Research Article Alkaloids Profiling of Fumaria capreolata by Analytical Platforms Based on the ...

17 Pages·2017·2.05 MB·English

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International Journal of Analytical Chemistry
Volume 2017, Article ID 5178729, 16 pages
https://doi.org/10.1155/2017/5178729
Research Article
Fumaria capreolata
Alkaloids Profiling of by Analytical
Platforms Based on the Hyphenation of Gas Chromatography
and Liquid Chromatography with Quadrupole-Time-of-Flight
Mass Spectrometry
MaríadelMarContreras,1,2NoureddineBribi,3,4AnaMaríaGómez-Caravaca,2
JulioGálvez,3andAntonioSegura-Carretero1,2
1ResearchandDevelopmentFunctionalFoodCentre(CIDAF),Bioregio´nBuilding,HealthScienceTechnologicalPark,
AvenidadelConocimientos/n,18016Granada,Spain
2DepartmentofAnalyticalChemistry,FacultyofSciences,UniversityofGranada,Avda.Fuentenuevas/n,18071Granada,Spain
3CIBER-EHD,DepartmentofPharmacology,ibs.GRANADA,CenterforBiomedicalResearch(CIBM),UniversityofGranada,
AvenidadelConocimientos/n,Armilla,18016Granada,Spain
4LaboratoiredeBiotechnologiesVe´ge´talesetEthnobotanique,Faculte´desSciencesdelaNatureetdelaVie,
Universite´deBejaia,06000Bejaia,Algeria
CorrespondenceshouldbeaddressedtoMar´ıadelMarContreras;[email protected]
AntonioSegura-Carretero;[email protected]
Received 29 April 2017; Accepted 2 July 2017; Published 28 November 2017
AcademicEditor:MohamedAbdel-Rehim
Copyright©2017Mar´ıadelMarContrerasetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Twoanalyticalplatforms,gaschromatography(GC)coupledtoquadrupole-time-of-flight(QTOF)massspectrometry(MS)and
reversed-phaseultrahighperformanceliquidchromatography(UHPLC)coupledtodiodearray(DAD)andQTOFdetection,were
appliedinordertostudythealkaloidprofileofFumariacapreolata.Theuseofthesemassanalyzersenabledtentativelyidentifying
thealkaloidsbymatchingtheiraccuratemasssignalsandsuggestedmolecularformulaewiththosepreviouslyreportedinlibraries
anddatabases.Moreover,theproposedstructureswerecorroboratedbystudyingtheirfragmentationpatternobtainedbyboth
platforms.Inthisway,8and26isoquinolinealkaloidswerecharacterizedusingGC-QTOF-MSandRP-UHPLC-DAD-QTOF-
MS, respectively, and they belonged to the following subclasses: protoberberine, protopine, aporphine, benzophenanthridine,
spirobenzylisoquinoline, morphinandienone, and benzylisoquinoline. Moreover, the latter analytical method was selected to
determineat280nmtheconcentrationofprotopine(9.6±0.7mg/g),apotentialactivecompoundoftheextract.Inconclusion,
althoughGC-MShasbeencommonlyusedfortheanalysisofthistypeofphytochemicals,RP-UHPLC-DAD-QTOF-MSprovided
essential complementary information. This analytical method can be applied for the quality control of phytopharmaceuticals
containingFumariaextractscurrentlyfoundinthemarket.
1.Introduction in the world, which grows in wheat fields, plains, and
low hills in Europe, Middle East, South Asia, and so on
In the “Conspiracy of Cortes” the Spanish novelist Matilde [3]. The biological activities of Fumaria spp. are linked
Asensi mentions the usage of Fumaria as a natural treat- to the presence of key compounds such as isoquinoline
ment for bubonic plague [1]. This fact is not far from alkaloids[4].Infact,alkaloids,includingtheaforementioned
fiction since the genus Fumaria (Fumarioideae, Papaver- type,haverecentlybeenreviewedtobeeffectivetreatments
aceae) has traditionally been used in traditional medicine of intestinal inflammation injury in animal models [5].
