica ion o componen s was pe o med by compa ing he mass spec a o hose o he a ailable NIST da abase (GC-MS) and by hei ela i e e en ion index (Ko acs Index, KI) owa ds a mix u e o alkanes C4 o C26, injec ed in he same expe imen al condi ions (GC-FID). 2.4. HPLC-DAD-ESI/MSnanalysis 2.4.1. Liquid ch oma og aphy S ock solu ions wi h concen a ions (m/ ) o 5 mg/mL we e p epa ed by dissol ing each d ied ex ac in ini ial HPLC mobile phase (Ace oni ile/Wa e (20/80, / )). These solu ions we e fil- e ed h ough 0.45 mm Nylon mic opo e memb anes p io o use and 10 ␮L we e injec ed o HPLC-DAD-ESI/MSnanalysis. Th ee independen assays we e pe o med o each sample. The HPLC sepa a ion was ca ied ou on a Dionex ul ima e 3000 se ies ins umen (Cali o nia, EUA) coupled o a bina y pump, a diode-a ay de ec o (DAD), an au osample and a column com- pa men . Samples we e sepa a ed on a Phenomenex Gemini C18 col- umn (5 ␮m, 250 mm × 3.0 mm i.d.; Phenomenex) wi h a sample injec ion olume o 10 ␮L. The mobile phase was composed o ace- oni ile (A) and wa e / o mic acid (100/0.1, / ) (B). A g adien p og am was used as ollows: 20% A (0 min), 25% A (10 min), 25% A (20 min), 50% A (40 min), 100% A (42–47 min), 20% A (49–55 min). The mobile phase flow a e was 0.4 mL/min; he ch oma og am was eco ded a 280 nm and 350 nm and spec al da a o all peaks we e accumula ed in he ange o 190–400 nm. Column empe a u e was con olled a 30 ◦C. 2.4.2. Mass spec ome y Fo HPLC-ESI/MSnanalysis, a model 6000 ion ap mass spec- ome e (B uke Esqui e, B emen, Ge many) fi ed wi h an ESI sou ce ope a ing in he nega i e mode was used. Da a acquisi ion and p ocessing we e pe o med using Esqui e con ol so wa e. Nega i e ion mass spec a o he column elua e we e eco ded in he ange m/z 100–1000 a a scan speed o 13,000 Da/s. High pu i y ni ogen (N2) was used bo h as d ying gas a a flow o 10.0 mL/min and as a nebulizing gas a a p essu e o 50 psi. The nebulize em- pe a u e was se a 365 ◦C and a po en ial o +400 V was used on he capilla y. Ul a-high-pu i y helium (He) was used as collision gas a a p essu e o 1 × 10−5mba and he collision ene gy was se a 40 V. The acquisi ion o MSnda a was made in au o MSnmode, wi h an isola ion wid h o 4.0 m/z. Fo MSnanalysis, he mass spec ome e was scanned om 10 o 1000 m/z wi h agmen a ion ampli ude o 1.0 V (MSnup o MS4) and wo p ecu so ions. 2.5. To al phenolic con en (TPC) The o al phenolic con en he ex ac s was de e mined ollow- ing he Folin-Ciocal eu me hod (Zheng and Wang, 2001) wi h some modifica ions (Gou eia and Cas ilho). Each sample aliquo o 50 ␮L (10 mg/mL, w/ ) was mixed wi h 1.25 mL o Folin-Ciocal eu eagen (dilu ed 1:10 old) and 1 mL o 7.5% sodium ca bona e solu ion. The mix u e was incuba ed o 30 min a oom empe a u e and hen abso bance was measu ed a  = 765 nm. The final esul s we e exp essed as millig ams o gallic acid equi alen s pe 100 g o d ied plan (mg GAE/100 g). 172 S.C. Gou eia, P.C. Cas ilho / Indus ial C ops and P oduc s 45 (2013) 170– 181 2.6. DPPH adical sca enging ac i i y Fo he DPPH assay, 100 ␮L o he sample solu ions (10 mg/mL) we e added o 3.5 mL o a 0.06 mM me hanol DPPH adical solu- ion (Gou eia and Cas ilho, 2012a, b). The dec ease in abso bance a  = 516 nm was measu ed du ing 30 min. The DPPH adical sca enging e ec o he ex ac s was exp essed as ␮mol T olox equi alen pe 100 g am o d ied plan (␮mol eq. T olox/100 g d ied plan ) o plan ex ac s and as ␮mol T olox equi alen pe mL o essen ial oil (␮mol eq. T olox/mL EO) o essen ial oils. 2.7. ABTS•+ adical sca enging ac i i y The ABTS•+ adical wo king solu ion was p epa ed by mixing 50 mL o 2 mM ABTS•+solu ion wi h 200 ␮L o 70 mM po assium pe sul a e solu ion (Gou eia and Cas ilho, 2012a,b). This mix u e was kep in he da k o 16 h a oom empe a u e. Fo each analysis, he ABTS•+solu ion was dilu ed wi h pH 7.4 phospha e bu e ed saline (PBS) solu ion o an ini ial abso bance o 0.700 ± 0.021 a 734 nm. This solu ion was eshly p epa ed o each analysis. Fo he assessmen o he adical sca enging ac i i y, an aliquo o 100 ␮L (10 mg/mL, w/ ) was added o 1.8 mL o ABTS•+wo king solu ion and he abso bance dec ease, a a  = 734 nm, was eco ded du ing 6 min. Resul s we e exp essed as ␮mol T olox