research article qualitative and quantitative analysis of...

Research ArticleQualitative and Quantitative Analysis of VolatileComponents of Zhengtian Pills Using Gas ChromatographyMass Spectrometry and Ultra-High PerformanceLiquid Chromatography

Cui-ting Liu1 Min Zhang1 Ping Yan1 Hai-chan Liu2 Xing-yun Liu1 and Ruo-ting Zhan1

1Research Center of Chinese Medicinal Resource Science and Engineering Key Laboratory of Chinese Medicinal Resources fromLingnan of Ministry of Education Joint Laboratory of National Engineering Research Center for the Pharmaceutics ofTraditional Chinese Medicines Guangzhou University of Traditional Chinese Medicine Guangzhou Guangdong 510006 China2School of Chinese Herbal Medicine Guangzhou University of Chinese Medicine Guangzhou Guangdong 510006 China

Correspondence should be addressed to Ping Yan ycyyp22hotmailcom and Ruo-ting Zhan ruotingzhanvip163com

Received 10 October 2015 Revised 23 December 2015 Accepted 24 December 2015

Academic Editor Mohamed Abdel-Rehim

Copyright copy 2016 Cui-ting Liu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Zhengtian pills (ZTPs) are traditional Chinese medicine (TCM) which have been commonly used to treat headaches Volatilecomponents of ZTPs extracted by ethyl acetate with an ultrasonicmethodwere analyzed by gas chromatographymass spectrometry(GC-MS) Twenty-two components were identified accounting for 78884 of the total components of volatile oil The three mainvolatile components including protocatechuic acid ferulic acid and ligustilide were simultaneously determined using ultra-highperformance liquid chromatography coupled with diode array detection (UHPLC-DAD) Baseline separation was achieved on anXB-C18 column with linear gradient elution of methanol-02 acetic acid aqueous solution The UHPLC-DAD method providedgood linearity (1198772 ge 09992) precision (RSD lt 3) accuracy (10068ndash10269) and robustness The UHPLC-DADGC-MSmethod was successfully utilized to analyze volatile components protocatechuic acid ferulic acid and ligustilide in 13 batchesof ZTPs which is suitable for discrimination and quality assessment of ZTPs

1 Introduction

In comparison with conventional fully porous particlecolumns core-shell particle columns have higher columnefficiency and sensitivity achieve better separation andremain constant over a wider linear range In addition thecolumns improve the speed of the mobile phase reduce anal-ysis time and improve throughput Moreover the backpres-sure produced by the columns at optimum linear velocity isvery low (lt400 bar) Sowe take advantage of this new columnto develop an efficient ultra-high performance liquid chro-matography (UHPLC) method [1ndash4] Gas chromatographymass spectrometry (GC-MS) has been employed in a broadrange of analytical applications due to its high sensitivityand capacity to separate compounds effectively GC-MS is

commonly used to characterize and identify volatile organiccompounds in complex mixtures [5ndash10] GC-MS has becomea popular and useful analytical tool in research on herbalmedicines especially in establishing chromatographic finger-prints for quality control of traditional Chinese medicines[11 12] such as Fructus Xanthii [13]

Zhengtian pills (ZTPs) are Chinese patentmedicine com-prised of 15 medicinal herbs Caulis Spatholobi Radix Angeli-cae Sinensis Rhizoma Chuanxiong Asari Radix et RhizomaUncariae Ramulus Cum Uncis Paeoniae Radix Alba RadixRehmanniae Radix Angelicae Dahuricae Radix Saposhniko-viae Notopterygii Rhizoma et Radix Persicae Semen Car-thami Flos Radix Angelicae Pubescentis Ephedrae Herba andAconiti Lateralis Radix Praeparata [14] ZTPs are used totreat tension headaches [15] headaches associatedwith spinal

Hindawi Publishing CorporationJournal of Analytical Methods in ChemistryVolume 2016 Article ID 1206391 8 pageshttpdxdoiorg10115520161206391

2 Journal of Analytical Methods in Chemistry

conditions and premenstrual headaches ZTPs are widelyused in China to treat migraine headaches a type of vascularheadache [16 17] According to the Chinese Pharmacopoeiapaeoniflorin is the marker compound used for ZTP qualitycontrol [14]

In a previous study HPLC was used to determine themajor active components of ZTP which included paeoni-florin ferulic acid prim-O-glucosylcimifugin and 41015840-O-beta-glucopyranosyl-5-O-methylvisamminol [18] howeverdetermination of these compounds is not sufficient forcomprehensive quality control of ZTPs which contain com-plex bioactive constituents including volatile componentsof essential oils alkaloids flavonoids and coumarin com-pounds Several compounds from ZTPs ingredients includ-ing protocatechuic acid from Spatholobi Caulis and ferulicacid and ligustilide from Rhizoma Chuanxiong and RadixAngelicae Sinensis have pharmacological activities includingantibacterial anti-inflammatory and analgesic effects aswell as protective effects on the cardiovascular system [19ndash21] Therefore an effective and reliable method capable ofqualitative and quantitative analysis of the diverse bioac-tive constituents of ZTPs is required to ensure their safetyand efficacy While significant research has been conductedon the volatile components of Rhizoma Chuanxiong andRadix Angelicae Sinensis such studies have mainly focusedon their cardiovascular cerebrovascular neuroprotectiveantinociceptive and anti-inflammatory effects [22 23] andthe volatile components of the other ZTPs constituents havenot been reported

In this study a combined UHPLC coupled with diodearray detection (UHPLC-DAD)GC-MS method was devel-oped to simultaneously identify volatile organic compoundsin ZTPs and qualitatively and quantitatively analyze 3 mainbioactive constituents present in various ZTPs ingredientsThe combinedUHPLC-DADGC-MSmethod can be utilizedto evaluate the quality of ZTPs

2 Experimental

21 Reagents and Chemicals HPLC grade anhydrous etherand methanol were purchased from Merck (DarmstadtGermany) Deionized water for the samples and mobilephase were prepared using a Milli-Q50 SP Reagent WaterSystem (Millipore France) Reference standards for proto-catechuic acid ferulic acid and ligustilide were obtainedfrom the National Institute for Food and Drug Control(Beijing China) The purity of all standards was at least98 Thirteen batches of ZTP were purchased from ChinaResources Sanjiu Medical amp Pharmaceutical Co Ltd (Shen-zhen China) Methanol acetic acid petroleum ether ethylether and ethyl acetate were purchased fromBaishi ChemicalIndustry Co Ltd (Tianjin China) and of analytical gradeAll solvents were filtered through 022120583m membrane filtersbefore analysis

22 Sample Preparation ZTPs were smashed into powder(40 mesh) For HPLC analysis 30 g of pulverized ZTP wasultrasonically extracted at room temperature with 50mL

methanol for 05 h in a 250 mL triangular flask and driedThe ZTP residue was dissolved with 20mL water andextracted 3 times with ethyl acetate (30 30 and 20mL) Theethyl acetate extracts were dried using an electrothermostaticwater bath Finally the extract was reconstituted in 5mLmethanol For GC-MS analysis 30 g of pulverized ZTPwas reflux-extracted twice for 1 h at 50∘C with 20mL ethylacetate in a 250-mL triangular flaskThe ethyl acetate extractswere dried and reconstituted in 5mL ethyl acetate All ZTPsolutions were filtered through a 022 120583m nylon membranefilter