[2]. This genus consists of above 46 species widespread Interestingly, recent studies have shown the low toxicity
2 InternationalJournalofAnalyticalChemistry
of a Fumaria capreolata alkaloid extract (white ramping- 2.2. Analysis by GC-QTOF-MS. Analyses were carried out
fumatory)invitroandinvivo[2],aswellastheirintestinal using an Agilent technologies (Palo Alto, CA, USA) gas
anti-inflammatoryeffectsinmicecolitis[6]andantinocicep- chromatography(GC)system7890B(G3440B)coupledtoa
tiveactivity[7]. 7200accuratemassquadrupole-time-of-flight(Q-TOF)mass
Isoquinolinealkaloidsderivefromtheaminoacidtyro- spectrometer. A capillary column was employed: HP-5MS
sine, after its conversion to 3,4-dihydroxyphenylethylamine (5% phenyl 95% dimethylpolysiloxane, 30m × 0.250mm
(dopamine)and4-hydroxyphenylacetaldehydethatactasin- i.d.; 0.25𝜇m film thickness). The column temperature was
∘ ∘
termediary molecules [8]. This type of alkaloids has been initially held at 200 C for 1min, increased at 25 C/min to
∘ ∘
characterized by thin layer chromatography [9], gas chro- 250 C, and then held at 250 C for 31min. Finally, a ramp
∘ ∘
matography(GC)coupledtoflameionizationdetector(FID) of 10 C/min was applied until 310 C and maintained for
[10], and mass spectrometry (MS) [11–14], as well as liq- 5min. The total run was 45min. The helium flow rate
uid chromatography (LC) coupled to UV/Vis, diode array was1.2mL/min.Theinjectionprogramincludedsequential
detection(DAD)[15],andmassspectrometry(MS)[16].In washingstepsofthesyringeandasamplepumpingstepfor
thecaseofGC-MS,thereisavailablestandardmassspectral removalofsmallairbubbles.Theinjectionwas1𝜇Lusinga
data library for the characterization of several phytochem- programmablePALGCsampler120andwithasplitratioof
∘
ical classes, but it is not at all complete for alkaloids in 10:1.Theinletandthetransferlinetemperatureswere250 C
∘
comparison with other phytochemicals and explained by and300 C,respectively.Theionsourcewaselectronimpact
∘
their relatively poor volatilization [17]. In the case of LC- (EI)carriedoutat70eVandthetemperaturewas250 C.The
MS, there are efforts to generate spectral libraries using mass range from m/z 125 to 700 was scanned at a rate of 3
different MS analyzers and collision energies due to the scans/s.TheTOFanalyzerwascalibratedpreviouslytoeach
little lack of consistency, standardization, or reproducibility analysisusinganAgilentcalibrationtunemix.
as compared with GC-MS or nuclear magnetic resonance
spectroscopy[18].MassBank(http://www.massbank.jp/)and 2.3.AnalysisbyReversed-PhaseUltrahighPerformanceLiquid
Metlin (https://metlin.scripps.edu/) are examples of public Chromatography Coupled to Diode Array Detection (DAD)
repositoriesoftandemmassspectraldata. and QTOF-MS. Analyses were made with an Agilent 1200
seriesrapidresolution(PaloAlto,CA,USA)equippedwith
Recent studies have demonstrated the potential of the
a binary pump, an autosampler, and a DAD. The mobile
hybridmassanalyzerquadrupole-time-of-flight(QTOF)for
phases consisted of a water with 0.2% formic acid (mobile
the profiling of hundreds of natural plant metabolites. This
phaseA)andacetonitrile(mobilephaseB),andamultistep
type of mass analyzers provides excellent selectivity and
linear gradient was applied: 0–5.5min, 1–7% B; 5.5–11min,
massaccuracyoverawidedynamicrangeandelucidatesthe
7–14% B; 11–17.5min, 14–24% B; 17.5–22.5min, 24–40%
molecularformulaofunknowncompounds.Italsoperforms
B; 22.5–27.5min, 40–100% B; 27.5–28.5min, 100-100% B;
tandem MS, which is useful for a preliminary structural
28.5–29.5min, 100-1% B. The latter value (99% A and 1%
elucidation[19–21]wheninformationindatabasesislimited.
B) was held for 5.5min to equilibrate the column with the
Thus,theobjectiveofthisstudywastoexplorethepotentialof
initialconditionspriortothenextinjection.Thetotalrunwas
GCandLCcoupledtoQTOFmassanalyzerfortheprofiling
35min.Theflowratewassetat0.5mL/minthroughoutthe
of isoquinoline alkaloids in F. capreolata. This plant was
gradient. Separation was carried out with a Zorbax Eclipse
selectedduetoitsaforesaidbiologicalproperties.Inaddition, XDB-C18 column (4.6 × 50mm, 1.8𝜇m of particle size)
to the best of our knowledge, there are not any reports in ∘
(Agilent) at 25 C. The UV-Vis spectra were recorded from
the literature accomplishing the comparison of these two 190to600nm,andawavelengthchannelof280±4nmwas
analyticalplatformsforthequalitativecharacterizationofthis
appliedforquantitativepurposes.Theinjectionvolumewas
typeofphytochemicals. 1𝜇L.