equi alen pe 100 g o d ied plan (␮mol eq. T olox/100 g d ied plan ) o plan ex ac s and as ␮mol T olox equi alen pe mL o essen ial oil (␮mol eq. T olox/mL EO) o essen ial oils. 2.8. Fe ic educing ac i i y (FRAP assay) The e ic educing abili y o he ex ac s was measu ed based on he FRAP assay (Benzie and S ain, 1996). FRAP eagen was p epa ed daily by mixing 2.5 mL o solu ion e ic ichlo ide hexa- hyd a e (20 mM), 2.5 mL o solu ion TPTZ (10 mM in 40 mM o hyd ochlo ic acid) and 25 mL o ace a e bu e 0.3 M (pH 3.6) and incuba ing a 37 ◦C. Fo each analysis, 30 ␮L o me hanolic solu- ion (1 mg/mL, w/ ) we e added o 180 ␮L o dis illed wa e and 1.8 mL o FRAP solu ion. The inc ease o abso bance was eco ded a  = 593 nm in 15 s in e als, du ing 30 min a 37 ◦C. The FRAP esul s we e exp essed as mmol I on(II) sul a e hep ahyd a e pe mg o d ied plan (mmol Fe(II)/mg) o plan ex ac s and as mmol I on(II) sul a e hep ahyd a e pe mL o essen ial oil (mmol Fe(II)/mL EO) o essen ial oils. 3. Resul s and discussion 3.1. Essen ial oil composi ion The chemical composi ion o essen ial oils ob ained om d ied lea es is p esen ed in Table 1. Simila esul s ha e been published by Magalhães e al. (2004), who p oduced his hyb id and cul i a ed i in B azil. Compa ing hese se s o esul s i was clea ha he composi ion o essen ial oil was no subs an ially a ec ed by clima e, loca ion o cul i a - ing condi ion bu i is in insic o he plan . E en d ying in di ec sunshine o shade did no a ec much he essen ial oil composi ion, wi h a li le loss o he mo e ola ile componen s being obse ed o he plan d ied unde sunligh . The CPQBA × POP hyb id has no hu- jone de i a i es o a emisia ke one, ha m ul compounds no mally p esen in wild- ype A emisia annua L. (´ Ca a e al., 2012; Reale e al., 2011). The only possible oublesome, in e ms o sa e y, com- ponen could be campho (ca. 40%). The pai 1,8-cineole/campho as he wo majo componen s in essen ial oils has been ound no only in A emisia annua (´ Ca a e al., 2012; Holm e al., 1997; M R, 2009) bu in se e al o he A emisia subspecies (Ko dali e al., 2005; Lopes-Lu z e al., 2008; Shang e al., 2012). 3.2. Phenolic composi ion by HPLC-DAD-ESI/MSn Compa ison be ween he HPLC-DAD-ESI/MSnsc eening o me hanolic and ace one ex ac s showed ha , o he me hanolic ex ac , he numbe o peaks de ec ed was lowe and wi h educed ch oma og aphic esolu ion leading o a poo agmen a ion o he de ec ed compounds. This can be associa ed o ma ix e ec s and high molecula weigh compounds common in mo e pola sol- en s ex ac ions. Se e al au ho s (Kalli h aka e al., 1995; Ko eka e al., 2011) ha e e alua ed me hanol e sus ace one as ex ac ion sol en s o phenolic compounds wi h clea ad an age o ace- one. Ou choice o his sol en esul ed om ou p e ious wo k whe e i was he bes o ex ac a emisinin. The ace one ex ac s om plan s cul i a ed in h ee di e en loca ions we e e y sim- ila be ween hem, hus we only p esen he ch oma og am and agmen a ion da a o one o hem (P eces). The HPLC-DAD-ESI/MSnbase peak ch oma og am p ofile o ace one ex ac is shown in Fig. 1. The iden ifica ion and cha ac e iza ion o he de ec ed com- pounds was made by compa ison o he HPLC e en ion, UV and mass spec a wi h hose o s anda d compounds. Since only a limi ed numbe o s anda d compounds was a ailable, s uc u es o unknown compounds we e es ablished la gely based on hei andem MSn agmen a ion beha iou . Among he de ec ed compounds, we ound ypical hyd oxycin- namic acid UV abso p ions (max 230–240, 300 sh. 320–340 nm) and fla onoids cha ac e is ic UV abso p ions: fla onols de i a- i es exhibi ed wo maximum abso p ions a 250–270 nm and 320–360 nm, de i ed om he aglycone A and B ings, espec i ely. Peaks co esponding o fla ones conjuga es showed h ee abso p- ions a 210–230 nm, 250–280 nm and 330–350 nm (Mab y e al., 1970). In ou s udy, 38 compounds we e en a i ely iden ified and some o hem a e epo ed o he fi s ime o A emisia annua. Fig. 2 ep esen s he chemical s uc u es o main classes o compounds de ec ed in A emisia annua. Table 2 shows he expe imen al analy ical da a: e en ion ime ( R), wa eleng h o maximum abso bance (max), dep o ona ed molecula ions [M–H]−, and mos impo an MSn agmen ions o each