w ww . e l s e v i e r . c o m / l o c a t e / b j p
Original
Article
Chemical
identification
and
quantification
of
Hu-Gu
capsule
by
UHPLC–Q-TOF-MS
and
HPLC-DAD
Qiang
Ren
a,∗,
Shi-song
Long
b,∗aDepartmentofPharmacy,JiningMedicalUniversity,Shandong,China
bGuangdongAnnolPharmaceuticalCo.,Ltd,Guangdong,China
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received7February2017 Accepted5June2017 Availableonline13July2017
Keywords: Hu-Gucapsule UHPLC–Q-TOF-MS HPLC-DAD Identification Quantification
a
b
s
t
r
a
c
t
Hu-GucapsuleisatraditionalChinesemedicalformulaforpreventingglucocorticoid-induced osteoporo-sis.Inthiswork,anultra-highperformanceliquidchromatographycoupledwithelectrosprayionization quadrupoletime-of-flightmassspectrometrywasdevelopedforchemicalidentificationofthe phyto-chemicalcompositionsinpositiveionmode.Atotalof41compoundsweredetectedinHu-Gucapsule, and25compoundswerecharacterizedandidentifiedbyretentiontime,accuratemasswithin5ppm errorandcharacteristicfragmentions.Amongofthem,fourmajorflavonoidswerefurtherquantifiedby highperformanceliquidchromatographydiodearraydetectioninthefourbatches.Themethodwas val-idatedintermsofcalibrationcurveregressioncoefficient(r2>0.9994),repeatability(RSD<3.5%),
intra-andinter-dayprecision(RSD<1.1%),recovery(93.0–106.4%),limitofdetection(0.1g/ml),andlimitof
quantification(1g/ml).Theresearchcouldprovidetheanalysisofchemicalcompositionandbehelpful
tocontrolthequalityofHGC.
©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Hu-Gucapsule(HGC)hasbeenusedasatraditionalmedicine
inChinaforpreventingglucocorticoid-inducedosteoporosis(GIO).
The formula is composedof ten Chineseherbs as described in
Box 1. According to Chinese National Drug Quality Standard
(volume82),thequalitycontrolofHGCmainlyconfinestoonly
an icariin (≥1.15mg/0.45g, per capsule) by high performance
liquidchromatographymethod(TheStateCommissionofChinese
Pharmacopoeia,2010),whichisfarfrommeetingthefurtherdepth
researchof HGConthe pharmacology.Toour bestknowledge,
theglobal chemical profileof HGC hasnot beenreported, and
fewreportsareavailableonitsqualitycontrol(Songetal.,2012). Therefore,it iscrucial todevelop aqualitative andquantitative
methodforelucidatingthesystematicchemicalcontourofHGC.
Nowadays,UHPLC–Q-TOF-MShasbecomeoneofdominanttools
toinvestigatethecomplexconstituentsofherbalformulas.Unlike
singlequadruple,iontraportandemmassspectrometers,aTOF
analyzerhasthehighresolution,highsensitivityandhighaccuracy
andadvantagetoprovidetheexcellentmassaccuracyfor
deter-minationofelementalcompositionandhighmassresolutionfor
∗ Correspondingauthors.
E-mails:ren2323@163.com(Q.Ren),long.shisong@annol.net(S.Long).
thedifficultychromatographicseparation(Liuetal.,2016;Xiang etal.,2015;Renetal.,2016).
Theaimofpresentstudyistodevelopacomprehensive
analy-sismethodforthechemicalprofileofHGCbyUHPLC–Q-TOF-MS.
The structures were elucidated on the basis of their accurate
massandcharacteristicfragmentationbehaviorofreference
sub-stances.Furthermore,fourflavonoidsinHGCwerequantifiedby
HPLC-DAD.
Experimental
Samples,chemicals,andreagents
HGC (batch number: 20160203, 20160501, 20160502, and
20160503)wereproducedbyGuangdongAnnolPharmaceutical
Co., Ltd.Epimedin A,epimedin B, epimedinC, and icariin were
purchasedfromtheNationalInstitutefortheControlof Pharmaceu-ticalandBiologicalProducts(NICPBP,BeijingChina).Thepurities
ofthereferencecompoundsweredeterminedtobeabove98%by
HPLC-DADanalysis.LC/MS-gradeacetonitrilewaspurchasedfrom
MallinckrodtBaker,Inc.(Phillipsburg,NJ,USA).HPLC-gradeacetic acid(AS1102-002)waspurchasedfromTEDIA,Inc(Fairfield,USA). DeionizedwaterwaspurifiedbyMilliporepurificationsystem (Mil-lipore,MA,USA).
http://dx.doi.org/10.1016/j.bjp.2017.06.002
Box1:CompositionofHu-Gucapsule.
