research article characterization of chemical composition of ...pericarpium citri reticulatae...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 237541 11 pageshttpdxdoiorg1011552013237541

Research ArticleCharacterization of Chemical Composition ofPericarpium Citri Reticulatae Volatile Oil by ComprehensiveTwo-Dimensional Gas Chromatography with High-ResolutionTime-of-Flight Mass Spectrometry

Kunming Qin12 Lijuan Zheng12 Hao Cai1 Gang Cao3 Yajing Lou1 Tulin Lu1

Yachun Shu14 Wei Zhou1 and Baochang Cai123

1 Engineering Center of Ministry of Education for Standardization of Chinese Medicine ProcessingNanjing University of Chinese Medicine Nanjing 210023 China

2Nanjing Haichang Chinese Medicine Group Co Ltd Nanjing 210061 China3 Research Center of TCM Processing Technology Zhejiang Chinese Medical University Hangzhou 310053 China4Affiliated Hospital of Nanjing University of Chinese Medicine Nanjing 210029 China

Correspondence should be addressed to Baochang Cai bccai126com

Received 28 January 2013 Accepted 2 April 2013

Academic Editor Carlo Ventura

Copyright copy 2013 Kunming Qin 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

Pericarpium Citri Reticulatae (Chenpi in Chinese) has been widely used as an herbal medicine in Korea China and Japan Chenpiextracts are used to treat indigestion and inflammatory syndromes of the respiratory tract such as bronchitis and asthmaThis thesiswill analyze chemical compositions of Chenpi volatile oil which was performed by comprehensive two-dimensional gas chroma-tography with high-resolution time-of-flight mass spectrometry (GC times GC-HR-TOFMS) One hundred and sixty-seven compo-nents were tentatively identified and terpene compounds are the main components of Chenpi volatile oil a significant larger num-ber than in previous studies The majority of the eluted compounds which were identified were well separated as a result of high-resolution capability of the GC times GC method which significantly reduces the coelution 120573-Elemene is tentatively qualified bymeans of GC times GC in tandem with high-resolution TOFMS detection which plays an important role in enhancing the effects ofmany anticancer drugs and in reducing the side effects of chemotherapyThis study suggests that GC timesGC-HR-TOFMS is suitablefor routine characterization of chemical composition of volatile oil in herbal medicines

1 Introduction

Pericarpium Citri Reticulatae (Chenpi in Chinese) has beenwidely used as an herbal medicine for a long time in ChinaKorea and Japan for its pharmacologic activity rich re-sources low toxicity and costs Chenpi is the dried ripe fruitpeel of Citrus reticulata Blanco and its cultivars gatheredfrom September to December [1] Their main cultivars areCitrus reticulata ldquoChachirdquo Citrus Reticulata ldquoDahongpaordquoand Citrus erythrosa Tanaka In Chinese peoplersquos traditionaluse Chenpi is mostly utilized to eliminate phlegm andstrengthen spleen [2] Moreover Chenpi is extensively addedto food as a condiment

It is well known that Chenpi contains various bioactivecompounds such as flavonoids phenolic acids and limo-noids [2 3] In the present study most reports on Chenpifocus on phenolic compounds and flavonoids [3ndash7] but fewfocus on volatile compounds which also have strong pharma-cologic bioactivities For example high-performance liquidchromatography (HPLC) high-speed countercurrent chro-matography (HSCCC) and capillary electrophoresis (CE)have been applied for the determination of phenolic com-pounds and flavonoids of Chenpi [8ndash10] However reviewingthe literature it seems that the chemical composition of thevolatile oil of Chenpi has been little investigated [11] Fur-thermore the volatile compounds of Chenpi may contribute

2 Evidence-Based Complementary and Alternative Medicine

to pharmacological effects of Chenpi extracts reported above[12 13] Therefore a method able to rapidly identify the vola-tile compounds of Chenpi could be a useful tool for the pur-pose of a complete phytochemical analysis

Gas chromatography-mass spectroscopy (GC-MS) hasbeen used for the qualitative analysis of the volatile consti-tuents in Chenpi [11] But it is difficult to achieve the completeseparation of minor volatile components andmany coelutionvolatile constituents To solve these problems it is neces-sary to use multidimensional gas chromatography Compre-hensive two-dimensional gas chromatography with high-resolution time-of-flight mass spectrometry (GC times GC-HR-TOFMS) is a new developed powerful and versatile analyticaltool which combines two powerful analytical technologieswith complementary attributes [14 15] GC times GC separateschemical species with two capillary columns interfaced by amodulator that traps and concentrates eluents from the firstcolumn and it then introduces them into the second columnproducing a full secondary chromatogram for each singledata point of a traditional one-dimensional separation [1617] HR-TOFMS provides mass precision that is fine enoughto distinguish elemental compositions providing amore defi-nitive basis for molecular identification GC times GC is impor-tant for HR-TOFMS because the better separations signifi-cantly reduce the coelution and the problems ofmass spectralmixing And HR-TOFMS is important for GC timesGC becausethe structural and compositional information available withHR-TOFMS aids in the interpretation of the rich complexdata from GC times GC separations [18] GC times GC-TOFMS hasbeen successfully applied in the volatile oil study and greatlyimproves the result of component separation and identifi-cation [19 20] In this study the volatile oil of Chenpi wasfirstly separated and detected with GC times GC-HR-TOFMS(Figure 1)

2 Materials and Methods

21 Samples Chenpi sample (fruit peels of Citrus reticulateldquoDahongpaordquo) was collected from Zigong in Sichuan pro-vince China The sample was authenticated by ProfessorChen Jianwei fromNanjingUniversity of Traditional ChineseMedicine China

22 Extraction of Volatile Oil After the sample was dried for2 h at 45∘C and smashed 50 g of sample was swollen with600mL of distilled water in a standard extractor for extract-ing volatile oil for 3 h Then the volatile oil was dried overanhydrous sodium sulphate until all the water was dried andthen stored in the dark glass bottle at 4∘C prior to GC times GC-HR-TOFMS analysis

