Screening and Identification of the Metabolites in Rat Plasma and Urine after Oral Administration of

16 Pages·2017·2.39 MB·English

molecules
Article
Screening and Identification of the Metabolites in
Rat Plasma and Urine after Oral Administration of
Areca catechu L. Nut Extract by Ultra-High-Pressure
Liquid Chromatography Coupled with Linear Ion
Trap–Orbitrap Tandem Mass Spectrometry
LuluLi1,†,ZhiqiangLuo1,†,YangLiu1,*,HaoWang1,AoxueLiu1,GuohuaYu1,MengweiLi1,
RuiruiYang1,XinjingChen1,JialianZhu1andBaoshengZhao2,*
1 SchoolofChineseMateriaMedica,BeijingUniversityofChineseMedicine,No.6,ZhonghuanSouthRoad,
Wangjing,ChaoyangDistrict,Beijing100102,China;[email protected](L.L.);[email protected](Z.L.);
[email protected](H.W.);[email protected](A.L.);[email protected](G.Y.);
[email protected](M.L.);[email protected](R.Y.);[email protected](X.C.);
[email protected](J.Z.)
2 ResearchInstituteofChineseMedicine,BeijingUniversityofChineseMedicine,No.11,
NorthThirdRingRoad,ChaoyangDistrict,Beijing100029,China
* Correspondence:[email protected](Y.L.);[email protected](B.Z.);
Tel./Fax:[phone](Y.L.);[phone](B.Z.)
† Theseauthorscontributedequallytothiswork.
Received:6June2017;Accepted:19June2017;Published:21June2017
Abstract: ArecacatechuL.nut,awell-knowntoxictraditionalherbalmedicine,hasbeenwidelyused
totreatvariousdiseasesinChinaandmanyotherAsiancountriesforcenturies. However,todate
the invivo absorption and metabolism of its multiple bioactive or toxic components still remain
unclear.Inthisstudy,liquidchromatographycoupledwithtandemmassspectrometrywasusedto
analyzethemajorcomponentsandtheirmetabolitesinratplasmaandurineafteroraladministration
ofArecacatechuL.nutextract(ACNE).Atotalof12compounds,including6alkaloids,3tanninsand
3aminoacids,wereconfirmedortentativelyidentifiedfromACNE.Invivo,40constituents,including
8prototypesand32metaboliteswereidentifiedinratplasmaandurinesamples. Insummary,this
studyshowedaninsightintothemetabolismofACNEinvivo,whichmayprovidehelpfulchemical
informationforbetterunderstandingofthetoxicologicalandpharmacologicalprofilesofACNE.
Keywords: ArecacatechuL.;majorcompounds;metabolites;UHPLC-LTQ-Orbitrap
1. Introduction
Traditional Chinese medicine (TCM) has played an important role in preventing or treating
avarietyofcomplicateddiseasesoverthousandsofyears[1–3]. Itisextensivelyacceptedthatthe
therapeuticortoxiceffectofTCMresultsfromtheprototypecomponentsand/ortheirmetabolites[4,5].
However,profilingtheabsorbedcomponentsandmetabolitesofanherbalmedicineinvivoisalways
agreatchallenge[6]. Metabolitesexistinavarietyofformsandareusuallypresentattracelevels;
therefore,thesignalsfromthemetabolitesareoftenmaskedbybackgroundnoisefromendogenous
interference[7]. Owingtoitshighselectivityandsensitivity,liquidchromatographycoupledwith
tandem mass spectrometry (LC–MS/MS) has been extensively used in drug metabolism studies.
LC–MS/MScombinedwithsophisticatedsoftwaretoolscanofferabundantstructuralinformationand
accuratemassmeasurementforbothprecursorandproductions,whichrepresentsapowerfuland
reliableanalyticaltechniquetodetectandidentifyunknownmetabolitesincomplexmatrixes[8–12].
Molecules2017,22,1026;doi:10.3390/molecules22061026 www.mdpi.com/journal/molecules
Molecules 2017, 22, 1026 2 of 15
MLoCle–cuMlesS2/0M17S, 2c2o,1m02b6ined with sophisticated software tools can offer abundant structural inform2aotfio16n
and accurate mass measurement for both precursor and product ions, which represents a powerful
and reliable analytical technique to detect and identify unknown metabolites in complex matrixes [8–12].