DrugLatinname Speciesname Chinesename Rate
PolygoniMultifloriRadixPraeparata Reynoutriamultiflora(Thunb.)Moldenke,Polygonaceae 1.67
EpimediumbrevicornuMaxim EpimediumbrevicornuMaxim,Berberidaceae 1.33
RehmanniaeRadixPraeparata Rehmanniaglutinosa(Gaertn.)DC.,Plantaginaceae 1.67
TestudinisCarapaxetPlastrum Chinemysreevesii(Gray),Emydidae 1
MorindaeOfficinalisRadix MorindaofficinalisF.C.How,Rubiaceae 1.33
EucommiaeCortex EucommiaulmoidesOliv.,Eucommiaceae 1.33
DipsaciRadix DipsacusasperWall.exC.B.Clarke,Caprifloiaceae 1.33
DrynariaeRhizoma DrynariaroosiiNakaike,Polypodiaceae 1.33
AngelicaeSinensisRadix Angelicasinensis(Oliv.)Diels,Apiaceae 1
DioscoreaeRhizoma DioscoreaoppositifoliaL.,Dioscoriaceae 1.33
Samplepreparation
Analiquot (500mg)ofHGCpowderwasaccuratelyweighed
andsoakedwith5ml80%methanolfor15min.Itwasextracted
byultrasonictreatment(140W,25◦C)for30min.After
centrifuga-tionat14,800×gfor10min,thesupernatantwasfilteredthrough a0.22mfilterpriortoinjectionintotheUHPLC–Q-TOF-MSand
HPLC-DADsystem.
AnalysisofHGCbyUHPLC–Q-TOF-MS
Agilent1290seriesRapidResolutionLCsystemwascoupledto
Agilent6530Accurate-MassQuadrupoleTimeof Flight(Q-TOF)
massspectrometer(AgilentTechnologies,CA,USA).It equipped
withanESIinterface.Thechromatographicseparationwas
per-formed on TOSOH TSK gel ODS-100Z (4.6×150mm, 3.0m)
column.Thecolumntemperaturewasmaintained30◦C,the
injec-tionvolumewas2l.Themobilephaseconsistedof0.1%aqueous aceticacid(v/v)(A)andacetonitrile(B).Thegradientprogramwas appliedasfollows:0–50minat10–45%B;50–60minat40–90%B; 60–62minat90–10%B;and62–70minat10%B.TheoutletofHPLC wassplit(1:5)andintroducedintotheESIsourceat4–62minand intowasteat0–4minand62–70min.
TheMSconditionswere setas follows:drying gasflowrate
10l/min;dryinggastemperature350◦C;pressureofnebulizergas
pressure42psig;sheathgastemperature335◦C;sheathgasflow
11l/min;capillaryvoltage3500Vpositiveionmodesandthemass
rangefromm/z200to1000.TheMS/MSspectrawereacquired
withautoMS/MSmodeatthescanrateof2spectra/s.Thedata
wasacquiredwithMassHunterAcquisitionB.06.00andanalyzed
bymeansofMass HunterQualitative Software,VersionB.06.00
(AgilentTechnologies,CA,USA).
QuantificationbyHPLC-DAD
QuantificationwasperformedonaShimadzuliquid
Chromatog-raphysystem.Theinstrumentwasequippedwithtwo LC-20AT
pumps, a SIL-20A auto sampler, a CTO-20AC column oven, a
SPD-M20Adiodearraydetector,aCBM-20Acommunicationsbus
module,andaDGU-20A3 degasser.LCsolutionsoftwareversion
1.24SP1wasusedtocontroltheHPLCsystemandprocessthedata.
A TOSOH TSK gel ODS-100Z (4.6×150mm, 3m) column
(Tosoh Bioscience, Japan) was used for the chromatographic
separation. Thecolumn temperaturewas maintained35◦C, the
injectionvolumewas5l,anddetectionwavelengthwassetat
269nm.Themobilephaseswerecomposedofwater(0.2%
aque-ousaceticacid,A)andacetonitrile(B).Thegradientprogramwas
appliedasfollows:0–50minat10–35%B;50–60minat35–95%
B;60–62minat95–10%B;and62–70minat10%B.Flowratewas
1ml/min.
Resultsanddiscussion
A total of 41 compounds were detected in HGC, and 25
compounds were tentatively identified and characterized by
UHPLC–Q-TOF-MS in the positive mode. They are summarized
alongwiththeirretentiontime,theoreticalmass,molecular for-mula,observedmass,MS/MSfragmentsasshowninTable1.Atotal
ionchromatogramandextractionchromatogramof25identified
compoundsareshowninFig.1.Tandemmassspectraandpossible
fragmentpathwaysof12typicalconstituentsinpositivemodeare presentedinFig.S1.