23 GC-MS System and GC times GC-HR-TOFMS ApparatusGC timesGC separations were performed by Tofwerk AG (ThunSwitzerland) on an Agilent 7890A GC and 7693 autosamplerwith 1120583L splitless injection column one DB-XLB (Agilent)15m times 025mm 025120583mfilm thickness column two BPX-50(SGE) 1m times 01mm 01120583mfilm thickness oven temperaturefrom 50 to 230∘C at 20∘Cminminus1 ramp inlet pressure from

35 PSI to 615 PSI at 028 PSIminminus1 injection temperature250∘C transfer line temperature 300∘C Zoex ZX2 thermalmodulator with a 7 s modulation period 300ms modulationduration 375∘C hot jet temperature 18 Lminminus1 cold jet nitro-gen flow rate and 40 PSI hot jet nitrogen pressure The ZoexFasTOF time-of-flight- (TOF-) HRMS system used 70 eV EIion source 280∘C ion source temperature amass range ofmz50ndash450 with 4000 FWHM resolution and 100 spectra persecond acquisition rate

24 Data Conversion and Peak Table Generation The finaldata for each chromatogram is an array of 1000 times 600 datapoints each data point with a HRMS vector of 40K inten-sities Thus each chromatogram has 24 billion values requir-ing 96 gigabytes for representing for single-precision floatingpoint numbers without compressionThe set of 18 chromato-grams has more than 17 terabytes of uncompressed dataThedata were compressed and stored by the Zoex FasTOF systemto HDF5-format files and were processed with GC ImageGC times GC Software R21 In order to manage such large fileson computers with limited random access memory (RAM)GC Image Software maintains a chromatogram with integermass or centroid-resampled spectra in RAM and accesses theHR-MS data from disk as needed GC Image can export rawdata and computed results to nonproprietary file formats forprocessing with external software The components can bequantified by Zoex software (Zoex Corp Lincoln NE USA)

All peaks with signal-to-noise ratio higher than 100 werefound in the raw GC times GC chromatogram The workstationcan automatically give the parameters such as similarityreverse and probability of peaks via comparing them withthe compounds in the library The results were combined ina peak table The NISTEPANIHMass Spectral Library Ver-sion 20 was used in this work

3 Results and Discussion

31 Qualitative Analysis of Chenpi Volatile Oil The columnsystem is nearly orthogonal and provides a structured sepa-ration A typical two-dimensional separationtotal ion chro-matogram (TIC) and three-dimensional chromatogram areshown in Figure 2 In the GC times GC system compounds areseparated by volatility difference on the first dimensionnonpolar column and by polarity on the second medium-polar column The GC times GC system accomplishes the trueorthogonal separation on account for both the change ofthe polarity of two fixed phases and the linear temperatureprogramming

Using GC timesGC-HR-TOFMS the quantity of the detectedcomponents was up to 834 Compared to the traditional iden-tification method such as GC-MS the analysis from GC timesGC-HR-TOFMS becomes more reliable relying on the com-bined identification information including retention timessimilarity reverse match factor and probability The similar-ity and reverse match factors indicate how well a mass spec-trummatches the library spectrum but the isomers have sim-ilar mass spectra In this case the probability is used to deter-mine whether the peaks with the same name belong to one

Evidence-Based Complementary and Alternative Medicine 3

Processing

Identification

Pericarpium Citri Reticulatae

Extraction

Analyzing

Zoex GC times GC-TOF-MS analysis

Steam distillation

Figure 1 Flow chart of the chemical composition study of Chenpi volatile oil by GC times GC-HR-TOFMS

6208

5173

4138

3104

2069

1035

0

0 6035 18105 24141 30176 36211 42246 48281 54316 60351 66387 72422

2-di

men

siona

l ret

entio

n tim

e (s)

1-dimensional retention time (min)1207

(a)

Column II

Column I

(b)

Figure 2 GC times GC-HR-TOFMS chromatogram (a) and three-dimensional chromatogram (b) of Chenpi volatile oil


A B

6208

5173

4138

3104

2069

1035

0

2-di

men

siona

l ret

entio

n tim

e (s)

75 13535 1957 25605 31641 43711 49746 55781 61816 67851 73887 79922

1-dimensional retention time (min)

TIC image

37676

Figure 3 The GC times GC contour plot of Chenpi volatile oil group separation result Regions marked by squares (A) and (B) were identifiedmainly as monoterpenes and sesquiterpenes respectively

compound or several compounds The GC times GC-HR-TOFMS software was used to find all the peaks in the raw GC timesGCchromatogramA library searchwas carried out for all thepeaks using the NISTEPANIH version 20 and the resultswere combined in a single peak table A similarity and reversematch factor above 583 and 612 respectively indicates thatan acquired mass spectrum usually shows a good match withthe library spectrum Because of the numerous isomers pre-sent in volatile oils especially within monoterpenes andsesquiterpenes more attention should be paid for identifica-tion using mass spectra In order to enhance the reliability ofthe identification by MS both similarity and reverse matchfactor should be used According to our experience and theliterature data [18ndash20] 167 compounds with good matchwere tentatively identified including 50 monoterpenes 36sesquiterpenes 31 esters and acids 9 aldehydes and ketones6 alcohols 3 ethers 12 phenyl compounds and 20 other com-ponents Compounds have lower search probabilities thanthese counted as unknowns and were disqualified for Kovatsindex comparison Table 1 listed 167 components identifiedin Chenpi volatile oil The volatile fraction is characterizedby high percentages of monoterpenes sesquiterpenes andesters including 120573-elemene p-mentha-1(7)8(10)-dien-9-oland limonene In this studymany components have also beententatively identified which were found in Chenpi volatile oilfor the first time such as globulol and isoledeneThere is highpossibility that they will be literally useful for further phar-maceutical research of Chenpi volatile oil

32 Group Separation of Chenpi Volatile Components InGC times GC-HR-TOFMS analysis the 167 identified volatilecomponents in Chenpi volatile oil were mainly classified intotwo groups that can be seen in Figure 3 Based on GC times GC-HR-TOFMS it can be found that the peaks in areas A andB are monoterpenes and sesquiterpenes respectively Thesemonoterpenes and sesquiterpenes are mainly alkenes alco-hols and ethers It was also found that a lot of saturatedand unsaturated fatty acid esters and phenyl compoundsconstitute the Chenpi volatile oil This study demonstrates

that GC times GC-HR-TOFMS is a powerful separation andidentification tool that allows for the identification and groupseparation of a much larger number of complex volatile oilcomponents