ArecacatechuL.oneofmostpopularmedicinalherbs,iswidelydistributedinthesouthofChina.
Areca catechu L. one of most popular medicinal herbs, is widely distributed in the south of China.
The nut of Areca catechu L. has been recorded in the pharmacopoeia of China for the treatment of
The nut of Areca catechu L. has been recorded in the pharmacopoeia of China for the treatment of
parasiticdiseases,dyspepsia,abdominalpain,etc.[13]. Modernpharmacologicalstudiesandclinical
parasitic diseases, dyspepsia, abdominal pain, etc. [13]. Modern pharmacological studies and clinical
practicerevealedthatArecacatechuL.nutshowsavarietyofpharmacologicalfunctions,including
practice revealed that Areca catechu L. nut shows a variety of pharmacological functions, including
wound healing [14], anti-migraine effect [15], anti-depressant effect [16], and hypoglycemic [17]
wound healing [14], anti-migraine effect [15], anti-depressant effect [16], and hypoglycemic [17] and
andantioxidanteffects[18]. Asatoxicherbalmedicine,ArecacatechuL.nutalsohassomepotential
antioxidant effects [18]. As a toxic herbal medicine, Areca catechu L. nut also has some potential
toxicities,suchasdeclineofspermcountsandmotilityandinductionofsubstantialabnormalities[19,20].
toxicities, such as decline of sperm count sand motility and induction of substantial abnormalities [19,20].
Althoughmanyreportsregardingthepharmacologicalactivitiesandpotentialtoxicitieshavebeen
Although many reports regarding the pharmacological activities and potential toxicities have been
publishedasdescribedabove,verylittleisknownaboutitsabsorbedcomponentsandmetabolites
published as described above, very little is known about its absorbed components and metabolites in vivo.
invivo.
Hence, the overall objective of this research was to investigate in detail the in vivo absorption
Hence,theoverallobjectiveofthisresearchwastoinvestigateindetailtheinvivoabsorption
and metabolism of Areca catechu L. nut extract (ACNE). As we all know, plasma and urine are
and metabolism of Areca catechu L. nut extract (ACNE). As we all know, plasma and urine are
recognized as ideal biological matrices in drug absorption and metabolism studies [21,22]. In this
recognized as ideal biological matrices in drug absorption and metabolism studies [21,22]. In this
work, a total of 26 components in the plasma and 31 components in the urine were characterized by
work, a total of 26 components in the plasma and 31 components in the urine were characterized
ultra-high-pressure liquid chromatography coupled with linear ion trap-Orbitrap tandem mass
byultra-high-pressureliquidchromatographycoupledwithlineariontrap-Orbitraptandemmass
spectrometry (UHPLC-LTQ-Orbitrap). From the results, we can know the probable metabolic
spectrometry(UHPLC-LTQ-Orbitrap).Fromtheresults,wecanknowtheprobablemetabolicpathways
pathways of chemical constituents in ACNE. To the best of our knowledge, this is the first study on
ofchemicalconstituentsinACNE.Tothebestofourknowledge,thisisthefirststudyonscreeningthe
screening the multiple absorbed and metabolic components of ACNE in vivo, which could provide a
multipleabsorbedandmetaboliccomponentsofACNEinvivo,whichcouldprovideascientificbasis
scientific basis for explaining the curative and toxicological mechanism of ACNE.
forexplainingthecurativeandtoxicologicalmechanismofACNE.
22.. RReessuullttss aanndd DDiissccuussssiioonn
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iinnffoorrmmaattiioonn iiss lliisstteedd iinn TTaabblele1 1.. TThhee ddeettaaiilleedd eexxttrraacctteedd iioonn cchhrroommaattooggrraapphhyy ooff AACCNNEE iiss sshhoowwnn iinn
FFiigguurree1 1a annddF FigiguurereS S11in int hteheS uSpupplpelmemenetnatlaml mataerteiarli.aTl. hTehset rsutrcutuctruesreosf othf eth1e2 1c2o mcopmoupnoudsndarse asrheo swhonwinn
Finig Fuirgeu2r.e 2.

Figure1.ExtractedionchromatogramofArecacatechuL.nutextract(ACNE)inpositiveionmode.
Figure 1. Extracted ion chromatogram of Areca catechu L. nut extract (ACNE) in positive ion mode.