23 GC-MS Analysis GC-MS analysis was performedon an Agilent 78905975C-GCMSD instrument (AgilentTechnologies USA) coupled with a HP-5MS fused silicacapillary column (30m times 025mm times 025120583m AgilentTechnologies Santa Clara CA USA) The GC oven temper-ature was initially increased from 60∘C to 120∘C at a rate of10∘Cmin then elevated at a rate of 3∘Cmin up to 175∘Cthen increased at a rate of 5∘Cmin up to 205∘C then wentup at a rate of 08∘Cmin up to 210∘C and then raised to280∘C at a rate of 5∘Cmin held for 5min giving a totalruntime of 55583min 1120583L volume of ethyl acetate extractswas injected into the GC Helium carrier gas at a constantflow rate of 10mLmin and a 30 1 split ratio were usedsimultaneously Mass spectrometer was operated in fullscan with an electron energy of 70 eV interface tempera-ture 280∘C MS source temperature 230∘C MS quadru-pole temperature 150∘C The scan range was from mz 30 to550

24 HPLC Analysis HPLC analysis was performed on aThermo Ultimate-3000 system (Thermo Scientific WalthamMA USA) coupled with a DAD A Phenomenex Kinetex XB-C18 column (100mm times 46mm 26 120583m id 25∘C columntemperature) with a guard column (21mm times 46mm 26 120583mid) was used (Phenomenex Torrance CAUSA)Themobilephase was 02 (vv) aqueous acetic acid solution (A) andmethanol (B) The linear gradient was as follows 0ndash7min10 B 7ndash15min 10ndash15 B 15ndash35min 15ndash40 B 35ndash65min40ndash50 B The flow rate was 15mLmin and the injectionvolume was 2 120583L

25 HPLC Method Validation All components were quanti-fied using chromatograms obtained at 260 nm The quantifi-cation was validated in terms of linearity limit of detection(LOD) limit of quantification (LOQ) accuracy and preci-sion

The stock solution containing the 3 markers wasprepared and diluted to appropriate concentration rangesfor the establishment of calibration curves The calibrationgraphs were plotted after linear regression of the peakareas versus the corresponding concentrations Good linearbehavior was observed with correlation coefficients (119903)between 09992 and 09994 LOD and LOQwere determinedat signal-to-noise (119878119873) ratios of approximately 3 and10 respectively Recovery experiments were performedat medium levels The concentrations of protocatechuicacid ferulic acid and ligustilide were 00204mgmL

Journal of Analytical Methods in Chemistry 3

Table 1 Regression equations correlation coefficients linearity ranges LODs LOQs and recoveries of investigated compounds

Analytes Regression equation 1198772 Linearity range120583gmLminus1 LOD120583g LOQ120583g Recovery RSD

Protocatechuic acid 119884 = 38403119883 + 00743 09994 41ndash286 00019 00037 10068 035Ferulic acid 119884 = 13889119883 + 03961 09992 802ndash5614 0025 0074 10098 056Ligustilide 119884 = 91687119883 + 11081 09994 110ndash770 0044 0084 10269 028

Pro FerLig

(min)

Abso

rban

ce(m

AU) 300

200

100

minus10

00 100 200 300 400 500 650

(a)

LigFer

Pro

(min)

Abso

rban

ce(m

AU) 100

minus10

00 100 200 300 400 500 650

(b)

Figure 1 Representative HPLC chromatograms of mixed standards and the ZTP extract at 260 nm (a) ZTP extract (sample 1209051H) (b)mixed standards of the 3 chemical constituents Peaks protocatechuic acid ferulic acid and ligustilide

01027mgmL and 022mgmL This procedure wasaccordingly repeated for six replicates The spiked sampleswere extracted processed and quantified in accordancewith the methods described above Recoveries variedfrom 10018 to 10339 with RSDs from 050 to 236(Table 1)

Precision was evaluated with the solution of sample1209051H under the selected optimal conditions 6 times in 1day to measure intraday variation The RSDs of the precisionresults were in the range of 054ndash089 Repeatability wasconfirmed with 6 different working solutions prepared fromsample 1209051H The RSDs of the repeatability results werein the range of 188ndash231 The stability of the solutions wastested by injecting them into the apparatus at 0 2 4 6 8 12and 24 h The RSDs of the stability results were in the rangeof 180ndash253

Moreover specificity was investigated by comparing thechromatograms of mixed standards and the ZTP extract(Figure 1) According to the three-dimensional plot of theabsorbance as a function of retention time and wavelengthin the HPLC-DAD data for sample number 1209051H noevidence of peak of impurities overlapping the markers wasfound

This is the first simultaneous analysis of protocatechuicacid ferulic acid and ligustilide with acceptable linearityprecision repeatability and accuracy

3 Results and Discussion

31 Optimization of the GC Method and Extraction In thisstudy the extraction efficiencies of petroleum ether ethylether and ethyl acetate were compared Total ion current(TIC) was greatest when ethyl acetate was used Investigationof the extraction efficiencies of the reflux and ultrasonicmethods indicated that total TIC was higher when thereflux method was used The GC results for the differentsolvent extracts and methods are described in Table 2

and Figure 2 In the ethyl acetate solvent extracts 22components were identified by artificial analysis and com-puter retrieval while relative content was determined bythe area normalization method The main components ofthe ethyl acetate solvent extracts were ligustilide (19381)oleic acid (10012) 912-octadecadienoic acid (9346)butylidene phthalide (5055) dibutyl phthalate (5891)xanthyletin (3813) methyl eugenol (3833) n-docosane(3545) asarinin (2085) and safrole (1555) In com-parison to the ethyl acetate solvent extract samples theethyl ether extract samples contained a greater amountof 26-di-tert-butyl-p-cresol (2667) while the petroleumether extract samples contained a greater amount of sulfur(3993)

32 Optimization of the HPLC Method and Extraction Dueto the existence of acidic components in the ZTP extractiona small amount of acid was added to the mobile phaseto ease ionization of these components with the goal ofimproving peak shape and restraining peak tailing Differentconcentrations of acetic acid phosphoric acid and formicacid were compared for this purpose The results showedthat all compounds could be baseline-separated when 02aqueous acetic acid solution was added

DAD detection was performed within a wavelengthrange of 190ndash400 nm When the chromatograms and char-acteristic UV spectra of the 3 reference compounds werecompared it was found that the 3 active compounds hadhigher absorbance better separation and a steady base-line at 260 nm in comparison with the other tested wave-lengths

The extraction procedure was optimized prior to sam-ple analysis The samples (30 g each) were extractedwith different volumes and percentages of methanol as wellas different volumes and percentages of ethanol respec-tively The optimum results were obtained using 50mLmethanol Investigation of the dependence of the yield on