The spectra were acquired in negative and positive ion
2.Experimental modes over a mass-to-charge (m/z) range from 70 to 1500.
The operation conditions were set in the adequate polarity
2.1. Preparation of the Alkaloid Extract. Aerial parts of F. as follows: gas temperature: 325∘C; drying gas: nitrogen at
capreolatawerecollectedfromBejaiaarea(Algeria)inMay 10L/min;nebulizerpressure:20psig;sheathgastemperature:
2012,whentheywereatthefloweringandfruitsettingstage. 400∘C;sheathgasflow:nitrogenat12L/min;capillaryvoltage:
Plants were authenticated by Dr. F. Maiza-Benabdesselam 4000V; skimmer: 45V; octapole radiofrequency voltage:
(Laboratory of Plant Biotechnology and Ethnobotany, Uni- 750V;focusingvoltage:500V,withthecorrespondingpolar-
versity of Bejaia, Algeria) and voucher specimen was ityautomaticallyset.TheacquisitionmodewasAutoMS2.
deposited (Reference number FC015). Aerial parts of the Internalmass correctionofeach sample was performed
∘
plants were dried in oven at 40 C overnight and ground. with a continuous infusion of Agilent TOF mixture con-
In brief, the powder samples were extracted as described taining two mass references for each ionization mode. In
before[2,7]withmethanolanddichloromethanetoafforda the positive ionization mode, the two reference mass ions
crude extract of alkaloids. For analysis, the alkaloid extract were at m/z 121.0509 (purine) and 922.0098 (hexakis (1H,
(10mg) was dissolved in methanol (1mL) (ThermoFisher, 1H, 3H-tetrafluoropropoxy) phosphazine). Alternatively, in
Waltham,MA,USA)andfilteredwitha0.20𝜇msyringefilter thenegativeionizationmodetrifluoroaceticacidammonium
ofpolytetrafluoroethylene(13mm)(ThermoFisher). salt (m/z 112.9856 that is trifluoroacetic acid) and hexakis
InternationalJournalofAnalyticalChemistry 3
(1H,1H,3H-tetrafluoropropoxy)phosphazine(m/z1033.9881 main fragments generated by EI from F. capreolata alka-
corresponding to the trifluoroacetic acid ammonium salt loids. Table S1 (in Supplementary Material available online
adduct)wereused.Thedetectionwindowwassetto100ppm. athttps://doi.org/10.1155/2017/5178729)additionallyprovides
Data acquisition (2.5Hz) in the profile mode was governed literatureabouttheiroccurrenceinF.capreolata,fragmenta-
viatheAgilentMassHunterWorkstationB.05.01. tionpatterninGC-MS,andinformationfoundindatabases.
Data analysis was performed on a Mass Hunter Quali- Using this approach, a total of eight isoquinoline alkaloids
tative Analysis B.06.00, as commented before. The isotope were characterized and eluted in the following order: sty-
modelselectedwas“commonorganicmolecules”withapeak lopine, protopine, cheilanthifoline, isoboldine, coreximine,
spacingtoleranceofm/z0.0025and7ppm. dihydrosanguinarine,fumariline,andparfumine.Thesealka-
All analyses by GC-QTOF-MS and RP-HPLC-DAD- loidswererepresentativesofprotoberberine,protopine,ben-
QTOF-MSweredoneintriplicate. zophenanthridine, aporphine, and spirobenzylisoquinoline
types.Theseresultsagreedwithpreviousstudiesonthisplant
using GC coupled to a quadrupole mass analyzer [11, 12]
2.4. Data Processing. Data analysis was performed on
and other studies [9, 25], while additionally we also detect
MassHunterQualitativeAnalysisB.06.00(Agilenttechnolo-
coreximine. Moreover, in agreement with previous studies
gies).Thecharacterizationofcompoundswasperformedby
[13, 14, 17, 24] our results suggest that the stationary phase
generationofcandidateformulawithamassaccuracylimit
5% phenyl 95% dimethyl polysiloxane is also an adequate
of 5ppm for the analysis of RP-UHPLC-DAD-QTOF-MS
alternativetothemoreapolarstationaryphasesconsistingof
and 10ppm for GC-QTOF-MS, and also considering the
100%dimethylpolysiloxane,whichisalsocommonlyusedfor
MS score that should be close to 100. The latter parameter
theseparationofisoquinolinealkaloids[11,12].