Molecules2017,22,1026 3of16
Molecules 2017, 22, 1026 3 of 15

Figure2.Thestructuresofthe12compounds.
Figure 2. The structures of the 12 compounds.
Molecules2017,22,1026 4of16
Table1.ThemassspectraldataofcomponentsobservedbyUHPLC-LTQ-Orbitrap.
Component tR(Min) [M+H]+Ion(m/z) Error(ppm) Formula FragmentIons(m/z) Identification
1 1.36 118.0861 −1.568 C5H11NO2 72.0806 Valine
2 1.70 128.0705 −0.743 C6H9NO2 110.0601,81.0334 Guvacine/Isoguvacine
3 1.74 142.0862 −0.085 C7H11NO2 128.0745,124.0758,96.0807,81.0333 Arecaidine
4 1.87 144.1018 −0.869 C7H13NO2 126.0915,114.0913,84.0806 Methylpiperidine-3-carboxylate
5 3.05 182.0810 −0.822 C9H11NO3 165.0548,136.0760 Tyrosine
6 3.47 142.0862 −0.115 C7H11NO2 113.9638,110.0602,81.0334 Guvacoline
7 4.09 156.1018 −0.115 C8H13NO2 124.0757,113.0597,96.0807,81.0333 Arecoline
8 7.81 156.1017 −0.255 C8H13NO2 128.1069,113.0597,81.0333 Arecolidine
9 8.68 579.1489 −1.334 C30H26O12 453.1197,427.1039,409.0937,291.0874 ProcyanidinB2
10 9.48 291.0861 −0.669 C15H14O6 273.0762,165.0548,151.0392,139.0392,123.0443 Catechin(C)
11 9.53 205.0970 −0.996 C11H12N2O2 188.0917,159.9331 Tryptophan
12 12.20 291.0860 −0.978 C15H14O6 273.0764,165.0549,151.0393,139.0393,123.0444 Epicatechin(EC)
tR:retentiontime.
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2.1.1. Alkaloids
2.1.1. Alkaloids
Component7(4.09min, −0.115ppm)showedtheaccurate[M+H]+ ionatm/z156.1018and
Component 7 (4.09 min, −0.115 ppm) showed the accurate [M + H]+ ion at m/z 156.1018 and
produced the characteristic ions at m/z 124.0757 [M + H-OCH -H ]+, 113.0597 [M + H-C H N]+,
produced the characteristic ions at m/z 124.0757 [M + H-OCH3-H2]+3, 1132.0597 [M + H-C2H5N]+, 962.08507
96.[0M80 +7 H[M-C2+H4HO-2C]+ 2aHnd4O 812.]0+3a33n d[M8 1+. H03-3C32H[M4O2+-CHH-3C]+.2 HBy4 Oco2m-CpHar3i]n+g. wBiythc tohme rpeaferirnengcwe ciothmtphoeurnedf earnedn ce
comthpe oluitnerdataunred dthaeta li[t2e3r]a, tucorempdoantaen[t2 37] ,wcoasm ipdoennetinfited7 wasa sariedceonlitniefi.e dThaes parroepcoosliende .fraTghmeepnrtoatpioonse d
frapgamthewntaayt ioofn apreactohlwinaey iso sfhaorwecno liinn Feiigsusrhe o3wa.n inFigure3a.

FigFuigreur3e. 3T. hTehpe rporpoopsoesdedf rfaraggmmeenntatatitoionnp paatthhwwaayyss ooff aarreeccoolliinnee ((aa)); ;ccaatetecchhinin (b(b) )anadn dvavlainlien (ec)( cin) ipnopsiotisvitei ve
ioniomn omdoed.e.
Component 8 (7.81 min, −0.255 ppm) displayed the molecular ion at m/z 156.1017. Moreover, it
Component8(7.81min,−0.255ppm)displayedthemolecularionatm/z156.1017. Moreover,
exhibited three major ions at m/z 128.1069 [M + H-2CH3]+, 113.0597 [M + H-3CH3]+ and 81.0333 [M +
itexhibitedthreemajorionsatm/z128.1069[M+H-2CH ]+,113.0597[M+H-3CH ]+ and81.0333
H-2OCH3-CH3]+. Accordingly [24], component 8 was tentati3vely identified as arecolidin3e.