Table2Th

erelativec

ontent

ofcompo

nentso

fZTP

sidentified

byGC-

MS

Peak

number

Retentiontim

e(min)

Com

poun

dname

Molecular

form

ula119898119911

Relativ

econ

tent

()

Ethylacetatereflu

xing

Ethylacetateultrason

icEtheru

ltrason

icPetro

leum

etheru

ltrason

ic1

2808

35-Dim

etho

xytoluene

C 9H12O2

15219

0637

1718

1372

1703

24312

Safro

leC 10H10O2

16219

1555

mdashmdash

mdash3

10059

2-Metho

xy-4-vinylph

enolph

enol

C 9H10O2

15017

1691

1373

0861

mdash4

12241

12-D

imetho

xy-4-(2-prop

enyl)benzene

C 12H18O3

21027

3833

2839

3238

0768

512442

345-Trim

etho

xytoluene

C 10H14O3

18222

0755

0571

0689

mdash6

14901

Pentadecane

C 15H32

21241

1319

1269

mdash0795

715384

26-Di-tert-b

utyl-4-m

ethylpheno

lC 15H24O

22035

mdashmdash

2667

mdash8

20422

Butylid

enep

hthalid

eC 12H12O2

18822

5055

4871

4086

3426

922455

Ligustilid

eC 12H14O2

19024

19381

1610

917654

15063

1024366

trans-Ligustilid

eC 12H14O2

19024

1007

1566

106

079

1126502

12-Benzenedicarboxylicacid

bis(2-methylpropyl)ester

C 16H22O4

27834

2724

0719

mdash18

81

1228946

Dibutylph

thalate

C 16H22O4

27834

5891

3172

5891

5193

1330251

Sulfu

rS 8

3206

mdashmdash

mdash3993

143318

8(ZZ)-91

2-Octadecadieno

icacid

C 18H32O2

28044

9346

10605

11484

5865

153332

7Oleicacid

C 18H34O2

28246

10012

1233

412347

16905

1633892

(ZZ)-91

2-Octadecadieno

icacid

ethyl

ester

C 20H36O2

3835

1181

071

1936

1268

173818

Tricosane

C 23H48

32463

0706

0678

0789

0888

1844

619

(S)-88-Dim

ethyl-2

-oxo-78-dihydro-

2H6H-pyrano(32-g)chromen-7-yl

3-methylbut-2-eno

ate

C 19H20O5

32836

1464

1209

2318

1962

194503

88-Dim

ethyl-2

H8H-benzo[12-11988754-

1198871015840]dipyran-2-one

C 14H12O3

22824

3813

4629

4166

7711

2046

766

Eicosane

C 20H42

28255

2005

239

2946

2839

2149893

n-Docosane

C 22H46

3106

3545

394

4272

5138

2253837

Asarin

inC 20H18O6

35435

2085

1916

71201


500 1500 2500 3500 45002000060000

100000140000180000220000260000300000340000

Retention time (min)

Inte

nsity

(a)

500 1500 2500 3500 4500

2000060000

100000140000180000220000260000300000340000


Inte

nsity

(b)

500 1500 2500 3500 45002000060000

100000140000180000220000260000300000340000


Inte

nsity

(c)

500 1500 2500 3500 45002000060000

100000140000180000220000260000300000340000


Inte

nsity

(d)

Figure 2 The total ion chromatogram (TIC) of ZTPs using different solvents and extraction methods (a) ethyl acetate extraction by refluxmethod (b) ethyl acetate extraction by ultrasonic wave (c) ether extraction by ultrasonic wave and (d) petroleum ether extraction byultrasonic wave

the duration of the extraction (15 30 and 60min) showedthat all of the investigated compounds were almost com-pletely extracted when 30 min extraction was used

33 Sample Analysis The UHPLC-DADGC-MS methodwas applied to analyze 13 batches of ZTP (Tables 3and 4) Protocatechuic acid content ranged from 607to 1730 120583gg ferulic acid content ranged from 7959 to11590 120583gg and ligustilide content ranged from 7959 to38869 120583gg The GC-MS results showed that the aver-age relative contents of ligustilide oleic acid asarininsafrole 2-methoxy-4-vinylphenol and 12-dimethoxy-4-(2-propenyl)benzene were 6991 23275 1631 05860575 and 0574 respectively 35-Dimethoxytoluene wasnot detected These results suggested that the contents ofeach component varied greatly among batches of ZTPs Thelargest observed difference in content was nearly 5-foldindicating large variations in the ZTP production processandor incorrectly identified herbal sources

The UHPLC-DADGC-MS method we established coulddetect as much volatile components as possible Com-pared to previous study using a two-dimensional liq-uid chromatography coupled to mass spectrometry therewere more compounds identified by the GC-MS methodsuch as 12-dimethoxy-4-(2-propenyl)benzene 2-methoxy-4-vinylphenol phenol butylidene phthalide oleic acid and(ZZ)-912-octadecadienoic acid ethyl ester which weremainly from Rhizoma Chuanxiong [24] It is very helpful

for a comprehensive understanding of volatile componentsof ZTPs At the same time the established UHPLC-DADmethod could determine protocatechuic acid ferulic acidand ligustilide simultaneously which was very helpful andpracticable for the quality control of ZTPs for companiesSince TCM is a complex system containing tens or evenhundreds of different chemical constituents the active com-pounds of most TCM still remain unknown It is reportedthat molecular biochromatography was a novel strategy forthe screening and analysis of biologically active compoundsin TCM [25 26] Thus in our further study we will makean attempt to screen active compounds of ZTPs by usingmolecular biochromatography

4 Conclusions

A UHPLC-DADGC-MS method was established for thecomprehensive analysis of ZTPs that allowed separationof complex constituents in a short time GC-MS providedaccurate masses of protonated molecules which were help-ful for compound identification The UHPLC-DADGC-MSmethod was successfully applied for simultaneous deter-mination of 3 bioactive compounds in ZTP The UHPLC-DADGC-MSmethod is readily available rapid and reliableTherefore the UHPLC-DADGC-MS method is suitable forroutine analysis original discrimination and effective qualitycontrol of ZTPs The amounts of protocatechuic acid ferulicacid and ligustilide in the ZTP batches varied considerably


Table3Re

lativec

onstitu

entcon

tent

determ

inations

from

13batcheso

fZTP

sSample

number

35-Dim

etho

xytoluene(

)

Safro

le(

)2-Metho

xy-4-vinylph

enolph

enol(

)12

-Dim

etho

xy-4-(2-prop

enyl)benzene

()

Ligustilid

e()

Oleicacid

()

Asarin

in(

)