33 Identification of Three Coelution Volatile Components inChenpi Volatile Oil The high-resolution mass spectra in theTIC can be used for accurate identification of volatile com-pounds in Chenpi volatile oil and these identified com-pounds will be significant to the further pharmaceuticalresearch For example Figure 4 compares the high resolutionmass spectrumof the blob (peak)markedwith 138 (4914min202 s) 104 (4926min 158 s) and 77 (4914min 145 s) head-to-tail with the mass spectrum of p-mentha-1(7)8(10)-dien-9-ol dodecanal and 120573-elemene TMS from the NISTEPANIH library mass spectra For p-mentha-1(7)8(10)-dien-9-ol the forward match factor is 806 reverse match factoris 855 and probability is 2012 For dodecanal the forwardmatch factor is 763 reverse match factor is 773 and pro-bability is 716 For 120573-elemene the forward match factoris 911 reverse match factor is 914 and probability is 1711The above three volatile components cannot be clearlyseparated or identified by traditional one-dimensional gaschromatography or GC-MS method because they are coelu-tion volatile components which have very similar chemicalproperties including volatility and polarity In this studythe three coelution volatile components in Chenpi volatileoil were well separated and identified by GC times GC-HR-TOFMS which have not been reported in other studies(Figure 4)

This study showed that GC timesGC-HR-TOFMS representsa powerful separation and analysis tool for the analysis ofcomplex volatile oils of herbal medicines GC times GC-HR-TOFMS can give the information about the formula andstructures can provide the opportunity for differentiating dif-ferent volatile oils can give the subtle differences of the oilsfrom different areas and can find new compounds that havethe possible pharmaceutical effect on some diseases


Table 1 167 main volatile components identified in the Chenpi volatile oil

No Compound name Peak Imin Peak IIs Volume Library formula Libraryprobability Library CAS no

(1) Nonanal 2862 16 2367153 C9H18O 501 124-19-6(2) Pyrrolizidine-3-one-5-ol ethyl ether 5349 274 531709 C9H15NO2 177 0-00-0(3) 2-Methoxy-4-vinylphenol 4238 273 2959926 C9H10O2 5939 7786-61-0(4) Styrene 1414 165 731272 C8H8 364 100-42-5(5) Octanal 2138 158 2235185 C8H16O 6219 124-13-0(6) Ethylbenzene 1257 139 4844996 C8H10 6486 100-41-4(7) Pterin-6-carboxylic acid 1124 082 503913 C7H5N5O3 4094 948-60-7(8) Hexadecane 11-bis(dodecyloxy)- 7727 12 1023988 C40H82O2 924 56554-64-4(9) 1-Heptatriacotanol 7509 276 1692274 C37H76O 5456 105794-58-9(10) Cholestan-3-ol 2-methylene- (31205735120572)- 6435 211 2684351 C28H48O 1309 22599-96-8

(11)[59-Dimethyl-1-(3-phenyl-oxiran-2-yl)-deca-48-dienylidene]-(2-phenyl-aziridin-1-yl)-amine

664 223 151311 C28H34N2O 4404 0-00-0

(12) 111015840-(4-Methyl-13-phenylene)bis[3-(5-benzyl-134-thiadiazol-2-yl)urea] 2355 215 1060574 C27H24N8O2S2 2495 0-00-0

(13) Morphinan-45-epoxy-36-di-ol 6-[7-nitro-benzofurazan-4-yl]amino- 4649 079 1055334 C26H27N5O6 948 0-00-0

(14) Benzene 111015840-[3-(3-cyclopentylpropyl)-15-pentanediyl]bis- 774 118 1492865 C25H34 1518 55191-62-3

(15) 2-(2-Azepan-1-yl-2-oxoethyl)-1-hydroxy-1-phenyl-octahydro-pyrido[12-a]azepin-4-one 7642 277 1421083 C24H34N2O3 5215 0-00-0

(16) 691215-Docosatetraenoic acid methyl ester 594 177 55943 C23H38O2 1928 17364-34-0

(17)2-[4-Methyl-6-(266-trimethylcyclohex-1-enyl)hexa-135-trienyl]cyclohex-1-en-1-carbo-xaldehyde

5349 215 644497 C23H32O 2557 0-00-0

(18) Naphthalen-2-yl-acetic acid 6-hydroxy-6-methyl-cyclodecyl ester 4154 263 655915 C23H30O3 273 0-00-0

(19) Z-5-Methyl-6-heneicosen-11-one 8089 111 1151736 C22H42O 827 0-00-0(20) 2H-Pyran 2-(7-heptadecynyloxy)tetrahydro- 6145 191 1336559 C22H40O2 144 56599-50-9(21) Doconexent 1788 163 1279331 C22H32O2 4098 6217-54-5

(22) 91215-Octadecatrienoic acid 23-dihydroxy-propyl ester (ZZZ)- 3116 222 1051463 C21H36O4 1978 18465-99-1

(23) 81114-Eicosatrienoic acid methyl ester(ZZZ)- 4275 202 2168234 C21H36O2 842 21061-10-9

(24) 5811-Eicosatrienoic acid methyl ester 5759 203 961455 C21H30O2 4395 0-00-0(25) cis-58111417-Eicosapentaenoic acid 2452 425 537632 C20H30O2 1399 10417-94-4(26) 581114-Eicosatetraynoic acid 588 317 1449613 C20H24O2 2978 1191-85-1

(27) 116-Cyclocorynan-17-oic acid 1920-di-dehydro- methyl ester (16S19E)- 1221 064 885548 C20H22N2O2 5345 6393-66-4