is related to the contribution to mass accuracy, isotope
EI source is considered a hard ionization method since
abundance,andisotopespacingforthegeneratedmolecular
it provokes extensive fragmentation by high energetic elec-
formulae.UsingGC-QTOF-MS,alkaloidswerecharacterized
trons. Interestingly, the TOF mass analyzer enabled us to
by direct comparison of their fragmentation pattern with
+
detect the molecule ions (M ) of each alkaloid in itself
data from the NIST MS library (NIST11.L) and NIST
and generate their molecular formulae with errors lower
chemistry WebBook (http://webbook.nist.gov/chem- istry/)
than 10ppm (Table 1 and Table S1). It seems that this
when possible, as well as literature about Papaveraceae. In
platformishighlyattractivesinceitcanprovidenotonlythe
the case of RP-UHPLC-DAD-QTOF-MS and –MS/MS,
fragmentationpatternofalkaloids,butalsotheirmolecular
MassBank and Metlin were consulted. Moreover, chemi-
formulae,abasicclueforstructureelucidationofunknown
calstructureinformationwasalsoretrievedfromSciFinder
compoundsthatarenotavailableincurrentGC-MSlibraries.
Scholar(https://scifinder.cas.org),Reaxys(http://www.reax-
This was the case of dihydrosanguinarine and parfumine
ys.com), and KNApSAcK Core System (http://kanaya.naist
(Table S1), whose structures were proposed based on both
.jp/knapsack jsp/top.html).
theirmolecularformulaandthefragmentationpatternthat
couldbefortunatelycomparedwithliterature[11,14,24].
2.5. Quality Control of Protopine. Protopine hydrochloride
Asanexampleofthestrategyfollowed,Figure2showsthe
stock solution was conveniently diluted with methanol to
fragmentationpatternofstylopine,protopine,andfumariline
prepare calibration points (8–260nmol/mL). The external
usingGC-QTOF-MS.
standardmethodwasusedandalinearregressionforthecali-
brationcurvewasestimatedusingtheareaunderthecurveof
3.2.AlkaloidalProfilingviaRP-HPLC-DAD-QTOF-MS. Pre-
protopineagainstconcentration.Repeatabilitywasassayedby
liminary F. capreolata alkaloids were studied using ESI in
threeconsecutiveinjectionsofthemethanolicsolutionsofat
bothnegativeandpositiveionizationmodes.Thesecondion-
threelevelsthreetimes(intradayrepeatability)andfivetimes
ization mode led to a richer and complex chromatographic
ontwodifferentdays(interdayrepeatability)[22].Thelimit profile (Figure S1) with more intense signals. Thus, it was
ofdetection(LOD)andquantification(LOQ)wereestimated
selectedforfurtherstudies.Thisisnotsurprisingsincemost
asprotopineconcentrationgivingasignalequaltotheblank
LC-MS methods have applied the latter ionization mode
signal plus three and ten standard deviations of the blanks,
for the analysis of alkaloids. As an example, the alkaloid
respectively[23].
profileofF.capreolataisshowninFigure1(b).Itdepictsthe
basepeakchromatogram(BPC)undertheselectedanalytical
3.ResultsandDiscussion conditions,aswellasthechromatogramsat280nm,atwhich
isoquinolinealkaloidsshowabsorption,and450nm,which
3.1. Alkaloidal Profiling via GC-TOF-MS. Briefly, for the isparticularofquaternaryprotoberberinealkaloids.
untargeted analysis of the alkaloidal extract via GC-TOF- Afterwards,usingthisanalyticalplatform,thecharacter-
MS the strategy followed consisted of the generation of ization of the alkaloids was based on the following strategy
the molecular formulae of the detected ions in the chro- describedinourpreviousstudiesonotherplantphytochem-
matographic profile (Figure 1(a)) and studying the frag- icals [20]. Firstly, the UV-Vis spectra and the generation
mentation pattern using NIST MS library when possible of the molecular formulae enabled proposing the chemical
or based on literature [11, 12, 14, 24]. The results provided structuresofthealkaloids.Forthat,literatureonFumariaspp.
by the GC–TOF-MS analyzer are given in Table 1, which [9,11,12,25,26]andtheaforementionedchemicaldatabases
shows MS experimental data, retention time (RT), and were consulted. Secondly, the MS/MS spectra were studied
4 InternationalJournalofAnalyticalChemistry
×106
×105 20
2.75
5 21 2.5 12 25
2.25
4
15 2
19
3 1.75
11 1.5
20
2 9 1.25
16 18 20 22 24 26 28 30
1
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
Counts versus Acquisition Time (min)
(a)
×106
8
1011 16 19 21
13
9 12 18 22
6 3 6 8 20
1 5 7
2 23 24 26
4 15
4
3 25
14 17 6
2
1 4,5
2
0
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Counts versus acquisition time (min)
(b)
×102
3
2
1
0
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Response Units versus acquisition time (min)
(c)
Figure1:Continued.