Characterizationandidentificationofprenylatedflavonoids
Fifteen prenylated flavonoids were identified in HGC. The
prenylatedflavonoidglycosidesarecharacteristicconstituentsof Epimediumbrevicornu.Theanhydroicaritinflavonoidsareusually 3-O,7-O,and3,7-di-O-glycosideswithamonosaccharideor
dis-accharide (Wang et al.,2010).The characteristic fragmentation
pathwaysindicated thatglucosesubstitutedatthe3-Oposition losteasierthanatthe7-Oposition.Theionofm/z369wasformed.
The rearrangement of theisopentane group in position C8 led
tothe formation of a product ion m/z 313(Shevlyakova et al.,
2016).
Compounds29,32,33,and34wereidentifiedbycomparison
withreferencesubstances.They wereidentifiedasepimedin A,
epimedinB, epimedinC,and icariin,respectively.Theepimedin
A,B,C,andicariinindicatedthesimilaritiesoffragmentation pat-terns.Allofthempresentedthreecharacteristicfragmentionsat m/z531,369,and313.Theionofm/z369wasthetypicalfragment foranhydroicaritinaglycone.Theionatm/z313wasformedby fur-therlossofC4H7(56Da)fromtheionofm/z369.Fortheprenylated
flavonoids,theglucosesubstitutedatthe3-Opositionlosteasier thanatthe7-Opositionaccordingtothecharacteristic
fragmenta-tionpathways.TheMS2spectrumgavethebasepeakatm/z531,
whichcorrespondedtoanhydroicaritinaglyconebearingO-linked monosaccharidesubstituent(Dingetal.,2011).
Compounds 31 (tR=32.70min), 36 (tR=39.40min), 38
(tR=52.02min),and41(tR=54.39min),[M+H]+ionsindicatedat
m/z 839.2971, 677.2455, 531.1874, and 515.1933, respectively.
The MS2 spectrum gave the same characteristic fragmentions
atm/z 369and 313.Compounds31,36, 38, and 41 possessan
anhydroicaritinaglyconeskeleton. Compound 31gave thebase
peakatm/z 531,due tothelossof bothglucoseand rhamnose
moieties(308Da).Theionswerecleavagedatm/z677[M–Glu+H]+,
531 [M–Glu–Rha+H]+, and 369[M–2Glu–Rha+H]+, respectively.
Compound31 wastentatively identified asepimedin A1.
Com-pound36yieldedamajorfragmentionatm/z369,duetothelossof bothglucoseandrhamnosemoieties(308Da).Theionswere
frag-mentedatm/z531[M–Rha+H]+and313[M–Glu–Rha–C
4H7+H]+,
Table1
IdentificationofthechemicalconstituentsfromHGCbyUHPLC–Q-TOF-MS.
PeakNo. tR(min) Theoreticalmass [M+H]+or[M+Na]+ Error(ppm) Observedmass max(nm) MS/MSfragments
(relativeion intensity,%)
Identification compounds
1 5.51 365.0867 C17H16O9 −3.00 365.0878 221,293 288.0345(43),
203.0509(100), 180.0639(50), 136.0570(30), 104.9880(69)
Unknown
2 5.74 379.0660 C17H14O10 0.19 379.0659 231,293 339.1278(14),
295.0779(15), 233.0214(100), 197.5969(10), 169.0289(17), 147.0468(63)
Unknown
3 5.82 399.1286 C18H22O10 0.18 399.1285 – 255.0827(58),
237.0741(100), 203.0459(23), 167.0317(31), 149.0169(14)
Unknown
4 6.12 441.1391 C20H24O11 1.45 441.1385 196,255 205.0092(100) Unknown
5 7.07 515.2639 C29H38O8 −2.05 515.2650 – 269.1270(100),
210.0535(67), 182.0557(16)
Unknown
6 7.36 359.1489 C20H22O6 2.55 359.1480 236,280 191.0669(100) Unknown
7 7.83 355.1024 C16H18O9 −4.07 355.1038 240,328 163.0393(100),
145.0297(17), 135.0436(6), 117.028(7)
Chlorogenic acid
8 8.06 355.1024 C16H18O9 −4.07 355.1038 242,328 163.0393(100),
145.0314(14), 135.0434(9), 117.0393(3)
Cryptochlorogenic acid
9 9.21 509.1290 C23H24O13 −0.06 509.1290 252,282 509.1291(100),
363.0718(13), 331.1036(46), 201.0154(11)
Unknown
10 9.55 413.1442 C19H24O10 1.51 413.1436 245 413.1426(100),
251.0932(11), 219.0611(20)
CnidiosideB methylester
11 9.72 739.2444 C34H42O18 1.61 739.2432 – 381.1175(100),