(28) Octadecane 6-methyl- 8209 125 1849235 C19H40 1841 10544-96-4(29) 2-Methyl-EE-313-octadecadien-1-ol 6483 201 1240941 C19H36O 974 0-00-0(30) ZZZ-469-Nonadecatriene 2681 17 109996 C19H34 2105 0-00-0(31) 6912-Octadecatrienoic acid methyl ester 5578 292 637153 C19H32O2 2852 2676-41-7(32) ZZZ-1469-Nonadecatetraene 215 18 654446 C19H32 1464 0-00-0(33) 1215-Octadecadienoic acid methyl ester 4914 235 1585734 C19H30O2 1933 57156-95-3(34) 1013-Octadecadienoic acid methyl ester 5047 247 1610184 C19H30O2 1545 18202-24-9(35) 25-Octadecadienoic acid methyl ester 5952 199 2547258 C19H30O2 1015 57156-91-9

(36) 2244-Tetramethyl-6-(1-oxo-3-phenylprop-2-enyl)-cyclohexane-135-trione 7027 049 832401 C19H20O4 2198 0-00-0

(37) Gentamicina 6363 321 626103 C18H36N4O10 2705 13291-74-2


Table 1 Continued


(38) Z-10-Methyl-11-tetradecen-1-ol propionate 4033 2 703719 C18H34O2 1053 0-00-0(39) 10-Heptadecen-8-ynoic acid methyl ester (E)- 3418 206 632456 C18H30O2 146 16714-85-5

(40) 120572-L-Fucopyranose 1234-bis(benzeneboro-nate) 6713 228 1805156 C18H18B2O5 2589 102281-26-5

(41) 3-(O-Anisidinomethyl)-5-(3-fluorobenzyli-dene)-24-thiazolidinedione 3466 246 829072 C18H15FN2O3S 943 302954-96-7

(42) 1-Hexadecanol 2-methyl- 4419 103 651528 C17H36O 1182 2490-48-4(43) 10-Methyl-E-11-tridecen-1-ol propionate 3333 156 1642353 C17H32O2 669 0-00-0(44) 13-Heptadecyn-1-ol 4009 225 785129 C17H32O 1204 56554-77-9(45) 4710-Hexadecatrienoic acid methyl ester 3297 205 835894 C17H28O2 798 17364-31-7(46) Methyl 57-hexadecadienoate 5361 17 58765 C17H26O2 1434 0-00-0

(47) 3-(5-Benzyloxy-3-methylpent-3-enyl)-22-dimethyloxirane 3502 261 96359 C17H24O2 895 0-00-0

(48) Falcarinol 3707 157 550178 C17H24O 3551 21852-80-2(49) tert-Hexadecanethiol 7027 103 1515898 C16H34S 1387 25360-09-2(50) n-Hexadecanoic acid 7908 174 7057247 C16H32O2 4305 10-3-1957(51) Cyclopentaneundecanoic acid 4649 162 94519 C16H30O2 1397 6053-49-2(52) 9-Hexadecenoic acid 6556 191 89144 C16H30O2 1329 2091-29-4

(53) Formic acid 3711-trimethyl-1610-dodecatri-en-3-yl ester 5192 171 991647 C16H26O2 2372 0-00-0

(54) 13-Dioxolane 2-heptyl-4-phenyl- 774 067 1371547 C16H24O2 2064 55668-40-1(55) Peyonine 2464 058 549972 C16H19NO5 2728 19717-25-0(56) 121414-Trimethyl-369-trioxapentadecan-1-ol 6652 118 585099 C15H32O4 1694 55489-54-8(57) Isocalamendiol 7365 276 523348 C15H26O2 2427 0-00-0(58) Geranyl isovalerate 5337 103 770183 C15H26O2 1172 109-20-6(59) Cubeduel 6326 188 2032965 C15H26O 248 0-00-0(60) 120572-Cadinol 6363 207 6493081 C15H26O 2436 481-34-5(61) Cubenol 6242 19 4084446 C15H26O 1617 21284-22-0(62) tau-Cadinol 6302 2 707983 C15H26O 117 11-1-5937(63) 120572-Acorenol 6363 217 1949884 C15H26O 972 0-00-0(64) Globulol 6013 188 2783377 C15H26O 64 51371-47-2(65) 7-Epi-cis-sesquisabinene hydrate 5457 13 812625 C15H26O 64 0-00-0(66) Limonen-6-ol pivalate 4456 185 788648 C15H24O2 2404 0-00-0(67) Aromadendrene oxide-(2) 6278 209 967312 C15H24O 1584 0-00-0(68) Caryophyllene oxide 5988 2 1615517 C15H24O 1315 1139-30-6

(69) Spiro[45]dec-6-en-8-one 17-dimethyl-4-(1-methylethyl)- 3816 227 732272 C15H24O 702 39510-36-6

(70) Humulene 53 161 5204711 C15H24 4449 6753-98-6(71) (ZE)-120572-farnesene 5578 152 310676 C15H24 4294 26560-14-5(72) 120572-Copaene 4842 14 7508707 C15H24 4237 0-00-0(73) 120575-Elemene 4588 133 5692139 C15H24 3801 20307-84-0

(74) Naphthalene 123568a-hexahydro- 47-di-methyl-1-(1-methylethyl)- (1S-cis)- 5675 168 1295664 C15H24 31 483-76-1

(75) 13610-Dodecatetraene 3711-trimethyl-(ZE)- 5494 151 1969983 C15H24 2901 26560-14-5

(76) Caryophyllene 5095 154 3006936 C15H24 179 87-44-5(77) 120573-Elemene 4914 145 2034787 C15H24 1711 515-13-9(78) 120572-Guaiene 5216 147 5565085 C15H24 1663 12-1-3691

(79) Cyclohexane 1-ethenyl-1-methyl-24-bis(1-methylethenyl)- 5276 123 1606527 C15H24 1642 110823-68-2


Table 1 Continued


(80) 16-Cyclodecadiene 1-Methyl-5-methylene-8-(1-methylethyl)- [S-(EE)]- 5445 165 14112 C15H24 1525 23986-74-5

(81) 120574-Elemene 5168 151 7427373 C15H24 1514 29873-99-2

(82) Naphthalene 12344a7- hexahydro-16-di-methyl-4-(1-methylethyl)- 5735 171 2007951 C15H24 1455 16728-99-7