peak. Usually, he base peak in he MS1spec um was assigned as he dep o ona ed molecula ion [M–H]−. When isome s we e obse ed, hei iden ifica ion was pe o med based on li e a u e da a. 3.2.1. Iden ifica ion o fla onoids The occu ence o fla onoids, in pa icula hose conjuga ed wi h one o mo e suga moie ies, in A emisia species has been p e iously epo ed (Fe ei a e al., 2010, Gou eia and Cas ilho, Han e al., 2008; Lai e al., 2007). In HPLC-ESI/MSnsc eening o A emisia annua, mainly apigenin and que ce in conjuga es ha e been epo ed. In he p esen wo k, in addi ion o hese wo ypes o aglycones, lu eolin, iso hamne in and kaemp e ol de i a i es compounds we e also iden ified. The andem MS/MS expe imen s o he fla onoids esul ed on he dep o ona ed molecula ion ([M–H]−) and he dep o ona ed aglycone ion (Y− 0) as a esul o he loss o he suga uni . Mos o he de ec ed fla onoids we e in hei glycosyla ed o m and/o es e ified wi h acyl g oups. Bu wo aglycones we e de ec ed on i s ee o m – lu eolin and apigenin. 3.2.1.1. F ee aglycones (36 and 40). Compound 36 ( R= 27.7 min) was iden ified as lu eolin by compa ison wi h a e e ence s anda d (da a no shown). In he MSnexpe imen s he [M–H]−ion a m/z 285 ga e se e al ypical lu eolin agmen ions a m/z 199, 217 ([M- H-C3O2]−), 175 ([M-H-C3O2-C2H2O]−) and 241 ([M-H-CO2]−). Compound 40 ( R= 33.9 min) exhibi ed a [M–H]−ion a m/z 269 and he i s MSn agmen a ion p esen ed main agmen ions a S.C. Gou eia, P.C. Cas ilho / Indus ial C ops and P oduc s 45 (2013) 170– 181 173 Table 1 Composi ion o he essen ial oil (EO) om lea es o A emisia annuag ow h in P eces, Po o San o and Ja dimBo ânico (d ied unde di ec sunligh and in he shade). KI e *KI Compound P eces Po o San o Ja dimBo ânico Shade Ja dimBo ânico Sunshine 937 937 ␣-pinene 3.58 1.55 3.19 2.79 953 954 camphene 0.28 0.01 0.23 0.31 990 902 ␤-pinene 0.60 0.27 1.35 0.88 1018 1017.0 ␣- e pinene 0.68 0.68 0.77 0.78 1026 1027.0 p-cymene 3.32 1.23 2.10 3.31 1030 1031.0 limonene 0.23 0.05 0.39 0.24 1030 1035.0 1,8-cineole 11.6 6.54 9.87 7.92 1059 1061.0 ␥- e pinene 1.27 1.22 1.35 1.28 1141 1141.0 ans-pinoca eol 0.51 0.88 0.86 0.64 1143 1149.0 campho 42.8 45.5 42.0 44.33 1165 1173.0 bo neol 2.50 8.96 2.58 4.74 1177 1180.0 e pinen-4-ol 2.80 2.59 2.45 2.51 1194 1196.0 my enol 1.23 1.21 1.00 1.08 1217 1222.0 ans-ca eol 1.77 1.76 1.79 1.55 1242 1247.0 l-ca one 1.03 0.52 0.30 0.78 1345 1346.0 ␣-cubebene 0.10 0.10 0.08 0.13 1351 1353.0 eugenol 1.13 0.66 1.23 1.09 1376 1373.0 ␣-copaene 1.75 0.17 0.82 1.15 1389 1388.0 ␤-cubebene 1.93 1.31 1.02 1.66 1394 1392.0 cis-jasmone 0.24 0.72 0.24 0.48 1428 1417.0 ␤-ca yophyllene 7.77 10.21 6.44 8.65 1444 1452.0 ␣-humulene 0.46 0.94 6.70 0.94 1480 1479.0 ge mac ene-D 7.15 8.41 8.61 7.77 1485 1486.0 ␤-selinene 1.93 3.09 2.78 3.04 1494 1493.0 bicycloge mac ene 3.29 1.4 1.86 1.95 *See h p://www.phe obase.com/da abase/ko a s/ko a s-index.php. m/z 225 ([M-.H-CO2]−), 151 (1,3A−) and 149 (1,4B−+ 2H). Compa ing hese esul s o hose ob ained o a s anda d solu ion o apigenin, 40 was iden ified as ee apigenin. Two ypes o fla onoid glycosila ion we e obse ed: O- glycosila ion and C-glycosila ion. The iden ifica ion o hese wo g oups was based on he andem MSn agmen a ion expe imen s. Fo C-glycosyla ed compounds he suga uni is linked o he aglycone wi h a ca bon-ca bon bond ha is esis an o up u e. The ep esen a i e MSn agmen s o C-glycosyla ed fla onoids a e ela ed o he suga pa and occu s a m/z 0,2 X−[M-H-120]−,0,3 X−[M-H-90]−and 0,2 X−[M-H-60]−(Cuyckens and Claeys, 2004). The non-obse a ion o dep o ona ed aglycone ion, Y− 0, makes iden- ifica ion p ocess ha de . Fo O-glycosyla ed compounds he suga moie ies a e los as neu al agmen s and he fla onoid s uc u e is mo e easily iden- ified due o aglycone bonds clea ages. 3.2.1.2. C-glycosides (7, 8, 9, 12 and 13). Fo hese compounds UV spec a showed maximum abso p ions bands a  = 225, 272 and 314 nm iden ical o cha ac e is ics abso p ions bands o fla ones (Mab y e al., 1970). Howe e , he andem MSnexpe imen s e ealed a di e en pa e n o ha known o O-glycosyla ed fla ones. Compound 7 ( R= 5.4 min) and 8 ( R= 5.8 min) showed a [M- H]−ions a m/z 563. The MS2 agmen a ion e ealed a beha iou ypical o he