1209051H

mdash0744

0321

0267

497

2616

60758

1303002H

mdash0393

0587

0753

7101

1676

918

061212023H

mdash0321

0393

0438

5747

32844

1084

1210019H

mdash0736

0556

mdash8905

20581

3120

1212024H

mdash0556

0420

0309

5903

34815

2163

1301023H

mdash0420

040

0mdash

4285

34989

1119

1302026H

mdash040

00753

mdash7558

18568

1334

1303036H

mdash0753

0973

mdash5916

20691

2074

1304

001H

mdash0973

0803

mdash10845

19593

1560

1306

019H

mdash0803

0588

mdash7396

20627

2164

1306

020H

mdash0588

0458

mdash5725

18016

1344

1306

027H

mdash0458

0475

0816

6517

23226

1154

1309014H

mdash0475

0744

0858

10012

15691

1523


Table 4 Determination of the contents of protocatechuic acid ferulic acid and ligustilide of 13 batches of ZTPs

Sample numberContent (120583gg)

Protocatechuic acid Ferulic acid LigustilideMean RSD () Mean RSD () Mean RSD ()

1209051H 1401 191 8011 278 17413 1651303002H 1443 126 7959 142 7959 2521303002H 1730 139 8037 184 17885 2651210019H 836 082 7806 202 36342 0391212024H 1064 275 8251 121 34878 2481301023H 1221 125 8642 088 25237 2571302026H 1008 167 11590 262 32311 2471303036H 607 251 8498 300 38869 1051304001H 1002 161 11197 235 26861 2421306019H 962 161 8668 132 30362 0811306020H 813 126 8093 289 35571 1721306027H 971 037 9314 049 26075 0521309014H 964 286 9650 289 30190 059

Therefore future studies should focus on evaluating theinfluence of bioactive constituent of ZTPs on their therapeu-tic effects using preclinical pharmacodynamic and clinicaltesting

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

This work was supported by the Chinese Medicinal Mate-rials Production and Construction Projects of the Ministryof Industry and Information Technology of China ([2015]no 282) and the Collaborative Innovation Center ResearchTeam Construction Project in Guangdong ProvincemdashtheInnovation Research Team of Traditional Chinese MedicineResources (A1-AFD01514A04)

References

[1] Kinetex core-shell particles column G F S I C httpwwwgzflmcomproductinfodetail 4 6 74aspx

[2] R Hayes A Ahmed T Edge and H Zhang ldquoCore-shell par-ticles preparation fundamentals and applications in high per-formance liquid chromatographyrdquo Journal of ChromatographyA vol 1357 pp 36ndash52 2014

[3] A Natale D Nardiello C Palermo M Muscarella M QuintoandD Centonze ldquoDevelopment of an analytical method for thedetermination of polyphenolic compounds in vegetable originsamples by liquid chromatography and pulsed amperometricdetection at a glassy carbon electroderdquo Journal of Chromatog-raphy A vol 1420 pp 66ndash73 2015

[4] R Preti M L Antonelli R Bernacchia and G Vinci ldquoFastdetermination of biogenic amines in beverages by a core-shellparticle columnrdquo Food Chemistry vol 187 pp 555ndash562 2015

[5] E A Nonato F Carazza F C Silva C R Carvalho and Z D LCardeal ldquoA headspace solid-phase microextraction method forthe determination of some secondary compounds of Braziliansugar cane spirits by gas chromatographyrdquo Journal of Agricul-tural and Food Chemistry vol 49 no 8 pp 3533ndash3539 2001

[6] T Gorecki J Harynuk and O Panic ldquoThe evolution of com-prehensive two-dimensional gas chromatography (GCtimesGC)rdquoJournal of Separation Science vol 27 no 5-6 pp 359ndash379 2004

[7] F Gong Y-Z Liang H Cui F-T Chau and B T-P ChanldquoDetermination of volatile components in peptic powder by gaschromatography-mass spectrometry and chemometric resolu-tionrdquo Journal of Chromatography A vol 909 no 2 pp 237ndash2472001

[8] S Shen Y Sha C Deng X Zhang D Fu and J ChenldquoQuality assessment of Flos Chrysanthemi Indici from differentgrowing areas in China by solid-phase microextraction-gaschromatography-mass spectrometryrdquo Journal of Chromatogra-phy A vol 1047 no 2 pp 281ndash287 2004

[9] CDeng YMaoN Yao andX Zhang ldquoDevelopment ofmicro-wave-assisted extraction followed by headspace solid-phasemicroextraction and gas chromatography-mass spectrometryfor quantification of camphor and borneol in Flos ChrysanthemiIndicirdquoAnalytica ChimicaActa vol 575 no 1 pp 120ndash125 2006

[10] C Deng N Yao B Wang and X Zhang ldquoDevelopment ofmicrowave-assisted extraction followed by headspace single-drop microextraction for fast determination of paeonol in tra-ditional Chinese medicinesrdquo Journal of Chromatography A vol1103 no 1 pp 15ndash21 2006

[11] H Zhu Y Wang H Liang Q Chen P Zhao and J Tao ldquoIden-tification of Portulaca oleracea L from different sources usingGC-MS and FT-IR spectroscopyrdquo Talanta vol 81 no 1-2 pp129ndash135 2010

[12] L Tong Y Wang J Xiong Y Cui YigangZhou and L YildquoSelection and fingerprints of the control substances for plantdrug Eucommia ulmodies Oliver by HPLC and LCndashMSrdquoTalanta vol 76 no 1 pp 80ndash84 2008


[13] G-H Ruan and G-K Li ldquoThe study on the chromatographicfingerprint of Fructus xanthii by microwave assisted extractioncoupled with GC-MSrdquo Journal of Chromatography B AnalyticalTechnologies in the Biomedical and Life Sciences vol 850 no 1-2pp 241ndash248 2007

[14] State Pharmacopoeia Commission of the Peoplersquos Republic ofChina Pharmacopoeia of Peoplersquos Republic of China ChemicalIndustry Press Beijing China 2010

[15] H R Qian X K Qi G Huang and D G Feng ldquoComparisonof effect of zhengtian pill and tongtian oral solution in treatingtension-type headacherdquo Chinese Journal of New Drugs andClinical Remedies vol 22 no 5 pp 279ndash282 2003

[16] Z Xie W L Li and G J Shi ldquoRandomized double-blinddontrolled clinical study of Zhengtianwan pill in treating 42cases of migrainerdquo Journal of Mathematical Medicine vol 24no 1 pp 70ndash73 2011

[17] C X Y Y Wang H C Shang M Ren and J Yuan ldquodouble-blind and double-dummy multi-center clinical study of Zheng-tian Pill for patients with migrainerdquo Chinese Traditional PatentMedicine vol 34 no 5 pp 791ndash794 2012

[18] L Huang H-L Chen and L-L Li ldquoSimultaneous determi-nation of paeoniflorin ferulic acid prim-O-glucosylcimifu-gin and 41015840-O-beta-glucopyranosyl-5-O-methylvisamminol inZhengtian pills by HPLCrdquo Zhongguo Zhongyao Zazhi vol 38no 13 pp 2114ndash2117 2013

[19] A G Z Q Lin J N Chen X P Lai S H Gui and C P FangldquoAnti-inflammatory and analgesic effects of ligustiliderdquo ChineseJournal of Experimental Traditional Medical Formulae vol 17no 77 pp 165ndash168 2011