(83) Cyclohexane 1-ethenyl-1-methyl- 24-bis(1-methylethenyl)- [1S-(112057221205734120573)]- 4866 147 5496758 C15H24 1066 515-13-9

(84) 120573-Copaene 5445 155 5614817 C15H24 1053 0-00-0(85) 120574-Elemene 588 182 1698139 C15H24 831 29873-99-2(86) 120573-Guaiene 5759 175 601417 C15H24 716 88-84-6(87) Guaia-1(10)11-diene 5482 167 1093799 C15H24 66 0-00-0(88) Isoledene 5421 158 4050377 C15H24 627 0-00-0(89) 45-Di-epi-aristolochene 4781 143 900937 C15H24 42 0-00-0(90) trans-calamenene 5651 185 84299 C15H22 3835 0-00-0(91) 120573-Vatirenene 7365 234 4374147 C15H22 2571 0-00-0

(92) 44-Dimethyl-3-(3-methylbut-3-enylidene)-2-methylenebicyclo[410]heptane 7268 232 2843733 C15H22 1128 79718-83-5

(93) 7-Hydroxy-69a-dimethyl-3-methylene-decahydro-azuleno[45-b]furan-29-dione 7521 361 616952 C15H20O4 1161 0-00-0

(94) Propanoic acid 3-(236-trimethyl- 14-dioxa-spiro[44]non-7-yl)- methyl ester 3393 27 848793 C14H24O4 1992 0-00-0

(95) 25-Furandione 3-(2-decenyl)dihydro- 4033 276 1541135 C14H22O3 1138 62568-81-4(96) trans-(2-Decenyl)succinic anhydride 7208 16 678793 C14H22O3 1039 81949-64-6(97) 14-Benzenediol 26-bis(11-dimethylethyl)- 7751 05 709573 C14H22O2 713 2444-28-2(98) Tetraacetyl-d-xylonic nitrile 5832 162 100275 C14H17NO9 1848 0-00-0(99) 120572-Ionol 3019 218 2779312 C13H22O 1516 25312-34-9

(100) 1-(2-Acetoxyethyl)-36-diazahomoadamantan-9-one oxime 7328 05 552116 C13H21N3O3 878 0-00-0

(101) 1b556a-Tetramethyl-octahydro-1-oxa-cyclo-propa[a]inden-6-one 3321 189 1386104 C13H20O2 645 0-00-0

(102) Pyrimidin-2-one 4-[N-methylureido]-1-[4-methylaminocarbonyloxymethyl 6894 038 543663 C13H19N5O5 2312 0-00-0

(103) 2H-Indeno[12-b]furan-2-one 33a456788b-octahydro-88-dimethyl 553 193 2032335 C13H18O2 2893 0-00-0

(104) Dodecanal 4926 158 1282104 C12H24O 66 112-54-9(105) 26-Octadien-1-ol 37-dimethyl- acetate (Z)- 4637 174 3059872 C12H20O2 3764 141-12-8(106) Geranyl acetate 4745 179 3772587 C12H20O2 287 105-87-3(107) (R)-Lavandulyl acetate 4637 225 817472 C12H20O2 1419 0-00-0(108) Geranyl vinyl ether 3683 177 760262 C12H20O 1431 0-00-0(109) 31015840-Hydroxyquinalbarbitone 3961 15 514505 C12H18N2O4 1072 839-21-4(110) Non-1-yn-5-en-9-aldehyde 4-carbethoxy- 4431 211 1151319 C12H16O3 1787 0-00-0(111) Undecanal 4275 159 7045031 C11H22O 3386 112-44-7

(112) Cyclohexane 2-ethenyl-11-dimethyl-3-methyl-ene- 3031 134 1352002 C11H18 2383 95452-08-7

(113) Cyclohexene 2-ethenyl-133-trimethyl- 4202 222 5912578 C11H18 1436 5293-90-3(114) Acetic acid octyl ester 3647 149 3236557 C10H20O2 1784 112-14-1(115) Decanal 3574 161 7031098 C10H20O 6162 112-31-2(116) Cephrol 3755 166 3282375 C10H20O 1234 40607-48-5(117) Linalol 2874 152 3645373 C10H18O 8159 78-70-6


Table 1 Continued


(118) 120572-Terpineol 349 189 7440985 C10H18O 6775 98-55-5(119) Terpinen-4-ol 3418 179 3293372 C10H18O 5003 562-74-3(120) Citronellal 3212 169 6153934 C10H18O 4259 106-23-0

(121) 2-Cyclohexen-1-ol 1-methyl-4-(1-methyl-ethyl)- cis- 3031 167 1592231 C10H18O 3911 29803-82-5

(122) Cyclohexanol 1-methyl-4-(1-methylethenyl)-cis- 3164 18 7268632 C10H18O 1059 7299-41-4

(123) 2-Cyclohexen-1-ol 2-methyl-5-(1-methyl-ethyl)- (1S-cis)- 3611 185 1012405 C10H18O 1053 536-30-1

(124) exo-277-trimethylbicyclo[221]heptan-2-ol 3297 189 1699902 C10H18O 815 0-00-0(125) 5-Hepten-1-ol 2-ethenyl-6-methyl- 4576 168 3971537 C10H18O 775 18479-48-6

(126) Bicyclo[410]heptane 377-trimethyl- [1S-(112057231205736120572)]- 4576 158 3688305 C10H18 972 2778-68-9

(127) Desulphosinigrin 6664 106 733518 C10H17NO6S 892 5115-81-1(128) R-Limonene 3937 273 970311 C10H16O3 4037 0-00-0(129) Limonene oxide trans- 3128 178 5708168 C10H16O 5424 4959-35-7(130) Limonene oxide cis- 3092 178 1068476 C10H16O 4043 13837-75-7(131) trans-p-Mentha-28-dienol 3007 178 4487691 C10H16O 3435 0-00-0(132) 3-Cyclohexene-1-acetaldehyde 1205724-dimethyl- 3635 209 1817561 C10H16O 3138 29548-14-9(133) cis-p-Mentha-1(7)8-dien-2-ol 3743 205 3985489 C10H16O 2642 0-00-0(134) (Z)-Carveol 355 197 4522402 C10H16O 2548 1197-06-4(135) trans-Carveol 3671 2 1591657 C10H16O 2266 1197-07-5(136) cis-p-Mentha-1(7)8-dien-2-ol 3393 191 3364337 C10H16O 215 0-00-0