219.0630(6), 149.0207(3)
Unknown
12 11.98 342.1700 C20H23NO4 −3.86 342.1713 – 342.1706(28),
297.1140(82), 282.0874(22), 265.0861(100), 237.0942(24)
Magnoflorine
13 13.77 413.2170 C21H32O8 0.96 413.2166 241 413.2163(100),
203.0536(15)
Unknown
14 16.85 547.2174 C28H34O11 1.99 547.2163 – 547.2151(100),
385.1600(18)
3-
Hydroxyicariine-O-glucoside 15 17.09 619.1633 C27H31O15Na −4.46 619.1661 230,285 619.1640(100),
473.1125(3), 331.1005(12)
Neoeriocitrin
16 17.56 363.1438 C19H22O7 1.19 363.1434 243,322 363.1366(100),
191.0420(35), 117.0669(45)
Unknown
17 18.13 407.1337 C20H22O9 −4.78 407.1356 230,320 245.0818(100),
227.0715(32), 199.0767(69), 181.0603(10), 151.0392(46), 125.0244(99), 121.0637(37), 107.0484(17)
Stilbene glucoside
18 18.69 355.1751 C18H26O7 1.78 355.1745 – 355.1726(100),
131.0513(7)
Unknown
19 19.36 518.2146 C27H33O10 1.25 518.2140 – 518.2109(98),
356.1590(100)
Unknown
20 19.87 435.2589 C21H38O9 1.98 435.2580 – 435.2529(100),
349.1808(34), 321.1940(9), 293.1556(9)
Table1(Continued)
PeakNo. tR(min) Theoreticalmass [M+H]+or[M+Na]+ Error(ppm) Observedmass max(nm) MS/MSfragments
(relativeion intensity,%)
Identification compounds
21 21.22 581.1865 C27H32O14 −2.27 581.1878 229,284 339.0889(17),
315.0855(16), 297.0741(20), 273.0772(100), 195.0279(55), 153.0171(13), 129.0547(22)
Naringin
22 22.12 769.2550 C35H44O19 1.24 769.2540 243,328 769.2556(100),
607.2022(5), 395.1308(5)
Unknown
23 23.09 247.0965 C14H14O4 2.79 247.0958 278 187.8379(100) Diallyl
phthalate
24 25.48 825.2812 C38H48O20 0.93 825.2804 – 663.2378(8),
517.1745(100), 355.1176(58)
DiphyllosideA
25 27.42 489.2119 C26H32O9 0.84 489.2115 – 489.2111(23),
309.1462(100), 281.1513(11)
Unknown
26 27.46 663.2283 C32H38O15 −2.35 663.2299 – 517.1756(23),
355.1194(100), 299.0526(6)
EpimedosideA
27 28.51 693.2389 C33H40O16 −1.86 693.2402 – 547.1816(38),
385.1317(100), 367.1011(11), 329.0609(18)
3-
Hydroxyicariine-O -glucose-rhamnose
28 31.29 431.1337 C22H22O9 1.53 431.1330 – 431.1257(100),
269.0802(57)
Ononin
29 31.81 839.2968 C39H50O20 −1.29 839.2979 270 677.2451(7),
531.1903(100), 369.1363(45), 313.0665(10)
EpimedinA
30 32.49 455.0973 C23H18O10 2.14 455.0963 – 455.1004(29),
293.0420(100), 185.0394(59)
Unknown
31 32.70 839.2968 C39H50O20 −0.33 839.2971 – 677.2454(6),
531.1862(100), 369.1313(55), 313.0797(4)
EpimedinA1
32 33.64 809.2863 C38H48O19 −2.41 809.2882 270 677.2454(8),
531.1878(100), 369.1329(52), 313.0665(7)
EpimedinB
33 34.17 823.3019 C39H50O19 −0.24 823.3021 270 677.2418(12),
531.1863(100), 369.1338(56), 313.0691(5)
EpimedinC
34 35.45 677.2440 C33H40O15 −1.78 677.2452 270 531.1874(26),
369.1336(100), 313.0702(17)
Icariin
35 38.64 469.1129 C24H20O10 3.25 469.1114 270 469.1089(47),
307.0555(100), 185.0364(67)
Epigallocatechin 3-O-caffeoate
36 39.40 677.2440 C33H40O15 −2.22 677.2455 – 531.1830(30),
369.1328(100), 313.0685(6)
SagittatosideA
37 47.17 523.1599 C26H27O10Na −0.63 523.1602 – 377.1013(100),
318.9111(9), 169.0395(8)
IkarisosideA (BaohuosideII)
38 52.02 531.1861 C27H30O11 −2.47 531.1874 – 369.1341(100),
313.0711(29)
IcarisideI
39 52.45 669.2154 C32H37O14Na −1.53 669.2164 – 391.1117(26),
301.0896(100)
SagittatosideB
40 52.70 683.2310 C33H40O14Na −2.08 683.2324 – 683.2299(100),
391.1118(41), 315.1050(92)
2′′-O -rhamnosyl icarisideII
41 54.39 515.1912 C27H30O10 −4.14 515.1933 – 369.1338(100),
313.0711(20)
IcarisideII (BaohuosideI)
–,UVspectrahavenotbeenobservedduetolowintensity.