[20] J M L M Zhai R An F L Wu and H H Xu ldquoContentdetermination of protocatechuic acid in Spatholobus suberectusdumnrdquo Traditional Chinese Drug Research amp Clinical Pharma-cology vol 20 no 5 pp 462ndash465 2009

[21] X Y X Y Y Hu ldquoChemical and pharmacological researchprogress of ferulic acidrdquo Chinese Traditional Patent Medicinevol 28 no 2 pp 253ndash255 2006

[22] X-P Chen W Li X-F Xiao L-L Zhang and C-X LiuldquoPhytochemical and pharmacological studies on RadixAngelicasinensisrdquo Chinese Journal of Natural Medicines vol 11 no 6 pp577ndash587 2013

[23] X Ran L Ma C Peng H Zhang and L-P Qin ldquoLigusticumchuanxiong Hort a review of chemistry and pharmacologyrdquoPharmaceutical Biology vol 49 no 11 pp 1180ndash1189 2011

[24] X Chen L Kong X Su et al ldquoSeparation and identification ofcompounds in Rhizoma chuanxiong by comprehensive two-dimensional liquid chromatography coupled tomass spectrom-etryrdquo Journal of Chromatography A vol 1040 no 2 pp 169ndash1782004

[25] H Wang L Kong H Zou J Ni and Y Zhang ldquoScreening andanalysis of biologically active compounds inAngelica sinensis bymolecular biochromatographyrdquo Chromatographia vol 50 no7-8 pp 439ndash445 1999

[26] H Wang H Zou L Kong J Ni and Y Zhang ldquoScreeningand analysis of biologically active components in traditionalChinese medicine by molecular biochromatographyrdquo ChineseJournal of Chromatography vol 17 no 2 pp 123ndash127 1999

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Physical Chemistry

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Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


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Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


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Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


conditions and premenstrual headaches ZTPs are widelyused in China to treat migraine headaches a type of vascularheadache [16 17] According to the Chinese Pharmacopoeiapaeoniflorin is the marker compound used for ZTP qualitycontrol [14]

In a previous study HPLC was used to determine themajor active components of ZTP which included paeoni-florin ferulic acid prim-O-glucosylcimifugin and 41015840-O-beta-glucopyranosyl-5-O-methylvisamminol [18] howeverdetermination of these compounds is not sufficient forcomprehensive quality control of ZTPs which contain com-plex bioactive constituents including volatile componentsof essential oils alkaloids flavonoids and coumarin com-pounds Several compounds from ZTPs ingredients includ-ing protocatechuic acid from Spatholobi Caulis and ferulicacid and ligustilide from Rhizoma Chuanxiong and RadixAngelicae Sinensis have pharmacological activities includingantibacterial anti-inflammatory and analgesic effects aswell as protective effects on the cardiovascular system [19ndash21] Therefore an effective and reliable method capable ofqualitative and quantitative analysis of the diverse bioac-tive constituents of ZTPs is required to ensure their safetyand efficacy While significant research has been conductedon the volatile components of Rhizoma Chuanxiong andRadix Angelicae Sinensis such studies have mainly focusedon their cardiovascular cerebrovascular neuroprotectiveantinociceptive and anti-inflammatory effects [22 23] andthe volatile components of the other ZTPs constituents havenot been reported

In this study a combined UHPLC coupled with diodearray detection (UHPLC-DAD)GC-MS method was devel-oped to simultaneously identify volatile organic compoundsin ZTPs and qualitatively and quantitatively analyze 3 mainbioactive constituents present in various ZTPs ingredientsThe combinedUHPLC-DADGC-MSmethod can be utilizedto evaluate the quality of ZTPs

2 Experimental

21 Reagents and Chemicals HPLC grade anhydrous etherand methanol were purchased from Merck (DarmstadtGermany) Deionized water for the samples and mobilephase were prepared using a Milli-Q50 SP Reagent WaterSystem (Millipore France) Reference standards for proto-catechuic acid ferulic acid and ligustilide were obtainedfrom the National Institute for Food and Drug Control(Beijing China) The purity of all standards was at least98 Thirteen batches of ZTP were purchased from ChinaResources Sanjiu Medical amp Pharmaceutical Co Ltd (Shen-zhen China) Methanol acetic acid petroleum ether ethylether and ethyl acetate were purchased fromBaishi ChemicalIndustry Co Ltd (Tianjin China) and of analytical gradeAll solvents were filtered through 022120583m membrane filtersbefore analysis

22 Sample Preparation ZTPs were smashed into powder(40 mesh) For HPLC analysis 30 g of pulverized ZTP wasultrasonically extracted at room temperature with 50mL

methanol for 05 h in a 250 mL triangular flask and driedThe ZTP residue was dissolved with 20mL water andextracted 3 times with ethyl acetate (30 30 and 20mL) Theethyl acetate extracts were dried using an electrothermostaticwater bath Finally the extract was reconstituted in 5mLmethanol For GC-MS analysis 30 g of pulverized ZTPwas reflux-extracted twice for 1 h at 50∘C with 20mL ethylacetate in a 250-mL triangular flaskThe ethyl acetate extractswere dried and reconstituted in 5mL ethyl acetate All ZTPsolutions were filtered through a 022 120583m nylon membranefilter

23 GC-MS Analysis GC-MS analysis was performedon an Agilent 78905975C-GCMSD instrument (AgilentTechnologies USA) coupled with a HP-5MS fused silicacapillary column (30m times 025mm times 025120583m AgilentTechnologies Santa Clara CA USA) The GC oven temper-ature was initially increased from 60∘C to 120∘C at a rate of10∘Cmin then elevated at a rate of 3∘Cmin up to 175∘Cthen increased at a rate of 5∘Cmin up to 205∘C then wentup at a rate of 08∘Cmin up to 210∘C and then raised to280∘C at a rate of 5∘Cmin held for 5min giving a totalruntime of 55583min 1120583L volume of ethyl acetate extractswas injected into the GC Helium carrier gas at a constantflow rate of 10mLmin and a 30 1 split ratio were usedsimultaneously Mass spectrometer was operated in fullscan with an electron energy of 70 eV interface tempera-ture 280∘C MS source temperature 230∘C MS quadru-pole temperature 150∘C The scan range was from mz 30 to550

24 HPLC Analysis HPLC analysis was performed on aThermo Ultimate-3000 system (Thermo Scientific WalthamMA USA) coupled with a DAD A Phenomenex Kinetex XB-C18 column (100mm times 46mm 26 120583m id 25∘C columntemperature) with a guard column (21mm times 46mm 26 120583mid) was used (Phenomenex Torrance CAUSA)Themobilephase was 02 (vv) aqueous acetic acid solution (A) andmethanol (B) The linear gradient was as follows 0ndash7min10 B 7ndash15min 10ndash15 B 15ndash35min 15ndash40 B 35ndash65min40ndash50 B The flow rate was 15mLmin and the injectionvolume was 2 120583L