(137) 1-Cyclohexene-1-methanol 4-(1-methyl-ethenyl)- 3562 189 1959113 C10H16O 2076 536-59-4

(138) p-Mentha-1(7)8(10)-dien-9-ol 4914 202 1162984 C10H16O 2012 29548-13-8(139) cis-p-Mentha-28-dien-1-ol 4347 191 990278 C10H16O 783 3886-78-0(140) 26-Dimethyl-357-octatriene-2-ol EE- 3188 198 1870291 C10H16O 76 0-00-0

(141) (S)-(-)-(4-Isopropenyl-1-cyclohexenyl)-methanol 3538 189 1727702 C10H16O 586 18457-55-1

(142) Bicyclo[310]hex-2-ene 4-methyl-1-(1-methyl-ethyl)- 174 111 7918412 C10H16 6504 28634-89-1

(143) 120573-Pinene 2066 131 1996434 C10H16 408 127-91-3(144) 120573-Ocimene 2548 134 4519345 C10H16 3756 13877-91-3(145) 120572-Phellandrene 2247 132 2085008 C10H16 3342 99-83-2(146) 120573-Myrcene 215 127 6926099 C10H16 313 123-35-3(147) 120572-Pinene 1788 116 4837696 C10H16 2382 80-56-8(148) Camphene 1885 122 4064708 C10H16 1993 79-92-5(149) 120573-Phellandrene 2029 127 4173575 C10H16 1968 555-10-2(150) D-Limonene 2452 169 1784450 C10H16 1959 5989-27-5(151) 120572-Terpinene 2343 133 4905547 C10H16 1758 99-86-5(152) Isoterpinene 2838 146 1460702 C10H16 1601 586-62-9(153) 120574-Terpinene 2633 15 1898081 C10H16 1528 99-85-4(154) 155-Trimethyl-6-methylene-cyclohexene 2633 186 3059981 C10H16 981 514-95-4

(155) Cyclohexene 1-methyl-4-(1-methylethenyl)-(S)- 2452 331 517426 C10H16 958 5989-54-8

(156) 120574-Pyronene 2307 127 603834 C10H16 533 514-95-4

(157) 5-Isopropenyl-2-methylcyclopent-1-enecarbo-xaldehyde 3611 23 7112013 C10H14O 3431 0-00-0

(158) (-)-Carvone 378 235 9098454 C10H14O 3276 6485-40-1


Table 1 Continued


(159) 1-Cyclohexene-1-carboxaldehyde 4-(1-methylethenyl)- 3985 243 1404104 C10H14O 3176 2111-75-3

(160) 35-Heptadienal 2-ethylidene-6-methyl- 3695 228 2380085 C10H14O 2828 99172-18-6(161) Benzenemethanol 1205721205724-trimethyl- 3405 228 2883869 C10H14O 1659 1197-01-9(162) Cyclohexanone 2-(2-butynyl)- 4238 318 591028 C10H14O 1502 54166-48-2(163) D-Verbenone 4142 216 4752141 C10H14O 1163 18309-32-5(164) 35-Heptadienal 2-ethylidene-6-methyl- 3611 25 18488 C10H14O 897 99172-18-6(165) o-Cymene 2355 155 3599109 C10H14 4751 527-84-4(166) 26-Dimethyl-1357-octatetraene EE- 3164 171 1648089 C10H14 2019 460-01-5(167) Benzene 1-methyl-4-(1-methylethenyl)- 279 18 2716784 C10H12 2042 1195-32-0

4138

3104

2069

1035

0

43711 49746 55781 61816 67851 73887

p-Mentha-1(7) 8(10)-dien-9-ol (138)

Dodecanal (104)

120573-Elemene (77)

2-di

men

siona

l ret

entio

n tim

e (s)


(a)

150

150

100

100

50

50

0

40 60 80 100 120 140 160 180

Inte

nsity

p-Mentha-1(7) 8(10)-dien-9-0l (138)

Peak true

119898119911

Library hit

55068946706999 7907414

9410049

1061051211912087

5567

79 93

119

134

152

45

55

57 65

67

77

79

84

91

106

119

134

41

(b)

Figure 4 Continued


55068946706999

7907414 9612316

4551

57

67

79

82

8596

105 110 119 133 140 147 161

55

7181

95

109121 135

150

150

100

100

50

50

0

10040 80 120 140 160 180

Inte

nsity

Dodecanal (104)

Library hit

Peak true

119898119911

41

60

(c)

2000

2000

1500

1500

500

500

0

40 60 80 100 120 160 180 200 220

1000

1000

Inte

nsity

120573-Elemene (77)

Peak true

119898119911

Library hit

53058426908714

8109600 930916310509300 11912087 13312993 14715757

53

67

8193

105

119133 147

161

204

189

140

55

68

8193

107121

133

147161

175

189

41

(d)

Figure 4 Details of three coelution volatile components (peak 77 104 138) in GC timesGC chromatogramThe spectra of 120573-elemene (peak 77)dodecanal (peak 104) and p-mentha-1(7)8(10)-dien-9-ol (peak 138) in sample and in NIST library respectively

4 Conclusions

In this study GCtimesGC-HR-TOFMSnot only tentatively iden-tified 167 volatile components in Chenpi volatile oil but alsoprovided several kinds of identification information thatmake the result more reliable Among 167 components thereare 50 monoterpenes 36 sesquiterpenes 31 esters and acids9 aldehydes and ketones 6 alcohols 3 ethers 12 phenyl com-pounds and 20 other components Monoterpenes andsesquiterpenes are the main components of Chenpi volatileoil This study demonstrates a dependable method for thequalitative analysis of volatiles which can achieve an accurateand comprehensive chromatographic profile with a low con-tamination risk and cost as well as shortened sample prepara-tion time GC times GC-HR-TOFMS will play an important rolein the analysis of volatile oils of herbalmedicines in the future