InternationalJournalofAnalyticalChemistry 5
×101
8
6
4
2
0
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
mAU versus acquisition time (min)
(d)
Figure 1: (a) Total ion chromatogram obtained by GC-QTOF-MS, (b) base peak chromatogram in the positive ionization mode,
(c) chromatograms at 280nm, and (d) 450nm of the alkaloid extract obtained by RP-UHPLC–DAD–QTOF-MS. Compounds are
numberedaccordingtoTable2:(1)pallidine;(2)isoboldinederivative;(3)N,N-dimethylcoclaurine(4)fumaritine;(5)coclaurine;(6)N-
methylcoclaurine;(7)magnoflorine;(8)reticuline;(9)parfumine;(10)parfumidine;(11)isoboldine;(12)coreximine;(13)methylcoreximine
1;(14)methylcoreximine2;(15)cheilanthifoline;(16)dehydrocheilanthifoline;(17)demethyleneberberine/jatrorubine;(18)cryptopine;(19)
protopine;(20)fumariline;(21)stylopine;(22)coptisine;(23)corysamine;(24)impatienC;(25)dihydrosanguinarine;(26)8-oxocoptisine.
󸀠 󸀠
1–6 unknowncompounds.
indepth.Inthisway,26alkaloidswerecharacterizedonthe thefragmentationpatternofisoboldine,protopine,coptisine,
basis of their spectrometric data and the results are shown and stylopine (tetrahydrocoptisine), as examples of apor-
in Table 2 and Table S2: RT, molecular formula, observed phine, protopine, and both protoberberine quaternary and
m/z, mass error, MS score, UV-Vis maximums, and main tertiaryalkaloids,respectively.Thefragmentationpatternsof
MS/MS fragments. As before, Table S2 includes additional isoboldine and coptisine were characterized by a cleavage
information.ThechemicalstructuresaredepictedinFigure3. of the substituted groups of the alkaloid core, whereas no
Usingthepositiveionizationmode,thetertiaryalkaloids ringfusionwasobserved.Inthissense,isoboldine(m/z328)
+ +
led[M+H] ions,whereasquaternaryalkaloidsyielded[M] shows the primary loss of CH3NH2 (31Da) at m/z 297 due
ions in the mass spectra that is in accordance with Ding et tothepresenceofamethylsubstituentintheaminogroup,
al. [27]. Moreover, the different types of alkaloids showed as it was observed for its derivative (m/z 314) at m/z 283.
different UVabsorptionspectrawithmaximumabsorption This is a characteristic of this type of aporphine alkaloids
between260and290nm(Table2andTableS2).Interestingly, [36]. Afterwards, the loss of CH3, CH3, OH, CH3OH, and
UV-Vis spectroscopy is particularly useful for the elucida- CO occurred. In the case of protopine, their product ions
tion of quaternary isoquinoline alkaloids. For example, UV weregeneratedbydehydration(m/z 336),retro-Diels-Alder
absorption spectra of quaternary protoberberine alkaloids, fragmentation (m/z 149 and 206), and subsequent losses of
16,17,22,and23,aredeterminedbytheauxochromicgroups H2O(m/z188)andOH(m/z189),inaccordancewithShim
boundtoringDwithaminimumat301–310nm,indicatinga et al. [37] and Schmidt et al. [30]. The fragmentation of
protoberberinecorewithsubstituentsoncarbonsC9andC10 the protopine backbone was also observed by GC-QTOF-
[28]andalsopresentedacharacteristicmaximumabsorption MSasitwasshowninFigure2.Similarly,thefragmentation
around450nm,inagreementwithGrycova´aetal.[29].Asan of the backbone of stylopine generated fragment ions at
example,seecoptisineinFigures3and4. m/z 176 and 149, which were the most abundant. These ion
OncethemolecularformulaeweregeneratedandtheUV- fragments were also observed in the MS/MS spectrum of
Vis results contrasted, the MS/MS fragmentation patterns thecompound24(m/z396,C22H21NO6)(Table2),together
werestudiedandcomparedwiththoseindatabases,butonly with the fragment at m/z 322 released after the primarily
experimental MS/MS spectra of protopine and cryptopine neutrallossofasubstituentwithmolecularformulaC3H6O2
were found (Table S2). However, most of the MS/MS spec- (74Da).Therefore,thiscompoundwastentativelyidentified
tra shared common ions with those found in studies on as impatien C, a protoberberine recently characterized in
plants containing alkaloids from Papaveraceae, Lauraceae, Corydalisimpatiens(Fumarioideae,Papaveraceae)[38].