etal.,2011).Compound38 gavethebasepeakat m/z369, due tothelossofaglucosemoiety(162Da).Theionofm/z369was
the characteristic fragment for anhydroicaritin aglycone. The
ionat m/z 313wasformedby theloss ofC4H7 (56Da).It was
tentativelyidentifiedasicarisideI(Zhaoetal.,2008).Compound
41 yieldedtwo fragmentions atm/z 369[M–Rha+H]+ and 313
[M–Rha–C4H7+H]+. Compound 41 wastentatively identified as
A
x107x106 1.2
+ESI TIC Frag 150.0V HGC ESI+ MS.d
+ESI EIC Frag 150.0V HGC ESI+ MS.d
1
0.8
0.6
0.4
0.2
3
2.5
2
1.5
1
0.5
0
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
0
2 4 6
78 10
12
141517 21 23
24 2627 282931 32
33 34
3536 37 3839
40 41
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
B
Fig.1. ThetotalionchromatogramofHGC(A)andextractedionchromatogramof25identifiedcompounds(B).
Compounds 14 and 27 (tR=16.85 and 28.51min), [M+H]+
pseudomolecularionspresentedatm/z547.2163 and693.2402,
whichcorrespondedtothemolecularformulaofC28H34O11and
C33H40O16,respectively.In theMS2 spectrathefragmentswere
atm/z 385, which wasattributedtotheloss ofa glucose
moi-ety(162Da)and bothglucose andrhamnose moieties(308Da).
Compound14wastentativelyidentifiedas3-hydroxyicariine-O -glucoside(Zhaoetal.,2008).Forcompound27,thefragmentions
wereatm/z547[M–Rha+H]+andm/z367[M–Rha–Glu–H
2O+H]+.
It was tentatively identified as 3-hydroxyicariine-O
-glucose-rhamnose(Zhaoetal.,2008).
Twocompounds24(tR=25.48min)and26(tR=27.46min)
indi-cated[M+H]+ionswereatm/z825.2804and663.2299.TheMS2
spectrum of compound 24 gave the fragment ions at m/z 663
[M–Glu+H]+(8%ofbasepeak),m/z517[M–Glu–Rha+H]+asbase
peak,andm/z355[M–Glu–Rha–Glu+H]+(58%ofbasepeak).
Com-pound24wastentativelyidentifiedasdiphyllosideAinagreement withtheliterature(Guanetal.,2011).IntheMS2spectraof
com-pound26,thefragmentionatm/z517wasattributedtothelossof arhamnosemoiety(146Da).Thebasepeakwasatm/z355,dueto thelossofbothrhamnoseandglucosemoieties.Itwastentatively identifiedasepimedosideA.
Compound37,tR=47.17min,[M+Na]+ionwasatm/z523.1602. TheMS2spectrumgavethebasepeakatm/z377,duetothelossofa
rhamnosemoiety(146Da).Itwastentativelyidentifiedas ikariso-sideAornamedbaohuosideII(Guanetal.,2011).
Compounds39(tR=52.45min)and40(tR=52.70min)exhibited [M+Na]+ionsatm/z669.2164and683.2324.IntheMS2spectrum,
thesamefragmentionatm/z391indicatedthelossofboth
rham-noseand xylosemoieties (278Da) and two rhamnose moieties
(292Da),respectively.Theyweretentativelyidentifiedas sagittato-sideBand2′′-O-rhamnosylicarisideII(Guanetal.,2011).