Conflict of Interests

The authors explicitly declare that this paper has no conflictof interests

Acknowledgments

This work was supported by the Jiangsu Provincial NaturalScience Foundation (no BK2011135) the Fund Project forTransformation of Scientific and Technological Achieve-ments of Jiangsu Province (no BA2011024)The InternationalScience and Technology Cooperation Project of Jiangsu Pro-vince (no BZ2011053) and the Open Project of National FirstClass Key Discipline for Science of Chinese Materia MedicaNanjing University of ChineseMedicine (no 2011ZYX2-013)


The authors would like to thank Miss Xiaoying Sun for herassistance in the revision of this paper

References

[1] Y J Shen Pharmacology of Traditional Chinese MedicineShanghai Publishing House of Science and Technology Shang-hai China 2002

[2] S Gorinstein O Martın-Belloso Y S Park et al ldquoComparisonof some biochemical characteristics of different citrus fruitsrdquoFood Chemistry vol 74 no 3 pp 309ndash315 2001

[3] A Bocco M E Cuvelier H Richard and C Berset ldquoAntioxi-dant activity and phenolic composition of citrus peel and seedextractsrdquo Journal of Agricultural and Food Chemistry vol 46no 6 pp 2123ndash2129 1998

[4] YQMaXQ Ye Z X Fang J C ChenGHXu andDH LiuldquoPhenolic compounds and antioxidant activity of extracts fromultrasonic treatment of satsuma mandarin (Citrus unshiuMarc) peelsrdquo Journal of Agricultural and Food Chemistry vol56 no 14 pp 5682ndash5690 2008

[5] Y QMa J C Chen DH Liu andXQ Ye ldquoEffect of ultrasonictreatment on the total phenolic and antioxidant activity ofextracts from citrus peelrdquo Journal of Food Science vol 73 no8 pp T115ndashT120 2008

[6] G Xu X Ye J Chen and D Liu ldquoEffect of heat treatment onthe phenolic compounds and antioxidant capacity of citrus peelextractrdquo Journal of Agricultural and Food Chemistry vol 55 no2 pp 330ndash335 2007

[7] J A Manthey N Guthrie and K Grohmann ldquoBiological pro-perties of citrus flavonoids pertaining to cancer and inflamma-tionrdquo Current Medicinal Chemistry vol 8 no 2 pp 135ndash1532001

[8] M Y Choi C Chai J H Park J Lim J Lee and S W KwonldquoEffects of storage period and heat treatment on phenolic com-pound composition in driedCitrus peels (Chenpi) and discrim-ination of Chenpi with different storage periods through tar-geted metabolomic study using HPLC-DAD analysisrdquo Journalof Pharmaceutical and Biomedical Analysis vol 54 no 4 pp638ndash645 2011

[9] Y Sun Z Liu J Wang Y Wang L Zhu and L Li ldquoPreparativeisolation and purification of flavones from Pericarpium CitriReticulatae by high-speed counter-current chromatographyrdquoChinese Journal of Chromatography vol 27 no 2 pp 244ndash2472009

[10] E Chizzali I Nischang andM Ganzera ldquoSeparation of adren-ergic amines in Citrus aurantium L var amara by capillaryelectrochromatography using a novel monolithic stationaryphaserdquo Journal of Separation Science vol 34 no 16-17 pp 2301ndash2304 2011

[11] A Yin Z Han J Shen et al ldquoComparison of essential oilenriched with ultrafiltration method and extraction methodrespectively from essential oil-in-water emulsion of Citri Retic-ulatae PericarpiumViride by GC-MSrdquoChina Journal of ChineseMateria Medica vol 36 no 19 pp 2653ndash2655 2011

[12] C A Phillips K Gkatzionis K Laird et al ldquoIdentification andquantification of the antimicrobial components of a citrusessential oil vaporrdquoNatural Product Communications vol 7 no1 pp 103ndash107 2012

[13] D Hamdan M Z El-Readi E Nibret et al ldquoChemical compo-sition of the essential oils of two Citrus species and their bio-logical activitiesrdquo Pharmazie vol 65 no 2 pp 141ndash147 2010

[14] S E Reichenbach X Tian Q Tao E B Ledford Z Wu and OFiehn ldquoInformatics for cross-sample analysis with comprehen-sive two-dimensional gas chromatography and high-resolutionmass spectrometry (GCtimesGC-HRMS)rdquo Talanta vol 83 no 4pp 1279ndash1288 2011

[15] G Cao Q Y Shan X M Li et al ldquoAnalysis of fresh Menthahaplocalyx volatile components by comprehensive two-dimen-sional gas chromatography and high - resolution time-of-flightmass spectrometryrdquo Analyst vol 136 no 22 pp 4653ndash46612011

[16] P Marriott and R Shellie ldquoPrinciples and applications of com-prehensive two-dimensional gas chromatographyrdquo Trends inAnalytical Chemistry vol 21 no 9-10 pp 573ndash583 2002

[17] R Shellie and P J Marriott ldquoComprehensive two-dimensionalgas chromatography with fast enantioseparationrdquo AnalyticalChemistry vol 74 no 20 pp 5426ndash5430 2002

[18] G Cao H Cai X D Cong et al ldquoGlobal detection and analysisof volatile components from sun-dried and sulfur-fumigatedherbal medicine by comprehensive two-dimensional gas chro-matographytime-of-flightmass spectrometryrdquoAnalyst vol 137no 16 pp 3828ndash3835 2012

[19] Y Qiu X Lu T Pang S Zhu H Kong and G Xu ldquoStudy oftraditional Chinese medicine volatile oils from different geo-graphical origins by comprehensive two-dimensional gas chro-matography-time-of-flight mass spectrometry (GCtimesGC-TOFMS) in combination with multivariate analysisrdquo Journal ofPharmaceutical and Biomedical Analysis vol 43 no 5 pp1721ndash1727 2007

[20] C Ma H Wang X Lu H Li B Liu and G Xu ldquoAnalysis ofArtemisia annua L volatile oil by comprehensive two-dimen-sional gas chromatography time-of-flight mass spectrometryrdquoJournal of Chromatography A vol 1150 no 1-2 pp 50ndash53 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


to pharmacological effects of Chenpi extracts reported above[12 13] Therefore a method able to rapidly identify the vola-tile compounds of Chenpi could be a useful tool for the pur-pose of a complete phytochemical analysis