25 HPLC Method Validation All components were quanti-fied using chromatograms obtained at 260 nm The quantifi-cation was validated in terms of linearity limit of detection(LOD) limit of quantification (LOQ) accuracy and preci-sion

The stock solution containing the 3 markers wasprepared and diluted to appropriate concentration rangesfor the establishment of calibration curves The calibrationgraphs were plotted after linear regression of the peakareas versus the corresponding concentrations Good linearbehavior was observed with correlation coefficients (119903)between 09992 and 09994 LOD and LOQwere determinedat signal-to-noise (119878119873) ratios of approximately 3 and10 respectively Recovery experiments were performedat medium levels The concentrations of protocatechuicacid ferulic acid and ligustilide were 00204mgmL





Pro FerLig

(min)

Abso

rban

ce(m

AU) 300

200

100

minus10

00 100 200 300 400 500 650

(a)

LigFer

Pro

(min)

Abso

rban

ce(m

AU) 100

minus10

00 100 200 300 400 500 650

(b)













Table2Th

erelativec

ontent

ofcompo

nentso

fZTP

sidentified

byGC-

MS

Peak

number

Retentiontim

e(min)

Com

poun

dname

Molecular

form

ula119898119911

Relativ

econ

tent

()

Ethylacetatereflu

xing


icEtheru

ltrason

icPetro

leum

etheru

ltrason

ic1

2808

35-Dim

etho

xytoluene

C 9H12O2

15219

0637

1718

1372

1703

24312

Safro

leC 10H10O2

16219

1555

mdashmdash

mdash3

10059

2-Metho

xy-4-vinylph

enolph

enol

C 9H10O2

15017

1691

1373

0861

mdash4

12241

12-D

imetho

xy-4-(2-prop

enyl)benzene

C 12H18O3

21027

3833

2839

3238

0768

512442

345-Trim

etho

xytoluene

C 10H14O3

18222

0755

0571

0689

mdash6

14901

Pentadecane

C 15H32

21241

1319

1269

mdash0795

715384

26-Di-tert-b

utyl-4-m

ethylpheno

lC 15H24O

22035

mdashmdash

2667

mdash8

20422

Butylid

enep

hthalid

eC 12H12O2

18822

5055

4871

4086

3426

922455

Ligustilid

eC 12H14O2

19024

19381

1610

917654

15063

1024366

trans-Ligustilid

eC 12H14O2

19024

1007

1566

106

079

1126502



C 16H22O4

27834

2724

0719

mdash18

81

1228946

Dibutylph

thalate

C 16H22O4

27834

5891

3172

5891

5193

1330251

Sulfu

rS 8

3206

mdashmdash

mdash3993

143318

8(ZZ)-91

2-Octadecadieno

icacid

C 18H32O2

28044

9346

10605

11484

5865

153332

7Oleicacid

C 18H34O2

28246

10012

1233

412347

16905

1633892

(ZZ)-91

2-Octadecadieno

icacid

ethyl

ester

C 20H36O2

3835

1181

071

1936

1268

173818

Tricosane

C 23H48

32463

0706

0678

0789

0888

1844

619

(S)-88-Dim

ethyl-2

-oxo-78-dihydro-


3-methylbut-2-eno

ate

C 19H20O5

32836

1464

1209

2318

1962

194503

88-Dim

ethyl-2

H8H-benzo[12-11988754-

1198871015840]dipyran-2-one

C 14H12O3

22824

3813

4629

4166

7711

2046

766

Eicosane

C 20H42

28255

2005

239

2946

2839

2149893

n-Docosane

C 22H46

3106

3545

394

4272

5138

2253837

Asarin

inC 20H18O6

35435

2085

1916

71201


500 1500 2500 3500 45002000060000

100000140000180000220000260000300000340000


Inte

nsity

(a)

500 1500 2500 3500 4500

2000060000

100000140000180000220000260000300000340000


Inte

nsity

(b)

500 1500 2500 3500 45002000060000

100000140000180000220000260000300000340000


Inte

nsity

(c)

500 1500 2500 3500 45002000060000

100000140000180000220000260000300000340000


Inte

nsity

(d)






4 Conclusions



Table3Re

lativec

onstitu

entcon

tent

determ

inations

from

13batcheso

fZTP

sSample

number

35-Dim

etho

xytoluene(

)

Safro

le(

)2-Metho

xy-4-vinylph

enolph

enol(

)12

-Dim

etho

xy-4-(2-prop

enyl)benzene

()

Ligustilid

e()

Oleicacid

()

Asarin

in(

)

1209051H

mdash0744

0321

0267

497

2616

60758

1303002H

mdash0393

0587

0753

7101

1676

918

061212023H

mdash0321

0393

0438

5747

32844

1084

1210019H

mdash0736

0556

mdash8905

20581

3120

1212024H

mdash0556

0420

0309

5903

34815

2163

1301023H

mdash0420

040

0mdash

4285

34989

1119

1302026H

mdash040

00753

mdash7558

18568

1334

1303036H

mdash0753

0973

mdash5916

20691

2074

1304

001H

mdash0973

0803

mdash10845

19593

1560

1306

019H

mdash0803

0588

mdash7396

20627

2164

1306

020H

mdash0588

0458

mdash5725

18016

1344

1306

027H

mdash0458

0475

0816

6517

23226

1154

1309014H

mdash0475

0744

0858

10012

15691

1523





1209051H 1401 191 8011 278 17413 1651303002H 1443 126 7959 142 7959 2521303002H 1730 139 8037 184 17885 2651210019H 836 082 7806 202 36342 0391212024H 1064 275 8251 121 34878 2481301023H 1221 125 8642 088 25237 2571302026H 1008 167 11590 262 32311 2471303036H 607 251 8498 300 38869 1051304001H 1002 161 11197 235 26861 2421306019H 962 161 8668 132 30362 0811306020H 813 126 8093 289 35571 1721306027H 971 037 9314 049 26075 0521309014H 964 286 9650 289 30190 059




Acknowledgments


References





































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





Pro FerLig

(min)

Abso

rban

ce(m

AU) 300

200

100

minus10

00 100 200 300 400 500 650

(a)

LigFer

Pro

(min)

Abso

rban

ce(m

AU) 100

minus10

00 100 200 300 400 500 650

(b)













Table2Th

erelativec

ontent

ofcompo

nentso

fZTP

sidentified

byGC-

MS

Peak

number

Retentiontim

e(min)

Com

poun

dname

Molecular

form

ula119898119911

Relativ

econ

tent

()