asymme ical di-C-glycosides wi h agmen ions a [M-H-210]−, [M-H-90]−and [M-H-60]−. The neu al loss o 60 Da (0,3X−) indica es he p esence o a pen ose moie y. Compound 8 has been p e iously desc ibed by ou g oup in A emisia a gen ea (Gou eia and Cas ilho, 2011a,b) and was cha - ac e ized as apigenin-6-C-hexoside-8-C-pen oside. Compound 7 simila o 8 had MS3 agmen ions a m/z 353 [0,2Xi0,2Xj]−and 383 [0,2Xi0,3Xj]−sugges ing apigenin as he aglycone (Vukics e al., 2008). Howe e , con a ily o compound 8, 7 did no p esen an in ense [M-H-60]−ion a m/z 503 (2.9% o base peak). Based on hese e idences and knowing ha 6-C-pen oside-8-C-hexoside elu es be o e he iso- me 6-C-hexoside-8-C-pen oside, compound 7 was iden ified as apigenin-6-C-pen oside-8-C-hexoside. Fig. 1. HPLC-DAD-ESI/MSnanalysis o he ace one ex ac s o A emisia annua o al ae ial pa s – HPLC-MS nega i e ion ESI/MSnbase peak ch oma og am (BPC). 174 S.C. Gou eia, P.C. Cas ilho / Indus ial C ops and P oduc s 45 (2013) 170– 181 Table 2 Cha ac e iza ion o phenolic componen s o he ace one ex ac om A emisia annua by HPLC-DAD-ESI/MSn. No. R(min) Iden ifica ion max (nm) [M-H]−(m/z) HPLC-DAD-ESI/MSnm/z (% base peak) 1 2.8 Ca eic acid hexoside de i a i e 275, 304 473 MS2[473]: 342 (14.4), 341 (100), 179 (13.8), 161 (13.8), 131 (29.2) MS3[473 → 341]: 179 (100), 161 (43.7), 143 (10.8), 119 (44.2), 101 (11.3) MS4[473 → 341 → 179]: 119 (92.0), 113 (54.0), 101 (63.2), 89 (100) 2 3.1 Quinic acid - 191 MS2[191]: 173 (74.9), 127 (100), 111 (47.0), 109 (69.7), 85 (70.7) MS3[191 → 127]: 163 (30.8), 109 (69.7), 99 (100) 3 4.0 Kaemp e ol-3,7-di-O- hexoside - 609 MS2[609]: 449 (18.2), 448 (11.6), 447 (100), 285 (34.9) MS3[609 → 447]: 327 (7.6), 284 (73.0), 285 (100), 255 (62.3) MS4[609 → 447 → 284]: 256 (31.1), 255 (100), 151 (34.2) 4 4.4 3-O-Ca eoylquinic acid 241, 300, 324 353 MS2[353]: 191 (100), 179 (51.4), 135 (15.9) MS3[353 → 191]: 173 (100), 171 (16.9), 127 (56.8), 111 (30.2), 109 (39.2), 85 (37.4) MS4[353 → 191 → 127]: 109 (100), 99 (90.9), 85 (12.9) 5*5.0 5-O-Ca eoylquinic acid 242, 300, 325 353 MS2[707]: 353 (100) MS2[353]: 191 (100) MS3[707 → 353]: 191 (100) MS3[353 → 191]: 173 (57.1), 127 (100), 109 (55.9), 85 (97.7) MS4[707 → 353 → 191]: 173 (100), 127 (63.7), 135 (21.8), 111 (17.4) MS4[353 → 191 → 127]: 109 (100) 6 5.2 4-O-Ca eoylquinic acid 240, 300, 323 353 MS2[353]: 191 (18.4), 179 (58.3), 173 (100), 135 (8) MS3[353 → 173]: 111 (30.8), 93 (100) 7 5.4 Apigenin-6-C- pen oside-8-C- hexoside. 224, 273, 314 563 MS2[563]: 503 (2.9), 473 (98.0), 443 (78.2), 383 (83.5), 353 (100) MS3[563 → 353]: 326 (13.4), 325 (100), 297 (78.2) MS4[563 → 352 → 325]: 298 (5.1), 297 (100) 8 5.9 Apigenin-6-C- hexoside-8-C- pen oside. 224, 273, 314 563 MS2[563]: 503 (59.3), 473 (68.6), 443 (100), 383 (63.0), 353 (63.2) MS3[563 → 443]: 383 (50.0), 354 (12.0), 353 (100) MS4[563 → 443 → 353]: 326 (35.1), 325 (100), 297 (56.2) 9 6.5 Apigenin-6-C- hamnoside-8-C- hexoside 224, 272, 313 577 MS2[577]: 559 (21.9), 503 (2.8), 487 (28.3), 473 (40.0), 457 (53.1), 353 (100) MS3[577 → 353]: 326 (18.3), 325 (100), 297 (71.2) MS4[577 → 353 → 325]: 297 (100) 10 6.9 3-O-Fe uloylquinic acid 326 367 MS2[367]: 193 (100), 191 (1.5), 173 (3.7) MS3[367 → 193]: 149 (31.9), 134 (100), 109 (10.3) 11 7.4 5-O-Fe uloylquinic acid 327 367 MS2[367]: 191 (100) MS3[367 → 191]: 179 (100), 134 (53.0), 111 (68.6), 127 (55.5), 109 (12.7), 85 (93.0) 12 7.9 Apigenin-6-C- hexoside-8-C- hamnoside 224, 272, 313 577 MS2[577]: 487 (41.2), 473 (2.7), 457 (100), 353 (36.9) MS3[577 → 457]: 383 (18.5), 354 (33.0), 353 (100) MS4[577 → 457 → 353]: 326 (55.5), 325 (100), 298 (22.0) 13 8.3 Apigenin-8-C-hexoside 224, 272, 314 431 MS2[431]: 341 (23.1), 311 (100) MS3[431 → 311]: 284 (38.1), 283 (100) MS4[431 → 311 → 283]: 283 (100), 224 (77.3), 163 (23.7) 14 8.8 Que ce in-O- dihexoside – 625 MS2[625]: 302 (17.1), 301 (100), 300 (19.1) MS3[625 → 301]: 271 (22.9), 212 (10.8), 179 (82.9), 151 (100) MS4[625 → 301 → 151]: 169 (100), 107 (20.2) 15 9.1 Lu eolin-7-O-hexoside – 447 MS2[447]: 286 (14.0), 285 (100) MS3 [447 → 285]: 285 (100), 241 (24.9), 176 (19.0), 175 (80.1), 149 (117.5) 16 9.3 Mea nse in-O- hexoside 257, 300, 342 493 MS2[493]: 332 (19.0), 331 (100), 330 (10.3), 316 (11.7) MS3[493 → 331]: 317 (12.8), 316 (100), 315 (16.3) MS4[493 → 331 → 316]: 287 (49.6), 271 (46.1), 229 (38.2), 166 (100) 17*9.5 Que ce in-3-O- glucoside 258, 353 463 MS2[463]: 302 (17.1), 301 (100), 300 (19.1) MS3[463 → 301]: 271 (22.9), 212 (10.8), 179 (82.9), 151 (100) MS4[463 → 301 → 151]: 169 (100), 107 (20.2) 18 9.8 Iso hamne in-O- hexoside 256, 270, 344 477 MS2[477]: 316 (17.1), 315 (100), 300 (29.3) S.C. Gou eia, P.C. Cas ilho / Indus ial C ops and P oduc s 45 (2013) 170– 181 175 Table 2 (Con inued) No. R(min) Iden ifica ion max (nm) [M-H]−(m/z) HPLC-DAD-ESI/MSnm/z (% base peak) MS3[477 → 315]: 301 (11.4), 300 (100) MS4[477 → 315 → 300]: 284 (62.1), 271 (67.5), 245 (63.1), 229 (100), 213 (61.9) 19*11.8 3,4-O-Dica eoylquinic acid 246, 299, 325 515 MS2[515]: 354 (17.5), 353 (100), 335 (12.3), 299 (12.2), 173 (21.8) MS3[515 → 353]: 191 (24.8), 179 (60.9), 173 (100), 135 (14.8) MS4[515 → 353 → 173]: 155 (20.8), 111 (100), 93 (61.1) 20*12.5 1,5-O-Dica eoylquinic acid 243, 300, 328 515 MS2[515]: 353 (100) MS3[515 → 353]: 191 (100) MS4[515 → 353 → 191]: 173 (43.8), 127 (100), 109 (22.1) 21*12.9 3,5-O-Dica eoylquinic acid 242, 300, 328 515 MS2[515]: 354 (10.7), 353 (100) MS3[515 → 353]: 191 (100), 179 (53.9) MS4[515 → 353 → 191]: 173 (100), 127 (90.9), 93 (34.5) 22 13.1 Dica eoylquinic acid isome 240, 326 515 MS2[515]: 354 (15.8), 353 (100) MS3[515 → 353]: 191 (100), 179 (52.1), 135 (15.2) MS4[515 → 353 → 191]: 85 (100), 173 (76.6), 127 (66.8), 109 (76.6) 23*14.2 4,5-O-dica eoylquinic acid 243, 300, 327 515 MS2[515]: 354 (14.5), 353 (100) MS3[515 → 353]: 191 (31.5), 179 (53.9), 173 (100), MS4[515 → 353 → 179]: 155 (33.6), 111 (57.0), 93 (100) 24 14.6 Lu eolin-7-O-pen oside 417 MS2[417]: 285 (100), 284 (22.4) MS3[417 → 285]: 257 (5.1), 243 (72.1), 241 (54.1), 217 (93.1), 199 (98.2), 175 (100), 151 (39.2) 25 15.3 Dihyd oxy- dime hoxyl-O- hexoside fla one 491 MS2[491]: 371 (12.1), 330 (12.5), 329 (100), 314 (10.3) MS3[491 → 329]: 315 (21.1), 314 (100) MS4[491 → 329 → 314]: 300 (22.8), 299 (100) 26 16.8 3-p-O-Couma oyl-5-O- ca eoylquinic acid 499 MS2[499]: 337 (100), 221 (11.4), 179 (), 163 (14.2) MS3[499 → 337]: 179 (28.2), 163 (100), 135 (15.8) MS4[499 → 337 → 163]: 119 (100) 27 18.1 Unknown 423 MS2[423]: 262 (10.6), 261 (100), 173 (100) MS3[423 → 261]: 175 (11.6), 173 (100) MS4[423 → 261 → 173]: 93 (100) 28 18.9 1-O-Ca eoyl-5-O- e uloylquinic acid 328 529 MS2[529]: 368 (12.9), 367 (100), 353 (30.0), 191 (8.5) MS3[529 → 367]: 191 (100) MS4[529 → 367 → 191]: 173 (69.0), 127 (100), 109 (52.9) 29 20.2 1 o 5-O-ca eoyl-4-O- e uloylquinic acid – 529 MS2[529]: 368 (42.4), 367 (100), 173 (23.3) MS3[529 → 367]: 193 (81.5), 191 (28.5), 173 (100) MS4[529 → 367 → 173]: 137 (21.5), 111 (14.6), 93 (100) 30 20.6 3-O- e uloyl-5-O- ca eoylquinic acid 529 MS2[529]: 368 (2.8), 367 (100), 353 (18.4), 193 (21.4) MS3[529 → 367]: 194 (8.4), 193 (100), 191 (42.2), 135 (2.4) 31 22.2 3,4-O-di e uloylquinic acid 543 MS2[543]: 367 (19.5), 349 (100), 193 (3.8), 173 (12.4) MS3[543 → 349]: 287 (38.6), 193 (28.4), 175 (100), 155 (34.2) 32 23.2 3,5-O-di e uloylquinic acid 543 MS2[543]: 367 (100), 349 (38.2), 191 (18.28) MS3[543 → 367]: 193 (100), 173 (2.4) 33 23.8 Kaemp e ol-3-O- ca eoylhexoside – 609 MS2[609]: 447 (20.1), 323 (17.1), 286 (11.5), 284 (100), 285 (98.2), 179 (29.4) MS3[609 → 285]: 213 (55.2), 151 (89.4), 107 (100) 34 26.5 Que ce in-O- ca eoylhexoside 253, 330 625 MS2[625]: 463 (57.1), 445 (24.2), 323 (14.3), 301 (100), 300 (5.1) MS3[625 → 301]: 273 (4.1), 271 (5.9), 255 (19.6), 176 S.C. Gou eia, P.C. Cas ilho / Indus ial C ops and P oduc s 45 (2013) 170– 181 Table 2 (Con inued) No. R(min) Iden ifica ion max (nm) [M-H]−(m/z) HPLC-DAD-ESI/MSnm/z (% base peak) 179 (45.9), 151 (100), 107 (7.0) MS4[625 → 301 → 151]: 107 (100) 35 27.2 Unknown 579 MS2[579]: 418 (24.7), 417 (100) MS3[579 → 417]: 243 (100), 179 (26.5) MS4[579 → 417 → 243]: 225 (16.7), 199 (100), 183 (31.0) 36*27.7 Lu eolin – 285 MS2[285]: 243 (38.2), 241 (92.1), 217 (12.5), 199 (69.5), 175 (100), 151 (40.2) MS3[285 → 175]: 147 (100) 37 28.5 Ca eoylcouma oyl a a ic acid 232, 300, 311 457 MS2[457]: 296 (10.5), 295 (100), 173 (12.9) MS3[457 → 295]: 163 (100), 121 (36.2) MS4[457 → 295 → 163]: 111 (38.8), 93 (100) 38*29.2 3,4,5-O- T ica eoylquinic acid 677 MS2[457]: 296 (10.5), 295 (100), 173 (12.9) MS3[457 → 295]: 163 (100), 121 (36.2) MS4[457 → 295 → 163]: 111 (38.8), 93 (100) 39 30.4 E iodic yol-7-O- hexoside 247, 329 449 MS2[449]: 287 (100), 173 (14.3) MS3[449 → 287]: 173 (100) MS4[449 → 287 → 173]: 111 (100), 83 (73.6) 40*33.9 Apigenin 260, 331 269 MS2[269]: 227 (33.4), 225 (100), 201 (59.2), 151 (32.6), 149 (80.3) MS3[269 → 225]: 198 (23.1), 183 (66.9), 181 (100) (–) Thei UV spec a ha e no been p ope ly obse ed due o low in ensi y. *Compa ison wi h e e ence s anda ds. Fig. 2. Chemical s uc u es o phenolic compounds cha ac e ized. S.C. Gou eia, P.C. Cas ilho / Indus ial C ops and P oduc s 45 (2013) 170– 181 177 Compounds 9 ( R= 6.5 min) and 12 ( R= 7.9 min) displayed a [M- H]−ion a m/z 577. MSnexpe imen s o hese wo peaks did no e ealed he agmen co esponding o he neu al loss o 60 Da and he p esence o a agmen ion a m/z 353 indica es ha he suga g oups mus be a hexoside o a me hylpen ose. Compound 9 p esen ed MS2ions a m/z 559 (21.9% o base peak) [M-H-H2O]−and m/z 503 [M-H-74]−sugges s ha he hamnoside g oup should be linked a 6-C-posi ion. Thus, 9 was iden ified as apigenin-6-C- hamnoside-8-C-hexoside. Compound 12 has been epo ed be o e and was iden ified as apigenin-6-C-hexoside-8-C- hamnoside. Compound 13 ( R= 8.3 min) showed a [M-H]−ion a m/z 431. The MS2spec um p esen ed main agmen ions a m/z 311 [M- H-120]− 0,2X−(base peak) and 341 0,3X−(23.1% o base peak). The loss o wa e molecules is indica i e o C-6 isome s. This ype o clea age was no obse ed o compound 13 and based on li e a u e epo s (Gou eia S. and Cas ilho) his compound was iden ified as apigenin-8-C-hexoside. 3.2.1.3. O-glycosides (3, 14, 15, 16, 17, 18, 24, 25, 33 and 34). The agmen s esul ed om he o O-glycosila ed fla onoids we e labelled as p oposed by Cuyckens and Claeys (2004). The i,jA−and i,jB−labels co espond o ions con aining in ac A- and B- ings, espec i ely, and i and j speci y he C- ing bonds ha ha e been b oken. Compound 3 ( R= 4.0 min) displayed a [M-H]−ion a m/z 609. I s analysis by MS2 agmen a ion esul ed in a neu al loss o 162 Da o ming a agmen ion a m/z 447 and an in ense agmen a m/z 285 (34.9% o base peak). MS3spec um displayed a agmen ion a m/z 285 (loss o 162 Da), as base peak, and i s subsequen agmen a ion ga e he ypical agmen s o kaemp e ol a m/z 255 [Y0−-CH2OH]− and 151 (1,3A−) (compa ison made wi h a s anda d solu ion o kaemp e ol). The absence o a agmen ion a m/z 323 excluded he hypo hesis o he wo 162 Da esidues being ca eoylhexo- sides and we e cha ac e ized as being wo hexosides esidues. Based on he p e ious epo s (Gou eia and Cas ilho, 2010) and acco ding o he ules desc ibed by Ablajan and co-wo ke s (Ablajan e al., 2006) 3 was iden ified as kaemp e ol-3,7-O- dihexoside. Compound 33 ( R= 23.8 min) also displayed a [M-H]−ion a m/z 609. Howe e , MSn agmen a ion was qui e di e en o ha ound o compound 4. The MS2spec a showed he adical aglycone ion [Y0-H]−a m/z 284, as base peak, and also an in ense agmen a m/z 285 (98.2% o base peak). The loss o a 324 Da esidue was a ibu ed o a combined loss o wo 162 Da g oups and was confi med by he p esence o a agmen ion a m/z 447 (20.1% o base peak) o med by he loss o 162 Da. The agmen ion a m/z 323 (17.1% o base peak) assigned as [ca eoylhexoside-H]−poin ou o a hexoside g oup es e ified wi h a ca eoyl g oup a he han wo hexosides moi- e ies. The aglycone was iden ified as being kaemp e ol based on he MSn agmen s and compa ison wi h a kaemp e ol e e ence solu ion. The a ou ed glycosila ion posi ions o fla onols, such as kaemp e ol a e 3-OH and 7-OH. When he aglycone adical is mo e abundan han he dep o ona ed aglycone ion, indica es an agly- cone subs i u ed a posi ion 3-OH (Cuyckens and Claeys, 2005). Fo compound 33, he MS2spec um base peak was he aglycone adical ion, a m/z 285, and he e o e i was iden ified as being kaemp e ol-3-O-ca eoylhexoside. Compound 17 ( R= 9.5 min) exhibi ed a [M-H]−ion a m/z 463 which easily los a 162 Da moie y, in MS2 agmen a ion, esul ing in a agmen ion a m/z 301. Fu he agmen a ion o his ion ga e he ep esen