[M+H-2OCH -CH ]+. Accordingly[24],component8wastentativelyidentifiedasarecolidine.
3 3
Molecules2017,22,1026 6of16
Component2(1.70min,−0.743ppm)providedthe[M+H]+ionatm/z128.0705,whichledtotwo
majorproductionsatm/z110.0601[M+H-H O]+and81.0334[M+H-COOH-H ]+.Inaccordancewith
2 2
theliteraturedata[25],component2wastentativelyconfirmedasguvacineoritsisomer,isoguvacine.
Component3(1.74min,−0.085ppm)exhibitedthemolecularionatm/z142.0862. Itgaveriseto
fourfragmentionsatm/z128.0745[M+H-CH ]+,124.0758[M+H-H O]+,96.0807[M+H-CH O ]+
2 2 2 2
and81.0333[M+H-CH O -CH ]+. Accordingtotheliteraturedata[26],component3wasidentified
2 2 3
asarecaidine.
Component6(3.47min,−0.115ppm)alsoshowedthemolecularionatm/z142.0862whichwas
a14-Da(CH )lossofcomponent7. Meanwhile,component6producedfragmentionsatm/z113.9638
2
[M+H-CH N]+,110.0602[M+H-OCH -H ]+and81.0334[M+H-C H O ]+. Bycomparingwiththe
3 3 2 2 4 2
referencecompoundandtheliteraturedata[25],component6wasconfirmedasguvacoline.
Component 4 (1.87 min, −0.869 ppm) yielded molecular ion at m/z 144.1018. Moreover, it
showedmajorfragmentionsatm/z126.0915[M+H-CH -H ]+,114.0913[M+H-CH N]+and84.0806
3 2 3
[M+H-C H O ]+. Thus,component4wastentativelyidentifiedasmethylpiperidine-3-carboxylate.
2 4 2
2.1.2. Tannins
Component10(9.48min,−0.669ppm)andcomponent12(12.20min,−0.978ppm)showedthe
same[M+H]+ionatm/z291.0861,andbothofthemproducedthesamefragmentionsatm/z273.0762
[M+H-H O]+,165.0548[M+H-C H O ]+,151.0392[M+H-C H O ]+,139.0392[M+H-C H O ]+
2 6 6 3 7 7 3 8 7 3
and 123.0443 [M + H-C H O -OH]+. According to the literature data [27], they were tentatively
8 7 3
designatedascatechin(C)andepicatechin(EC),respectively. Theproposedfragmentationpathwayof
catechinisshowninFigure3b.
Compound9(8.68min,−1.334ppm)exhibitedthe[M+H]+ionatm/z579.1489,andgaveriseto
fourmajorproductionsatm/z453.1197,427.1039,409.0937and291.0874. Thefragmentionatm/z
409.0937wasgeneratedthroughthelossofH Ofromtheionatm/z427.1039;thefragmentionatm/z
2
427.1039wasattributedtotheretroDiels-Alder(RDA).Theionatm/z291.0874[M+H-C H O ]+
15 13 6
was a prototype losing an aggregate unit. According to the literature data [28], component 9 was
tentativelycharacterizedasprocyanidinB2.
2.1.3. AminoAcid
Component1(1.36min,–1.568ppm)displayedthemolecularionatm/z118.0861whichledthe
majorproductionatm/z72.0806[M+H-HCOOH]+. Bycomparingwiththereferencecompound,
component1wasidentifiedasvaline. Theproposedfragmentationpathwayofvalineisshownin
Figure3c.
Compound 5 (3.05 min, −0.822 ppm) exhibited [M + H]+ ion at m/z 182.0810 and the main
fragmentionsofitwereatm/z165.0548and136.0760. Theformerionatm/z165.0548wasproduced
fromtheneutrallossofNH ,andthelatterionatm/z136.0760wasgeneratedthroughthelossof
3
HCOOHfromthemolecularion. Hence,compound5wastentativelyidentifiedastyrosine.
Compound11(9.53min,−0.996ppm)showedthe[M+H]+ionatm/z205.0970andityielded
major ions at m/z 188.0917 [M + H-NH ]+ and 159.9331 [M + H-HCOOH]+. Based on these data,
3
component11wastentativelyassignedastryptophan.