Ethylacetatereflu

xing


icEtheru

ltrason

icPetro

leum

etheru

ltrason

ic1

2808

35-Dim

etho

xytoluene

C 9H12O2

15219

0637

1718

1372

1703

24312

Safro

leC 10H10O2

16219

1555

mdashmdash

mdash3

10059

2-Metho

xy-4-vinylph

enolph

enol

C 9H10O2

15017

1691

1373

0861

mdash4

12241

12-D

imetho

xy-4-(2-prop

enyl)benzene

C 12H18O3

21027

3833

2839

3238

0768

512442

345-Trim

etho

xytoluene

C 10H14O3

18222

0755

0571

0689

mdash6

14901

Pentadecane

C 15H32

21241

1319

1269

mdash0795

715384

26-Di-tert-b

utyl-4-m

ethylpheno

lC 15H24O

22035

mdashmdash

2667

mdash8

20422

Butylid

enep

hthalid

eC 12H12O2

18822

5055

4871

4086

3426

922455

Ligustilid

eC 12H14O2

19024

19381

1610

917654

15063

1024366

trans-Ligustilid

eC 12H14O2

19024

1007

1566

106

079

1126502



C 16H22O4

27834

2724

0719

mdash18

81

1228946

Dibutylph

thalate

C 16H22O4

27834

5891

3172

5891

5193

1330251

Sulfu

rS 8

3206

mdashmdash

mdash3993

143318

8(ZZ)-91

2-Octadecadieno

icacid

C 18H32O2

28044

9346

10605

11484

5865

153332

7Oleicacid

C 18H34O2

28246

10012

1233

412347

16905

1633892

(ZZ)-91

2-Octadecadieno

icacid

ethyl

ester

C 20H36O2

3835

1181

071

1936

1268

173818

Tricosane

C 23H48

32463

0706

0678

0789

0888

1844

619

(S)-88-Dim

ethyl-2

-oxo-78-dihydro-


3-methylbut-2-eno

ate

C 19H20O5

32836

1464

1209

2318

1962

194503

88-Dim

ethyl-2

H8H-benzo[12-11988754-

1198871015840]dipyran-2-one

C 14H12O3

22824

3813

4629

4166

7711

2046

766

Eicosane

C 20H42

28255

2005

239

2946

2839

2149893

n-Docosane

C 22H46

3106

3545

394

4272

5138

2253837

Asarin

inC 20H18O6

35435

2085

1916

71201


500 1500 2500 3500 45002000060000

100000140000180000220000260000300000340000


Inte

nsity

(a)

500 1500 2500 3500 4500

2000060000

100000140000180000220000260000300000340000


Inte

nsity

(b)

500 1500 2500 3500 45002000060000

100000140000180000220000260000300000340000


Inte

nsity

(c)

500 1500 2500 3500 45002000060000

100000140000180000220000260000300000340000


Inte

nsity

(d)






4 Conclusions



Table3Re

lativec

onstitu

entcon

tent

determ

inations

from

13batcheso

fZTP

sSample

number

35-Dim

etho

xytoluene(

)

Safro

le(

)2-Metho

xy-4-vinylph

enolph

enol(

)12

-Dim

etho

xy-4-(2-prop

enyl)benzene

()

Ligustilid

e()

Oleicacid

()

Asarin

in(

)

1209051H

mdash0744

0321

0267

497

2616

60758

1303002H

mdash0393

0587

0753

7101

1676

918

061212023H

mdash0321

0393

0438

5747

32844

1084

1210019H

mdash0736

0556

mdash8905

20581

3120

1212024H

mdash0556

0420

0309

5903

34815

2163

1301023H

mdash0420

040

0mdash

4285

34989

1119

1302026H

mdash040

00753

mdash7558

18568

1334

1303036H

mdash0753

0973

mdash5916

20691

2074

1304

001H

mdash0973

0803

mdash10845

19593

1560

1306

019H

mdash0803

0588

mdash7396

20627

2164

1306

020H

mdash0588

0458

mdash5725

18016

1344

1306

027H

mdash0458

0475

0816

6517

23226

1154

1309014H

mdash0475

0744

0858

10012

15691

1523





1209051H 1401 191 8011 278 17413 1651303002H 1443 126 7959 142 7959 2521303002H 1730 139 8037 184 17885 2651210019H 836 082 7806 202 36342 0391212024H 1064 275 8251 121 34878 2481301023H 1221 125 8642 088 25237 2571302026H 1008 167 11590 262 32311 2471303036H 607 251 8498 300 38869 1051304001H 1002 161 11197 235 26861 2421306019H 962 161 8668 132 30362 0811306020H 813 126 8093 289 35571 1721306027H 971 037 9314 049 26075 0521309014H 964 286 9650 289 30190 059




Acknowledgments


References





































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Table2Th

erelativec

ontent

ofcompo

nentso

fZTP

sidentified

byGC-

MS

Peak

number

Retentiontim

e(min)

Com

poun

dname

Molecular

form

ula119898119911

Relativ

econ

tent

()

Ethylacetatereflu

xing


icEtheru

ltrason

icPetro

leum

etheru

ltrason

ic1

2808

35-Dim

etho

xytoluene

C 9H12O2

15219

0637

1718

1372

1703

24312

Safro

leC 10H10O2

16219

1555

mdashmdash

mdash3

10059

2-Metho

xy-4-vinylph

enolph

enol

C 9H10O2

15017

1691

1373

0861

mdash4

12241

12-D

imetho

xy-4-(2-prop

enyl)benzene

C 12H18O3

21027

3833

2839

3238

0768

512442

345-Trim

etho

xytoluene

C 10H14O3

18222

0755

0571

0689

mdash6

14901

Pentadecane

C 15H32

21241

1319

1269

mdash0795

715384

26-Di-tert-b

utyl-4-m

ethylpheno

lC 15H24O

22035

mdashmdash

2667

mdash8

20422

Butylid

enep

hthalid

eC 12H12O2

18822

5055

4871

4086

3426

922455

Ligustilid

eC 12H14O2

19024

19381

1610

917654

15063

1024366

trans-Ligustilid

eC 12H14O2

19024

1007

1566

106

079

1126502



C 16H22O4

27834

2724

0719

mdash18

81

1228946

Dibutylph

thalate

C 16H22O4

27834

5891

3172

5891

5193

1330251

Sulfu

rS 8

3206

mdashmdash

mdash3993

143318

8(ZZ)-91

2-Octadecadieno

icacid

C 18H32O2

28044

9346

10605

11484

5865

153332

7Oleicacid

C 18H34O2

28246

10012

1233

412347

16905

1633892

(ZZ)-91

2-Octadecadieno

icacid

ethyl

ester

C 20H36O2

3835

1181

071

1936

1268

173818

Tricosane

C 23H48

32463

0706

0678

0789

0888

1844

619

(S)-88-Dim

ethyl-2

-oxo-78-dihydro-


3-methylbut-2-eno

ate

C 19H20O5

32836

1464

1209

2318

1962

194503

88-Dim

ethyl-2

H8H-benzo[12-11988754-

1198871015840]dipyran-2-one

C 14H12O3

22824

3813

4629

4166

7711

2046

766

Eicosane

C 20H42

28255

2005

239

2946

2839

2149893

n-Docosane

C 22H46

3106

3545

394

4272

5138

2253837

Asarin

inC 20H18O6

35435

2085

1916

71201


500 1500 2500 3500 45002000060000

100000140000180000220000260000300000340000


Inte

nsity

(a)

500 1500 2500 3500 4500

2000060000

100000140000180000220000260000300000340000


Inte

nsity

(b)