Otherchemicalcomponents
Compounds7and8(tR=7.83minand8.06min)showed[M+H]+ ionatm/z355.1038,whichcorrespondedtothemolecularformula ofC16H18O9.TheMS2spectrumgaveabasepeakatm/z163
[caf-feicacid–H2O+H]+,correspondingtothelossofquinicacidmoiety
(192Da).Thecompounds7and8indicatethesameelemental com-position.TheMS2spectrademonstratethesimilarfragmentation
andintensity.However,thedifferenceinretentiontimeislikelyto identifythetwopeaksduetolinkage.Generally,cryptochlorogenic
acidhasstrongerretentionontheC18columnthanchlorogenic
acidbycomparison withthepublisheddata(Dinget al.,2011;
Zhaoetal.,2008).Thecompound7and8wastentatively iden-tifiedaschlorogenicacidandcryptochlorogenicacid.Theymight befromEucommiaeCortexandEpimediumbrevicornuMaxim(Zhu etal.,2016;Zhaoetal.,2008).
Compound 10 (tR=9.55min), [M+H]+ ion presented at m/z
413.1436, which corresponded to the molecular formula of
C19H24O10.Thefragmentionatm/z251indicatedthelossofa
glu-cosemoiety.Compound10wastentativelyidentifiedascnidioside Bmethylester.
Compound 12 (tR=11.98min), [M+H]+ ion indicated at m/z
342.1713, which corresponded to the molecular formula of
C20H23NO4.Thefragmentionsatm/z297and265indicatedthe
lossof–N(CH3)2and–OCH3.Compound12wastentatively
iden-tifiedasmagnoflorinefromEpimediumbrevicornu(Naseeretal., 2015).
Compound 15 (tR=17.09min), [M+Na]+ ion showed at m/z
619.1661, which corresponded to the molecular formula of
C27H31O15Na.Thefragmentionatm/z473indicatedthe
elimina-tionofarhamnoseresidue(146Da)fromtheprecursorion.Itwas tentativelyidentifiedasneoeriocitrin(Liuetal.,2012).
Compound 17 (tR=18.13min), [M+H]+ ion presented at m/z
407.1356withidenticalmolecularformulaC20H22O9.The
elemen-talcompositionwasobtainedwithanerroroflessthan5ppm.The MS2spectrumofionatm/z407presentedthesuccessivefragments
ofm/z245,227,199,181,151,125,121,and107.Hence,itwas tentativelyidentifiedasstilbeneglucosidefromPolygoniMultiflori RadixPraeparatabycomparisontotheliterature(Qiuetal.,2013). Compound21,tR=21.22min,[M+H]+ionatm/z581.1878gave themolecularformulaofC27H32O14.TheMS2spectrumgavethe
fragmentionsatm/z339, 315,297,273,195,153,and129.The
aglyconeionatm/z273indicated theconsecutivelossof
rham-noseandglucosefragment(308Da).Theionatm/z153wasyielded fromretro Diels–Alderfragmentationpattern oftheC-ring.The
neutral loss of 120Da was attributed to the characteristic ion
[M–C4H8O4+H]+.Compound21fromDrynariaeRhizomawas
ten-tativelyidentifiedasnaringin(Lietal.,2010;Wuetal.,2015). Compound23,tR=23.09min,[M+H]+ionatm/z247.0958gave themolecularformulaofC14H14O4.Theprominentfragmentionin
theMS/MSspectrumwasatm/z187[M–C3H5O+H]+.Itwas
tenta-tivelyidentifiedasdiallylphthalate.Thecompoundmaybefroma contaminationofthecolumnorthesolventsusedintheextraction.
Compound 28, tR=31.29min, [M+H]+ ion exhibited at m/z
whichwasattributedtothelossofaglucosemoiety(162Da).Itwas
tentativelyidentifiedasononinfromEpimediumbrevicornu(Luo
etal.,2014).
Compound 35, tR=38.64min, [M+H]+ ion displayed at m/z
469.1114.IntheMS2 spectra,thefragmentionwasatm/z307,
whichwasattributedtothelossofacaffeicacidgroup(162Da).It wastentativelyidentifiedasepigallocatechin-O-caffeoate.
Calibrationcurves,limitsofdetection(LOD)andquantification (LOQ)
ForHPLC-DAD quantification,fourstandardsubstances were
accuratelyweighedanddissolvedin 80%methanoltomakethe
concentrationof1000g/mlforepimedinA,epimedinB,epimedin C,andicariin.Mixstocksolutionwasdilutedinthe10–500g/ml concentrationrange.Thesixconcentrationsfortheconstruction ofcalibrationcurveswereanalyzedintriplicate.Thecalibration curveswereconstructedbyplottingthepeakareasversusthe con-centrations.Peakareas(x)ofserialworkingsolutionswereplotted againstthecorrespondingconcentrations(y,g/ml).Allthe cali-brationcurvesshowedgoodlinearitywithcorrelationcoefficients (r2)nolessthan0.9994.Limitofdetection(LOD)andlimitof
quan-tification (LOQ) weredetermined byserial dilution ofstandard
solutionuntil thesignalto noise ratio(S/N) for each reference substancegotto3and10,respectively.Thecalibrationcurves, cor-relationcoefficients(r2),linearranges,LOD,andLOQarepresented
inTableS1.