2.2. IdentificationoftheMajorMetabolitesinACNEbyUHPLC-LTQ-Qrbitrap
The identification of the compounds in rat urine and plasma were also performed by
UHPLC-LTQ-Qrbitrap. Altogether,40compounds,including8prototypeand32metaboliteswere
identifiedbycomparingtheretentiontimeandthemassdata(Table2). Amongthem,31ofthemwere
fromraturinewhile26werefromratplasma. Detailedextractedionchromatogramsoftheurine
sampleareshowninFigure4,andtheplasmasampleareshowninFigure5. Inaddition,themetabolic
pathwaysofcatechin(C)andarecolinearesummarizedinFigure6.
Molecules2017,22,1026 7of16
Table2.ThemassspectraldataofmetabolitesobservedbyUHPLC-LTQ-Orbitrap.
Metabolite tR Selected Calculated Error Formula Fragmentation Identification Source
(Min) Ion Mass(m/z) (ppm)
M1 1.75 [M+H]+ 128.0706 −0.352 C6H9NO2 110.0601,81.0332 Guvacine/Isoguvacine Urine;Plasma
M2 1.76 [M+H]+ 142.0862 −0.740 C7H11NO2 124.0759,96.0809,81.0334 Arecaidine Urine;Plasma
M3 1.78 [M+H]+ 144.1018 −0.661 C7H13NO2 126.0915,114.0915,84.0806 Methylpiperidine-3-carboxylate Urine;Plasma
M4 1.96 [M+H]+ 158.0811 −0.315 C7H11NO3 140.0529,112.0870 ArecaidineN-oxide Urine;Plasma
M5 3.28 [M+H]+ 142.0862 −0.388 C7H11NO2 113.0597,110.0601,81.0333 Guvacoline Urine;Plasma
M6 3.58 [M+H]+ 156.1018 −0.418 C8H13NO2 124.0758,113.0598,96.0807,81.0333 Arecoline Urine
M7 4.31 [M+H]+ 319.1319 −1.000 C13H22N2O5S 287.1068,277.1223,190.0901,156.1022 Arecolinemercapturicacid Urine;Plasma
M8 5.58 [M+H]+ 343.1862 −0.417 C16H26N2O6 172.0974 ArecolineN-oxidedimer Urine;Plasma
M9 5.79 [M+H]+ 172.0966 −1.103 C8H13NO3 154.0866,140.0710,112.0759 ArecolineN-oxide Urine;Plasma
M10a 7.60 [M−H]− 465.1025 −0.586 C21H22O12 289.0719 GlucuronidationofC/EC Urine
M11 7.67 [M+H]+ 144.1019 −0.175 C7H13NO2 126.0916,98.0965,84.0807 N-methylnipecoticacid Urine;Plasma
M12a 7.90 [M−H]− 369.0282 1.872 C15H14O9S 289.0723 SulfationofC/EC Urine
M10b 8.12 [M−H]− 465.1037 1.973 C21H22O12 289.0721 GlucuronidationofC/EC Urine
M12b 8.22 [M−H]− 369.0282 2.279 C15H14O9S 289.0723 SulfationofC/EC Urine
M13 8.33 [M+H]+ 335.1270 −0.369 C13H22N2O6S 317.1172,303.1019,293.1173,206.0851,172.0973 ArecolineN-oxidemercapturicacid Urine;Plasma
M14 8.41 [M+H]+ 170.0812 −0.057 C8H11NO3 152.0711 Norepinephrine Urine;Plasma
M10c 8.47 [M−H]− 465.1024 −0.715 C21H22O12 289.0716 GlucuronidationofC/EC Plasma
M15 8.60 [M+H]+ 579.1491 −1.023 C30H26O12 453.1199,427.1041,409.0938,291.0875 ProcyanidinB2 Urine;Plasma
M16a 9.20 [M−H]− 383.0435 1.020 C16H16O9S 303.0879,137.0249 Sulfation+methylationofC/EC Urine
M17a 9.31 [M+H]+ 291.0865 0.602 C15H14O6 273.0766,165.0550,151.0394,139.0394,123.0444 Catechin(C) Urine;Plasma
M10d 9.53 [M−H]− 465.1031 0.790 C21H22O12 289.0721 GlucuronidationofC/EC Plasma
M18a 9.86 [M−H]− 479.1187 0.579 C22H24O12 303.0877,289.0716,175.0249 Methylation+glucuronidationofC/EC Urine