andRutaceae[27,30–35]beingusefultodefinethechemical Dihydrosanguinarine (benzophenanthridine type) was
structure of the alkaloids. As an example, Figure 4 shows alsocharacterizedbythefragmentationoftheirsubstituents,
6 InternationalJournalofAnalyticalChemistry
e e
e n n
Alkaloidtype Protoberberine Protopine Protoberberine Aporphine Protoberberine nzophenanthridin obenzylisoquinoli obenzylisoquinoli
Be pir pir
S S
EI-MSm/z(relativeintensity,%)323.1139(44),322.1071(52),148.0523(100),91.0541(26)163.0396(19),149.0564(11),148.0518(100),91.0539(14)325.1289(40),324.1215(46),149.0581(26),148.0518(100)327.1438(54),326.1375(100),284.1048(32),253.0833(27)327.1436(57),326.1376(63),178.0841(100),176.069(45)334.1014(18),333.0971(88),332.0899(100),105.0669(15)336.0862(28),323.1104(25),322.1061(100),264.1006(29)353.1246(17),338.1006(29),325.1252(26),324.1215(100),322.086(31),308.126(31)
S.
M
)
- m
QTOF Massr(pp 3.96 3.53 3.86 8.09 3.57 7.01 3.5 4.4
o
C- rr
G e
y
b
a
at
preol MScore 94 94 91 80 69 83 96 89
a s
c
F.
n
i
d
e
z
scharacteri Molecularformula CHNO19174 CHNO20195 CHNO19194 CHNO19214 CHNO19214 CHNO20154 CHNO20175 CHNO20195
d
oi
al
k
Al
1:
Table Precursor+m/z(M) 323.1139 353.1247 325.1296 327.1438 327.1428 333.0971 351.1089 353.1246 metry.
o
ctr
e
p
e s
Proposedalkaloid Stylopine Protopine Cheilanthifoline Isoboldine Coreximine hydrosanguinarin Fumariline Parfumine ectronimpact-mass
Di el
S:
M
EI-
d
aRTmin) 17.5 18.7 20.4 21.2 23.8 24.7 25.2 29.8 mean
( ti
n
o
nti
e
et
k r
a T:
Pe∘N 1 2 3 4 5 6 7 8 aR
InternationalJournalofAnalyticalChemistry 7
×102
1 Library
148
0.5
322
91
174 323
51 63 77 102 116 131 193 206 219 232 248 262 277 290 308
0
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340
(a)
×102
GC-QTOF-MS
＃ （ ／ + 148
9 8 2
1 148.0523 O
O
0.8 N
0.6 322.1071
0.4 9＃17.0（574+1 ＃10（8．／2+
0.2 174.0549 323.1139 174 O
O
0
100 150 200 250 300 350 400 450 500
Counts (%) versus mass-to-charge(m/z)
(b)
×102
1 Library
148
0.5
91
163
89 134 190
51 63 76 104 118 177 209 223 237 252 267 281 295 309 323 338 353
0
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360
(c)
×102
GC-QTOF-MS
＃ （ ／ +
9 8 2
148.0518
1 ＃（3
0.8 N
O
89.0382
0.6 O
0.4 ＃ （ + ＃9（7／3+ O
7 7 O
163.0396
0.2 91.0539 O
163
0 148
100 150 200 250 300 350 400 450 500
Counts (%) versus mass-to-charge(m/z)
(d)
Figure2:Continued.