Precision,recovery,repeatability,andstability
Thestandardworkingsolutionsusedfortheintra-dayand inter-dayprecisionwerethesameasthoseusedinthecalibrationcurve
experiment.Theintra-andinter-dayprecisionsweredetermined
bytherelativestandarddeviation(RSD<1.1%).Therecoverywas carriedoutbyaddingaknownamountofstandardsubstancesinto
HGC(sample20160502).Thespikedsampleswereextractedusing
theproceduredescribedforsamplepreparation.Themethod recov-erywascalculatedusingtheequationsbelow:
Recovery%=
Csample+standard−Csample
Cstandard
×100.
Therecoveryfor each compound atdifferent concentrations
rangedfrom93.0%to106.4%.TheHGC(sample20160502)was
appliedtodeterminetherepeatabilityand stability.Thesample
wasprepared according tothe procedure described as sample
preparation. It wasanalyzed for six replicates in a dayfor the repeatability.TherepeatabilityofRSDwaslessthan3.5%.The sam-plewasstoredat25◦
Candanalyzedat0,2,4,6,12and24hfor stabilitytest.ThestabilitiesofRSDwerelessthan0.68%.The pre-cision,recovery,repeatability,andstabilityareindicatedinTable S2.Themethodwasvalidatedaccordingtotheguidancefor Inter-nationalCouncilforHarmonisationguidelinesQ2(R1).
Sampleanalysis
TheproposedHPLC-DADhasbeenusedtodeterminethefour
majorflavonoids inHGC. Thequantitative resultsindicated the
contentsofepimedinA,epimedinB,epimedinC,andicariinper cap-suleinfourbatchesofsamplesasshowninTableS3.Thecontents
offourmajorcompoundsinHGCwererelativelyconsistentamong
fourbatches.AccordingtoChineseNationalDrugQualityStandard, thecontentoficariinshouldbenolessthan1.15mg/450mgorper
capsule.Ourresultsshowedthatthefourbatchescompliedwith
thisqualitystandard.
Conclusion
TheUHPLC–Q-TOF-MSandHPLC-DADmethodswereapplied
toqualitativeandquantitativeforthequalitycontrolofHGC. Com-paredwithpreviousinvestigations,atotalof41compoundswere
detectedinHGC.Amongofthem,25compoundsincludingfifteen
prenylatedflavonoidsandtenotherchemical componentswere
identifiedonthebasisofdiagnosticmassdataandfragment
pat-ternbyUHPLC–Q-TOF-MSinpositivemode.TheHPLC-DADmethod
wasappliedtodetermineinthefourbatchesofHGC.Prenylated
flavonoidsarerevealedtobethemainbioactiveingredientsofHGC, whichmightplayimportantrolesinthebiologicaland pharmaco-logicaleffects.ThefourpotentialbioactivecompoundsinHGCwere
determinedbythedevelopedHPLC-DADmethod.Theinvestigation
washelpforthequalityevaluationofHGC.
Authorship
QRdesignedandperformedtheexperiments;S-SLonganalyzed
thedata.Allauthorsreadandapprovedthefinalmanuscript.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
This investigation was supportedby grants from the Jining
Medical Universitydoctor scientificresearchfoundation project
(No. JY14QD05). This investigation was supported by grants
fromShandongProvincialNaturalScienceFoundation,China(No.
ZR2015HL124).WethanktheMedicineandHealthScience
Tech-nology Development Plan Funds from the Health and Family
PlanningCommissionofShandongProvince(No.2014WS0508).
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2017.06.002.
References
Ding,X.P.,Wang,X.T.,Chen,L.L.,Guo,Q.,Wang,H.,Qi,J.,Yu,B.Y.,2011.On-line high-performanceliquidchromatography-diodearraydetection-electrospray ionization-massspectrometry-chemiluminescenceassayofradicalscavengers inEpimedium.J.Chromatogr.A1218,1227–1235.
Guan,X.Y.,Li,H.F.,Yang,W.Z.,Lin,C.H.,Sun,C.,2011.HPLC-DAD-MSn analysis
andHPLCquantitationofchemicalconstituentsinXian-ling-gu-baocapsules. J.Pharm.Biomed.Anal.55,932–933.