M18b 10.12 [M−H]− 479.1179 −1.132 C22H24O12 303.0873,289.0715,175.0247 Methylation+glucuronidationofC/EC Urine
M19a 10.16 [M+H]+ 305.1020 0.083 C16H16O6 287.0921,179.0707 MethylationofC/EC Urine;Plasma
M19b 10.50 [M+H]+ 305.1021 0.280 C16H16O6 287.0923,179.0708 MethylationofC/EC Urine;Plasma
M16b 10.73 [M−H]− 383.0430 −0.259 C16H16O9S 303.0876,137.0249 Sulfation+methylationofC/EC Urine
M16c 11.73 [M−H]− 383.0427 −1.225 C16H16O9S 303.0882,137.0250 Sulfation+methylationofC/EC Urine
M17b 11.99 [M+H]+ 291.0864 0.293 C15H14O6 273.0767,165.0551,151.0394,139.0394,123.0445 Epicatechin(EC) Urine;Plasma
M16d 12.72 [M−H]− 383.0429 −0.729 C16H16O9S 303.0882,137.0250 Sulfation+methylationofC/EC Urine
M19c 12.94 [M+H]+ 305.1020 −0.015 C16H16O6 287.0923,179.0708 MethylationofC/EC Urine;Plasma
M16e 13.01 [M−H]− 383.0436 1.334 C16H16O9S 303.0876,137.0250 Sulfation+methylationofC/EC Plasma
M20 13.06 [M+H]+ 184.0968 −0.271 C9H13NO3 166.0865,152.0710 Epinephrine(EP) Urine
M16f 13.29 [M−H]− 383.0432 0.289 C16H16O9S 303.0875,137.0249 Sulfation+methylationofC/EC Plasma
M18c 13.70 [M−H]− 479.1183 −0.297 C22H24O12 303.0876,289.0718,175.0249 Methylation+glucuronidationofC/EC Plasma
M12c 13.89 [M−H]− 369.0271 −0.946 C15H14O9S 289.0722 SulfationofC/EC Plasma
M18d 13.93 [M−H]− 479.1179 −1.007 C22H24O12 303.0876,289.0717,175.0249 Methylation+glucuronidationofC/EC Plasma
M21 14.27 [M+H]+ 321.0943 −2.549 C16H16O7 303.1331,289.0689 Methylationofepigallocatechin(EGC) Urine
M16g 14.34 [M−H]− 383.0432 2.378 C16H16O9S 303.0883,137.0250 Sulfation+methylationofC/EC Plasma
M19d 14.35 [M+H]+ 305.1018 −0.507 C16H16O6 287.0920,179.0706 MethylationofC/EC Urine
M12d 14.68 [M−H]− 369.0270 −1.352 C15H14O9S 289.0722 SulfationofC/EC Plasma
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Molecules 2017, 22, 1026 8 of 15

FFigiguurere4 4..E Exxtrtaraccteteddi oionnc chhrorommaatotoggrarammsso offt htheeu urirninees asammpplelei ninp poosistiitviveei oionnm mooddee( a(a););t htheeb blalannkku urirninee
sampleinpositiveionmode(b);theurinesampleinnegativeionmode(c);theblankurinesamplein
sample in positive ion mode (b); the urine sample in negative ion mode (c); the blank urine sample in
negativeionmode(d).
negative ion mode (d).
MMoolelcecuulelses2 2001177,,2 222,,1 1002266 99o off1 165

Figure5.Extractedionchromatogramsoftheplasmasampleinpositiveionmode(a);theblankplasma
Figure 5. Extracted ion chromatograms of the plasma sample in positive ion mode (a); the blank
sampleinpositiveionmode(b);theplasmasampleinnegativeionmode(c);theblankplasmasample
plasma sample in positive ion mode (b); the plasma sample in negative ion mode (c); the blank plasma
innegativeionmode(d).
sample in negative ion mode (d).
Molecules2017,22,1026 10of16
Molecules 2017, 22, 1026 10 of 15

FiFgiugruere6 .6.T Thheep prrooppoosseedd mmeettaabboolliicc ppaatthhwwaayyss ooff aarreeccoolilninee (a()a )anadn dcactaetcehcihni n(b()b. ).