8 InternationalJournalofAnalyticalChemistry
×102
1 Library
322
0.5
351
135 264 336
175 293
103
206 236
80 120 160 200 240 280 320 360 400 440 480
(e)
×102
＃ （ ．／ + GC-QTOF-MS
19 16 4
322.1061
＃ （ ．／ + O
1 19 14 5 351.1089
336.0862
0.8 ＃（3 N O
0.6 ＃8（7／2+ ＃ （ ．／ + O 135
17 14 2
0.4 264.1006 Lossof:
O 336:＃（3
0.2 135.0468 322: CHO
O 264:＃（3+2＃／+／
0
100 150 200 250 300 350 400 450 500
Counts (%) versus mass-to-charge(m/z)
(f)
Figure2:Fragmentationpatternsofstylopine((a)and(b)),protopine((c)and(d)),andfumariline((e)and(f))describedinthelibraryand
determinedbyGC-QTOF-MS,respectively.
being the most abundant ions formed by the loss of CH3 authors(e.g.,compounds2,3,5,10,13,14,17,23,24,and26).
(m/z 319), CH2O (m/z 304), and CO (m/z 276) (Table 2). Thiscanbeexplainedbythefactthatmostofthestudieson
Benzylisoquinolines presented the loss of NH3 (coclau- Fumariaspp.usedGC-MSforstudyingtheiralkaloidsprofile.
rine), CH3NH2 (N-methylcoclaurine), or C2H6NH (N,N- A recent study has shown the trends of using ESI, EI,
dimethylcoclaurine)atm/z269,substituentsoftheisoquino- andmatrix-assistedlaserdesorption/ionization(MALDI)as
+
linecore,andalsosharedacommonionatm/z107(C7H7O ), ionization sources for natural products investigations; the
which correspond to the methylphenol moiety generated application of ESI source is continuously growing, while
byinductivecleavage[37].Finally,spirobenzylisoquinolines MALDI and EI remain constant [39]. This trend possibly
showednotonlytheneutrallossofsubstituentsofthealkaloid reflects the huge expansion of ESI applications in this area
corebutalsothebreakageattheisoquinolinecoregenerating explainedbythefactthatMSisaversatiledetectionsystem
ionsatm/z177(C10H11NO2)(fumariline),179(C10H13NO2) and ESI source enables the analysis of a wide range of
(parfumidine), and 193 (C11H15NO2) (parfumine), and its chemicalstructures,asourresultsrevealed.
subsequentfragmentation(e.g.,m/z135and137).
3.4. Quantification of Protopine. Protopine exhibits anti-
3.3.ComparisonoftheQTOFPlatforms. Figure5represents inflammatory activity both in vitro and in vivo [40, 41].
acomparativeplotoftheresultsdescribedabove.Alltheiso- This alkaloid can contribute, at least in part, to explaining
quinolinealkaloidsfoundbyGC-TOF-MSintheextractofF. the antinociceptive and anti-inflammatory effects of the F.
capreolatawerealsodetectedusingRP-HPLC-DAD-QTOF- capreolata extract showed in our previous studies [2, 6, 7].
MS.Morepolaralkaloids,suchasquaternaryalkaloids,were Thus, for the quality control of this alkaloid extract, pro-
notdetectedbythefirstanalyticalplatformprobablydueto topine was preliminary selected as analytical/active marker
theirpoorvolatilitythatisitsmajordrawback[17].Moreover, since it is also commercially available and the RP-HPLC
the use of RP-HPLC-DAD-QTOF-MS enables us to find method was selected since it was shorter than the GC
somealkaloids,whichwerenotreportedinthisplantbyother method.Moreover,thequantificationofthiscompoundwas
InternationalJournalofAnalyticalChemistry 9
e e e
n n n
ne ne oli ne ne ne oli oli
Alkaloidtype Morphinandieno Aporphine Benzylisoquinoli robenzylisoquin Benzylisoquinoli Benzylisoquinoli Aporphine Benzylisoquinoli robenzylisoquin robenzylisoquin Aporphine
pi pi pi
S S S