500 1500 2500 3500 45002000060000

100000140000180000220000260000300000340000


Inte

nsity

(c)

500 1500 2500 3500 45002000060000

100000140000180000220000260000300000340000


Inte

nsity

(d)






4 Conclusions



Table3Re

lativec

onstitu

entcon

tent

determ

inations

from

13batcheso

fZTP

sSample

number

35-Dim

etho

xytoluene(

)

Safro

le(

)2-Metho

xy-4-vinylph

enolph

enol(

)12

-Dim

etho

xy-4-(2-prop

enyl)benzene

()

Ligustilid

e()

Oleicacid

()

Asarin

in(

)

1209051H

mdash0744

0321

0267

497

2616

60758

1303002H

mdash0393

0587

0753

7101

1676

918

061212023H

mdash0321

0393

0438

5747

32844

1084

1210019H

mdash0736

0556

mdash8905

20581

3120

1212024H

mdash0556

0420

0309

5903

34815

2163

1301023H

mdash0420

040

0mdash

4285

34989

1119

1302026H

mdash040

00753

mdash7558

18568

1334

1303036H

mdash0753

0973

mdash5916

20691

2074

1304

001H

mdash0973

0803

mdash10845

19593

1560

1306

019H

mdash0803

0588

mdash7396

20627

2164

1306

020H

mdash0588

0458

mdash5725

18016

1344

1306

027H

mdash0458

0475

0816

6517

23226

1154

1309014H

mdash0475

0744

0858

10012

15691

1523





1209051H 1401 191 8011 278 17413 1651303002H 1443 126 7959 142 7959 2521303002H 1730 139 8037 184 17885 2651210019H 836 082 7806 202 36342 0391212024H 1064 275 8251 121 34878 2481301023H 1221 125 8642 088 25237 2571302026H 1008 167 11590 262 32311 2471303036H 607 251 8498 300 38869 1051304001H 1002 161 11197 235 26861 2421306019H 962 161 8668 132 30362 0811306020H 813 126 8093 289 35571 1721306027H 971 037 9314 049 26075 0521309014H 964 286 9650 289 30190 059




Acknowledgments


References





































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


500 1500 2500 3500 45002000060000

100000140000180000220000260000300000340000


Inte

nsity

(a)

500 1500 2500 3500 4500

2000060000

100000140000180000220000260000300000340000


Inte

nsity

(b)

500 1500 2500 3500 45002000060000

100000140000180000220000260000300000340000


Inte

nsity

(c)

500 1500 2500 3500 45002000060000

100000140000180000220000260000300000340000


Inte

nsity

(d)






4 Conclusions



Table3Re

lativec

onstitu

entcon

tent

determ

inations

from

13batcheso

fZTP

sSample

number

35-Dim

etho

xytoluene(

)

Safro

le(

)2-Metho

xy-4-vinylph

enolph

enol(

)12

-Dim

etho

xy-4-(2-prop

enyl)benzene

()

Ligustilid

e()

Oleicacid

()

Asarin

in(

)

1209051H

mdash0744

0321

0267

497

2616

60758

1303002H

mdash0393

0587

0753

7101

1676

918

061212023H

mdash0321

0393

0438

5747

32844

1084

1210019H

mdash0736

0556

mdash8905

20581

3120

1212024H

mdash0556

0420

0309

5903

34815

2163

1301023H

mdash0420

040

0mdash

4285

34989

1119

1302026H

mdash040

00753

mdash7558

18568

1334

1303036H

mdash0753

0973

mdash5916

20691

2074

1304

001H

mdash0973

0803

mdash10845

19593

1560

1306

019H

mdash0803

0588

mdash7396

20627

2164

1306

020H

mdash0588

0458

mdash5725

18016

1344

1306

027H

mdash0458

0475

0816

6517

23226

1154

1309014H

mdash0475

0744

0858

10012

15691

1523





1209051H 1401 191 8011 278 17413 1651303002H 1443 126 7959 142 7959 2521303002H 1730 139 8037 184 17885 2651210019H 836 082 7806 202 36342 0391212024H 1064 275 8251 121 34878 2481301023H 1221 125 8642 088 25237 2571302026H 1008 167 11590 262 32311 2471303036H 607 251 8498 300 38869 1051304001H 1002 161 11197 235 26861 2421306019H 962 161 8668 132 30362 0811306020H 813 126 8093 289 35571 1721306027H 971 037 9314 049 26075 0521309014H 964 286 9650 289 30190 059




Acknowledgments


References





































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Table3Re

lativec

onstitu

entcon

tent

determ

inations

from

13batcheso

fZTP

sSample

number

35-Dim

etho

xytoluene(

)

Safro

le(

)2-Metho

xy-4-vinylph

enolph

enol(

)12

-Dim

etho

xy-4-(2-prop

enyl)benzene

()

Ligustilid

e()

Oleicacid

()

Asarin

in(

)

1209051H

mdash0744

0321

0267

497

2616

60758

1303002H

mdash0393

0587

0753

7101

1676

918

061212023H

mdash0321

0393

0438

5747

32844

1084

1210019H

mdash0736

0556

mdash8905

20581

3120

1212024H

mdash0556

0420

0309

5903

34815

2163

1301023H

mdash0420

040

0mdash

4285

34989

1119

1302026H

mdash040

00753

mdash7558

18568

1334

1303036H

mdash0753

0973

mdash5916

20691

2074

1304

001H

mdash0973

0803

mdash10845

19593

1560

1306

019H

mdash0803

0588

mdash7396

20627

2164

1306

020H

mdash0588

0458

mdash5725

18016

1344

1306

027H

mdash0458

0475

0816

6517

23226

1154

1309014H

mdash0475

0744

0858

10012

15691

1523





1209051H 1401 191 8011 278 17413 1651303002H 1443 126 7959 142 7959 2521303002H 1730 139 8037 184 17885 2651210019H 836 082 7806 202 36342 0391212024H 1064 275 8251 121 34878 2481301023H 1221 125 8642 088 25237 2571302026H 1008 167 11590 262 32311 2471303036H 607 251 8498 300 38869 1051304001H 1002 161 11197 235 26861 2421306019H 962 161 8668 132 30362 0811306020H 813 126 8093 289 35571 1721306027H 971 037 9314 049 26075 0521309014H 964 286 9650 289 30190 059




Acknowledgments


References





































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





1209051H 1401 191 8011 278 17413 1651303002H 1443 126 7959 142 7959 2521303002H 1730 139 8037 184 17885 2651210019H 836 082 7806 202 36342 0391212024H 1064 275 8251 121 34878 2481301023H 1221 125 8642 088 25237 2571302026H 1008 167 11590 262 32311 2471303036H 607 251 8498 300 38869 1051304001H 1002 161 11197 235 26861 2421306019H 962 161 8668 132 30362 0811306020H 813 126 8093 289 35571 1721306027H 971 037 9314 049 26075 0521309014H 964 286 9650 289 30190 059




Acknowledgments


References





































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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