2.22.1.2..1P.r Portoottoytpyepses(M (M1,1M, M22,,M M33,,M M55,,M M66,, MM1155,, MM1177))
MM1 (11 (.17.575m minin,, −−00.3.35522 ppppmm) s)hsohwoewde tdhet h[Me [+M H]++ ioHn] +ati mon/z a1t28m.0/7z061,2 a8n.d07 g0a6v,ea rnisde tgoa tvweor pisreodtoucttw o
proiodnusc taito mns/za t11m0/.0z610110 a.0n6d0 18a1n.0d33821,. 0w33h2ic,hw whiecrhe wcoenresicsotennsti swteintht wthiteh pthroedpurcotd iuocntsi oonf scoofmcpomonpeonnt e2n. t2.
ThTehreefroerfeo,reM, M1w1 wasaisd iednetnitfiifeideda sasg guuvvaaccinineeo orr iissoogguuvvaacciinnee..
MM2(21 .(716.7m6 mini,n−, −00.7.74400p pppmm)) ddiissppllaayyeedd tthhee [[MM ++ HH]]++ ioionn aat tmm/z/ 1z4124.028.06826, 2a,nadn pdrpodroudcuedce fdrafgrmagemnte nt
ions at m/z 124.0759, 96.0809 and 81.0334 corresponding to component 3. Thus, M2 was tentatively
ionsatm/z124.0759,96.0809and81.0334correspondingtocomponent3. Thus,M2wastentatively
identified as arecaidine.
identifiedasarecaidine.
M3 (1.78 min, −0.661 ppm) showed the [M + H]+ ion at m/z 144.1018, and it produced the fragment
M3 (1.78 min, −0.661 ppm) showed the [M + H]+ ion at m/z 144.1018, and it produced the
ions at m/z 126.0915, 114.0915 and 84.0806 corresponding to component 4. Based on these data, M3
fragmentionsatm/z126.0915,114.0915and84.0806correspondingtocomponent4. Basedonthese
was identified as methyl piperidine-3-carboxylate.
data,M3wasidentifiedasmethylpiperidine-3-carboxylate.
M5 (3.28 min, −0.388 ppm) displayed the same [M + H]+ ion at m/z 142.0862. In the MS/MS
M5(3.28min, −0.388ppm)displayedthesame[M+H]+ ionatm/z142.0862. IntheMS/MS
spectrum, M5 led three major fragment ions at m/z 113.0597, 110.0601 and 81.0333 which were
spectrum, M5 led three major fragment ions at m/z 113.0597, 110.0601 and 81.0333 which were
consistent with component 6. Therefore, M5 was tentatively identified as guvacoline.
consistentwithcomponent6. Therefore,M5wastentativelyidentifiedasguvacoline.
M6 (3.58 min, −0.418 ppm) displayed the [M + H]+ ion at m/z 156.1018. In the MS/MS spectrum,
M6(3.58min,−0.418ppm)displayedthe[M+H]+ionatm/z156.1018. IntheMS/MSspectrum,
M6 yielded ions at m/z 124.0758, 113.0598, 96.0807 and 81.0333, which were the diagnostic ions of
M6coymieplodneednito 7n. sThatusm, /Mz61 w24a.s0 c7o5n8f,ir1m13e.d0 5a9s8 a,r9ec6o.0li8n0e7. and81.0333,whichwerethediagnosticionsof
componMe1n5t (78..6T0h umsi,nM, −61.w02a3s pcopnmfi)r mexehdibaitsedar tehceo l[iMne .+ H]+ ion at m/z 579.1491, and led to four major
proMdu15ct( i8o.n6s0 amt min/,z −4513.0.121399p,p 4m27).1e0x4h1i,b 4i0te9d.09t3h8e a[nMd +29H1.]0+87io5,n wahtimch/ wz5er7e9 t.1h4e9 c1h,aarnacdtelreidstitco ffroagumrmenat jor
proiodnusc otfi ocnomsaptomne/nzt 495. 3C.1o1n9s9e,q4u2e7n.t1l0y4, 1M,1450 9w.0a9s3 t8enatnadtiv2e9l1y. 0c8o7n5s,idwehreicdh tow beere ptrhoecycahnairdaicnt eBr2is. ticfragment
ionsofcomponent9. Consequently,M15wastentativelyconsideredtobeprocyanidinB2.