a i e agmen s o que ce in a m/z 151 (1,2 A−-CO), 179 ([1,2A−-H]−) and 271 [M-H-CH2O]−. The e o e, compound 17 was unequi ocally iden ified as que ce in-3-O-glucoside by compa ison wi h a e e ence s anda d solu ion. Compounds 14 ( R= 8.8 min) and 34 ( R= 26.5 min) ga e he same [M-H]−ion a m/z 625 and hei MSn agmen a ion was simila . In he MS2spec a, he same base peak a m/z 301 was obse ed (loss o 324 Da). The occu ence o a MS1ion a m/z 463 (loss o 162 Da) sugges s ha he esidue o 324 Da is composed o wo uni s o 162 Da linked. Howe e , he na u e o hese wo g oups appea s o be dis inc o each compound. Fo 34, a MS1 ion a m/z 323 (14.3% o base peak) was obse ed indica ing a ca - eoylhexoside g oup which is in good ag eemen wi h he long e en ion ime o his compound. The lowe e en ion ime o 14 and he absence o a agmen ion a m/z 323 indica e a dihexoside esidue. F agmen a ion o he dep o ona ed aglycone ion, Y− 0a m/z 301 allowed o iden i y common agmen ions o que ce in, as desc ibed o compound 17. Since he Y− 0ion is he MS2base peak, he subs i u ion g oups mus be linked o only one OH g oup o aglycone s uc u e. The aglycone adical ion was no obse ed, so he 3-OH posi- ion is excluded bu no o he agmen s we e ound o suppo in which posi ion he subs i u ion occu s. Thus, 14 was en a i ely iden ified as que ce in-O-dihexoside and 34 was as que ce in-O- ca eoylhexoside. Compound 15 ( R= 9.1 min) displayed a [M-H]−in a m/z 447 and i s MS2 agmen a ion e ealed he loss o 162 Da o ming a ag- men ion a m/z 285, Y− 0. This compound was al eady desc ibed o A emisia a gen ea (Gou eia and Cas ilho, 2011a, b) and iden ified as lu eolin-7-O-hexoside. Ano he lu eolin de i a i e was de ec ed a a e en ion ime o 14.6 min (compound 24). I ga e a [M-H]−ion a m/z 417 easily los a neu al g oup o 132 Da (pen oside) ising he dep o ona ed aglycone ion a m/z 285. F agmen a ion o he ion a m/z 285 ga e he cha ac e is ics agmen s o lu eolin a m/z 175 ([M-H-C3O2- C2H2O]−), 217 ([M-H-C3O2]−) and 241 ([M-H-CO2]−) (Fig. 3). The a ou ed subs i u ion posi ion o fla ones such as lu eolin is he 7- OH posi ion (Cuyckens and Claeys, 2004). Consequen ly, compound 24 was cha ac e ized as lu eolin 7-O-pen oside. To ou knowledge i is he fi s ime ha his compound is epo ed o A emisia species. Compound 16 ( R= 9.3 min) showed a [M-H]−ion a m/z 493. I s MSn agmen a ion esul ed in he aglycone ion a m/z 331 due o he loss o 162 Da. The MS3 adical ion a m/z 316 is simila o he agmen a ion beha iou desc ibed o mea nse in-O-hexoside, also de ec ed in A emisia a gen ea and A emisia annua (Gou eia and Cas ilho, 2011a,b; Han e al., 2008). Compound 18 ( R= 9.8 min) displayed a [M-H]−ion a m/z 477 and was cha ac e ized as iso hamne in-O-hexoside by compa ison wi h li e a u e da a (Gou eia and Cas ilho, 2009). This compound was no epo ed be o e o A emisia annua bu was de ec ed in A emisia species (Gou eia and Cas ilho, 2011a,b). Compound 25 ( R= 15.3 min) showed a [M-H]−ion a m/z 491. A loss o 162 Da was obse ed in he MS2 agmen- a ion, o ming a agmen ion a m/z 329. In he u he MSn agmen a ions, wo losses o 15 Da each we e obse ed and associa ed o wo me hoxyl g oups. So, 25 was cha - ac e ized as dihyd oxy-dime hoxyl-O-hexoside fla ones. This compound was also epo ed o A emisia a gen ea (Gou eia and Cas ilho, 2011a,b) bu i was no epo ed o A emisia annua be o e. One compound (39) om he g oup o fla anones is epo ed o A emisia annua o he fi s ime. I was ound a a e en ion ime o 30.4 min and iden ified as a e iodic yol-7-O-hexoside. The [M- H]−ion appea ed a m/z 449 and he hexoside esidue was easily expelled in he MS2 agmen a ion esul ing in he dep o ona ed aglycone ion, Y− 0, a m/z 287. 178 S.C. Gou eia, P.C. Cas ilho / Indus ial C ops and P oduc s 45 (2013) 170– 181 Fig. 3. P oposed agmen a ion pa hway o compound 24–lu eolin-O-pen oside. 