MainfragmentsinMS/MS297.1051,265.0834,251.0635,243.0954,237.0846,211.0704,192.0962283.0920,268.0680,251.0664,233.0555,223.0716,205.0617299.1133,298.1061,269.1159,237.0903,209.0957,175.0762,121.0610,107.0497338.1313,277.0788,249.0847,137.0541269.1131,237.0864,209.0921,175.0723,145.0631,143.0457,107.0460269.1105,237.0846,209.0902,177.0489,175.0692,145.0232,107.0445297.1081,282.0853,265.0828,237.0915,191.0832192.0990,175.0719,143.0460,137.0564336.1191,323.0927,305.0774,295.0930,279.0611,179.0912,137.0567323.0859,305.0756,293.0754,261.0502,193.1053,137.0556297.1125,282.0840,265.0826,237.0877,233.0559,205.0610
MS. aVmax.(nm) 282 68,306 60,358 283 286 282 68,305 281 71,372 290 79,304
F- U 2 2 2 2 2
O
T
AD-Q Score 98.9 91.27 95.1 94.4 88.0 96.0 99.5 96.6 97.2 93.5 98.4
D
-
C
L
P
P-UH Error(ppm) −0.6 3.79 −3.87 0.6 2.0 3.0 −0.4 1.6 −1.4 3.7 −0.4
R
y
b
a
nF.capreolat a.m/zExp++M+H]/M) 328.1544 314.1381 314.1763 356.1481 286.1427 300.1585 342.1701 330.1696 354.1341 368.1478 328.1546
di ([
e
z
ri
e
ct
a
r
a
h
2:Alkaloidsc Molecularformula CHNO19214 CHNO18194 +CHNO19243 CHNO20215 CHNO17193 CHNO18213 +CHNO20244 CHNO19234 CHNO20195 CHNO21215 CHNO19214
e
l
b
a
T
Proposedalkaloid Pallidine vativeofisoboldine(demethyl) Dimethylcoclaurine Fumaritine Coclaurine Methylcoclaurine Magnoflorine Reticuline Parfumine Parfumidine Isoboldine
eri N- N-
D N,
aRT(min) 11.59 11.77 12.14 12.33 13.32 13.44 14.12 14.80 15.05 15.29 15.42
k
a
Pe∘N 1 2 3 4 5 6 7 8 9 10 11
10 InternationalJournalofAnalyticalChemistry
e
n e
Alkaloidtype Protoberberine Protoberberine Protoberberine Protoberberine Protoberberine Protoberberine Protopine Protopine obenzylisoquinoli Protoberberine Protoberberine Protoberberine Protoberberine nzophenanthridin Protoberberine
pir Be
S
MainfragmentsinMS/MS313.1226,178.0803,151.0693,119.0436,91.0493327.1431,192.0989,177.0754,151.0728,137.0573192.0976,177.0743,151.0712311.1157,192.0988,178.0829,151.0721,119.0461,91.0517307.0755,294.1039,279.0808,264.0663,250.0778,222.0839309.1003,294.0765,266.0814,210.0910291.1008,222.1115,204.1006,190.0853,165.0898,165.0536,149.0582206.0780,189.0755,188.0678,165.0518,149.0603334.1026,309.0703,279.0601,263.0653,177.0740,135.0404176.0678,149.0563,119.0459,91.0515305.0668,292.0912,277.0725,262.0855,249.0779,234.0905320.0909,306.1083,291.0843,276.0974,261.0741,248.1019322.1019,176.0671,149.0557,119.0452,91.0507319.0792,318.0723,317.0703,304.0958,276.0972,274.0812320.0532,308.0889,278.0784,280.0966,174.0520
aUVmax.(nm) 282 282 280 285 267,355,458 276,355 283 288 270,372 288 265,357,459 268,345,449 288 277 277,330
b
2
Score 96.5 99.1 97.3 90.6 99.3 92.4 97.2 96.4 93.0 99.3 90.1 95.6 96.3 90.7 5/92.2
1.
7
b
2
Error(ppm) 2.4 −0.3 −0.4 −1.1 0.3 3.8 1.2 −0.9 −2.8 −0.4 −1.2 3.4 −2.9 −2.8 −6/4.8
ed. 9.1
u
n
onti +) b703
Table2:Ca.m/zExp+M+H]/M 328.1535 342.1701 342.1701 326.1405 322.1074 324.1217 370.1603 354.1342 352.1187 324.1232 320.0917 334.1068 396.1454 334.1079 0858/358.0
([ 6.
3
3
ara O4 O4 O4 O4 +O4 +O4 O5 O5 O5 O4 +O4 +O4 O6 O4 O5
ulul N N N N N N N N N N N N N N N
Molecform CH1921 CH2023 CH2023 CH1919 CH1916 CH1918 CH2123 CH2019 CH2017 CH1917 CH1914 CH2016 CH2221 CH2015 CH1913
e
n
bi
u
Proposedalkaloid Coreximine Methylcoreximine1 Methylcoreximine2 Cheilanthifoline ehydrocheilanthifoline hyleneberberine/jatror Cryptopine Protopine Fumariline Stylopine Coptisine Corysamine ImpatienC Dihydrosanguinarine 8-Oxocoptisine
D et
m
e
D
aRT(min) 15.73 16.03 16.47 16.53 17.58 18.11 18.32 18.69 19.24 19.86 20.05 21.47 23.44 29.62 27.15
k
a
Pe∘N 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26