Li,X.H.,Xiong,Z.L.,Lu,S.,Zhang,Y.,Li,F.M.,2010.Pharmacokineticsofnaringinand itsmetabolitenaringenininratsafteroraladministrationofrhizomedrynariae extractassayedbyUPLC–MS/MS.Chin.J.Nat.Med.8,40–46.
Liu,Q.F.,Jiao,Z.,Liu,Y.,Li,Z.D.,Shi,X.J.,Wang,W.J.,Wang,B.,Zhong,M.K.,2016.
ChemicalprofilingofSan-HuangdecoctionbyUPLC-ESI-Q-TOF-MS.J.Pharm. Biomed.Anal.131,20–32.
Liu,W.Y.,Zhou,C.,Yan,C.M.,Xie,S.L.,Feng,F.,Wu,C.Y.,Xie,N.,2012.Characterization andsimultaneousquantificationofmultipleconstituentsinAurantiiFructus ImmaturusextractsbyHPLC-DAD-ESI-MS/MS.Chin.J.Nat.Med.10,456–463.
Luo,Q.L.,Nurahmat,M.,Li,M.H.,Sun,J.,Chen,M.X.,Liu,F.,Wei,Y.,Dong,J.C., 2014.PharmacologicalinvestigationofaHPLC/MSstandardizedthreeherbal extractscontainingformulae(Bu-Shen-Yi-Qi-Tang)onairwayinflammationand hypothalamic-pituitary-adrenalaxisactivityinasthmaticmice.Phytomedicine 21,1439–1450.
Naseer,S.,Lone,S.H.,Lone,J.A.,Khuroo,M.A.,Bhat,K.A.,2015.LC–MSguided iso-lation,quantificationandantioxidantevaluationofbioactiveprinciplesfrom Epimediumelatum.J.Chromatogr.B989,62–70.
Qiu,X.H.,Zhang,J.,Huang, Z.H.,Zhu,D.Y., Xu,W.,2013.Profilingofphenolic constituents in Polygonum multiflorum Thunb. by combination of ultra-high-pressure liquid chromatography with linear ion trap-Orbitrap mass spectrometry.J.Chromatogr.A1292,121–131.
UHPLC–Q-TOF-MS mass spectrometry and HPLC-DAD. Int. J. Food Prop.,
http://dx.doi.org/10.1080/10942912.2016.1256303.
Shevlyakova,O.A.,Vasil’ev,K.Y.,Ikhalainen,A.A.,Antokhin,A.M.,Taranchenko,V.F., Goncharov,V.M.,Mitrofanov,D.A.,Aksenov,A.V.,Rodin,I.A.,Shpigun,O.A.,2016.
Identificationofbarrenwortflavonoidsbyhigh-resolutiontandemmass spec-trometry.J.Anal.Chem.71,768–776.
Song,L.J.,Zhao,W.C.,Zeng,Q.L.,Wu,J.D.,Li,B.H.,2012.Simultaneousdetermination of5majorbioactivecomponentsinHuguCapsulebyHPLC-DAD.J.Chin.Med. Mater.35,2019–2023.
TheStateCommissionofChinesePharmacopoeia,2010.Chin.Natl.DrugQual.Stand. 82,141–143.
Wang,Y.Q.,Guo,Z.M.,Jin,Y.,Zhang,X.L.,Wang,L.,Xue,X.Y.,Liang,X.M.,2010.
IdentificationofprenylflavonoidglycosidesandphenolicacidsinEpimedium koreanumNakaibyQ-TOF-MScombinedwithselectiveenrichmenton“click oligo(ethyleneglycol)”column.J.Pharm.Biomed.Anal.51,606–616.
Wu,H.,Li,X.X.,Yan,X.M.,An,L.,Luo,K.W.,Shao,M.J.,Jiang,Y.,Xie,R.,Feng,F., 2015.Anuntargetedmetabolomics-drivenapproachbasedonLC-TOF/MSand LC–MS/MSforthescreeningofxenobioticsandmetabolitesofZhi-Zi-Da-Huang decoctioninratplasma.J.Pharm.Biomed.Anal.115,315–322.
Xiang,H.,Xu,H.Y.,Zhan,L.B.,2015.Fingerprintanalysisandmulti-component deter-minationofZibuPiyinrecipebyHPLCwithDADandQ-TOF/MSmethod.Nat. Prod.Res.30,1081–1084.
Zhao,H.Y.,Sun,J.H.,Fan,M.X.,Fan,L.,Zhou,L.,Li,Z.,Han,J.,Wang,B.R.,Guo, D.A.,2008.AnalysisofphenoliccompoundsinEpimediumplantsusing liq-uidchromatographycoupledwithelectrosprayionizationmassspectrometry. J.Chromatogr.A1190,157–181.