3.2.2. Iden ifica ion o hyd oxycinnamic acids (2, 4, 5, 6, 10, 11, 19, 20, 21, 22, 23, 26, 28, 29, 30, 31, 32 and 38) Compound 2 ( R= 3.1 min) displayed a [M-H]−ion a m/z 191 and was iden ified as quinic acid (Gou eia and Cas ilho, 2011a,b). Despi e ha his compound was ound as a ela i e in ense com- ponen o he A emisia annua ex ac , i was no epo ed be o e o his plan . A o al o 19 quinic acid de i a i es we e de ec ed and iden ified in his s udy, mos o hem quinic acid es e ified wi h acyl g oups. The iden ifica ion o each compound was made based on he main agmen ions ob ained in he MSnexpe imen s. The hie a chical key o he iden ifica ion by LC-MSno quinic acid de i a i es p o- posed by Cli o d e al. (Cli o d e al., 2003, 2005) was used o iden i y his class o compounds. Mono-, di- and ica eoylquinic acids we e iden ified by com- pa ison o he e en ion ime, MSn agmen a ion beha iou and UV spec a wi h hose o s anda d compounds. I was he case o com- pounds 5 ( R= 4.4 min) as 5-O-ca eoylquinic acid; 19 ( R= 11.8 min) as 3,4-O-dica eoylquinic acid; 20 ( R= 12.5 min) as 1,5-O- dica eoylquinic acid; 21 ( R= 12.9 min) as 3,5-O-dica eoylquinic acid; 23 ( R= 14.2 min) as 4,5-O-dica eoylquinic acid and 38 ( R= 29.2 min) as 3,4,5-O- ica eoylquinic acid. Addi ionally, wo monoca eoylquinic acids, compounds 4 and 6, we e iden ified based on MSn agmen a ion o he [M-H]−ion a m/z 353. Compound 4 ( R= 4.4 min) ga e a MS2ion a m/z 191 (base peak) and an in ense agmen ion a m/z 179 (51.4% o base peak) which is ep esen a i e o a ca eoyl g oup linked o he 3-OH posi ion o quinic acid. Thus, 4 was iden ified as 3-O-ca eoyquinic acid. Compound 6 ( R= 5.2 min) showed a MS2ion a m/z 173 (base peak) poin ing o a quinic acid es e ified a posi ion 4-OH (Fig. 4). The e o e, 6 was classified as 4-O-ca eoylquinic acid. Rema kably, his compound ga e a highe in ensi y han he isome 5-O-CQA commonly ound in o he As e aceae plan s. Compound 22 ( R= 13.1 min) ga e a [M-H]−ion a m/z 515 and u he MSn agmen a ion ga e he cha ac e is ic agmen ions o dica eoylquinic acid isome s a m/z 191, 173 and 353. This should be a cis isome o a dica eoylquinic acid wi h no subs i u ion posi ion 4-OH (Jaiswal e al., 2011; Ma e al., 2008). The unequi - ocally iden ifica ion o his compound can only be es ablished by NRM s udies o compa ison wi h a e e ence s anda d since e en UV-i adia ion s udies would no cla i y i i is a mono-cis o a di-cis compound. Compounds 10 ( R= 6.9 min) and 11 ( R= 7.4 min) exhibi ed he same [M-H]−ion a m/z 367 bu a di e en MSn agmen a- ion beha iou . Fo 10, he MS2spec um showed a agmen ion a m/z 193 (base peak). Based on he ules epo ed by Cli o d e al. (Cli o d e al., 2003) his compound was iden ified as 3-O- e uloylquinic acid. Compound 11 ga e, as base peak o he MS2 spec um, a agmen ion a m/z 191 [quinic acid-H]−. This pa e n is consis en o ha epo ed o 5-O- e uloylquinic acid (Cli o d e al., 2005). Compound 26 ( R= 16.8 min) displayed a [M-H]−ion a m/z 499. The MS2spec um showed a agmen ion a m/z 337, as base peak, indica ing he loss o 162 Da and sugges ing a couma oylquinic acid de i a i e. MS3 agmen a ion o he ion a m/z 337 esul ed in a agmen ion a m/z 163 and a MS4ion a m/z 119, as base peaks, poin ing o a 3-p-O-couma oylquinic acid s uc u e, acco ding o li e a u e epo s (Cli o d e al., 2003). The absence o a s ong agmen a ion a m/z 173 sugges s a 3,5-O-p-couma oyl-ca eoylquinic acid. Since he ca eoyl g oup is he fi s o be los i should be linked o he 5-OH posi ion. Taking in o accoun hese da a, 26 was iden ified as 3-p-O-couma oyl-5-O-ca eoylquinic acid. To ou knowledge, quinic acids es e ified wi h couma oyl g oups ha e no been epo ed be o e in phenolic sc eenings o A emisia annua. Th ee compounds wi h [M-H]−ions a m/z 529 we e obse ed (compound 28 ( R= 18.9 min), 29 ( R= 20.2 min) and 30 ( R= 20.7 min)). In he MS2 agmen a ion all compounds ga e MS2 base peaks a m/z 367 [ e uloylquinic acid-H]−, due o he loss o a ca eoyl esidue, and a seconda y agmen ion a m/z 353 (ca. 20% o base peak). The dis inc ion o hese h ee isome s was based on he MS3 agmen a ion. Compound 28 ga e a MS3ion a m/z 191, as base peak, which is consis en wi h a 5-O- e uloylquinic acid s uc u e. Acco ding o he agmen a ion da a, a agmen ion a m/z 179 was no obse ed, hus he ca eoyl g oup mus be linked o he 1-OH posi ion o quinic acid. So, compound 28 was iden ified as 1-O-ca eoyl-5-O- e uloylquinic acid. Compound 29 ( R= 20.2 min) exhibi ed a MS3base peak a m/z 173 which indica es a 4-O- e uloylquinic acid s uc u e. The exac