Gas chromatography-mass spectroscopy (GC-MS) hasbeen used for the qualitative analysis of the volatile consti-tuents in Chenpi [11] But it is difficult to achieve the completeseparation of minor volatile components andmany coelutionvolatile constituents To solve these problems it is neces-sary to use multidimensional gas chromatography Compre-hensive two-dimensional gas chromatography with high-resolution time-of-flight mass spectrometry (GC times GC-HR-TOFMS) is a new developed powerful and versatile analyticaltool which combines two powerful analytical technologieswith complementary attributes [14 15] GC times GC separateschemical species with two capillary columns interfaced by amodulator that traps and concentrates eluents from the firstcolumn and it then introduces them into the second columnproducing a full secondary chromatogram for each singledata point of a traditional one-dimensional separation [1617] HR-TOFMS provides mass precision that is fine enoughto distinguish elemental compositions providing amore defi-nitive basis for molecular identification GC times GC is impor-tant for HR-TOFMS because the better separations signifi-cantly reduce the coelution and the problems ofmass spectralmixing And HR-TOFMS is important for GC timesGC becausethe structural and compositional information available withHR-TOFMS aids in the interpretation of the rich complexdata from GC times GC separations [18] GC times GC-TOFMS hasbeen successfully applied in the volatile oil study and greatlyimproves the result of component separation and identifi-cation [19 20] In this study the volatile oil of Chenpi wasfirstly separated and detected with GC times GC-HR-TOFMS(Figure 1)

2 Materials and Methods

21 Samples Chenpi sample (fruit peels of Citrus reticulateldquoDahongpaordquo) was collected from Zigong in Sichuan pro-vince China The sample was authenticated by ProfessorChen Jianwei fromNanjingUniversity of Traditional ChineseMedicine China

22 Extraction of Volatile Oil After the sample was dried for2 h at 45∘C and smashed 50 g of sample was swollen with600mL of distilled water in a standard extractor for extract-ing volatile oil for 3 h Then the volatile oil was dried overanhydrous sodium sulphate until all the water was dried andthen stored in the dark glass bottle at 4∘C prior to GC times GC-HR-TOFMS analysis

23 GC-MS System and GC times GC-HR-TOFMS ApparatusGC timesGC separations were performed by Tofwerk AG (ThunSwitzerland) on an Agilent 7890A GC and 7693 autosamplerwith 1120583L splitless injection column one DB-XLB (Agilent)15m times 025mm 025120583mfilm thickness column two BPX-50(SGE) 1m times 01mm 01120583mfilm thickness oven temperaturefrom 50 to 230∘C at 20∘Cminminus1 ramp inlet pressure from

35 PSI to 615 PSI at 028 PSIminminus1 injection temperature250∘C transfer line temperature 300∘C Zoex ZX2 thermalmodulator with a 7 s modulation period 300ms modulationduration 375∘C hot jet temperature 18 Lminminus1 cold jet nitro-gen flow rate and 40 PSI hot jet nitrogen pressure The ZoexFasTOF time-of-flight- (TOF-) HRMS system used 70 eV EIion source 280∘C ion source temperature amass range ofmz50ndash450 with 4000 FWHM resolution and 100 spectra persecond acquisition rate

24 Data Conversion and Peak Table Generation The finaldata for each chromatogram is an array of 1000 times 600 datapoints each data point with a HRMS vector of 40K inten-sities Thus each chromatogram has 24 billion values requir-ing 96 gigabytes for representing for single-precision floatingpoint numbers without compressionThe set of 18 chromato-grams has more than 17 terabytes of uncompressed dataThedata were compressed and stored by the Zoex FasTOF systemto HDF5-format files and were processed with GC ImageGC times GC Software R21 In order to manage such large fileson computers with limited random access memory (RAM)GC Image Software maintains a chromatogram with integermass or centroid-resampled spectra in RAM and accesses theHR-MS data from disk as needed GC Image can export rawdata and computed results to nonproprietary file formats forprocessing with external software The components can bequantified by Zoex software (Zoex Corp Lincoln NE USA)

All peaks with signal-to-noise ratio higher than 100 werefound in the raw GC times GC chromatogram The workstationcan automatically give the parameters such as similarityreverse and probability of peaks via comparing them withthe compounds in the library The results were combined ina peak table The NISTEPANIHMass Spectral Library Ver-sion 20 was used in this work

3 Results and Discussion

31 Qualitative Analysis of Chenpi Volatile Oil The columnsystem is nearly orthogonal and provides a structured sepa-ration A typical two-dimensional separationtotal ion chro-matogram (TIC) and three-dimensional chromatogram areshown in Figure 2 In the GC times GC system compounds areseparated by volatility difference on the first dimensionnonpolar column and by polarity on the second medium-polar column The GC times GC system accomplishes the trueorthogonal separation on account for both the change ofthe polarity of two fixed phases and the linear temperatureprogramming

Using GC timesGC-HR-TOFMS the quantity of the detectedcomponents was up to 834 Compared to the traditional iden-tification method such as GC-MS the analysis from GC timesGC-HR-TOFMS becomes more reliable relying on the com-bined identification information including retention timessimilarity reverse match factor and probability The similar-ity and reverse match factors indicate how well a mass spec-trummatches the library spectrum but the isomers have sim-ilar mass spectra In this case the probability is used to deter-mine whether the peaks with the same name belong to one


Processing

Identification


Extraction

Analyzing


Steam distillation


6208

5173

4138

3104

2069

1035

0

0 6035 18105 24141 30176 36211 42246 48281 54316 60351 66387 72422

2-di

men

siona

l ret

entio

n tim

e (s)


(a)

Column II

Column I

(b)



A B

6208

5173

4138

3104

2069

1035

0

2-di

men

siona

l ret

entio

n tim

e (s)

75 13535 1957 25605 31641 43711 49746 55781 61816 67851 73887 79922


TIC image

37676












664 223 151311 C28H34N2O 4404 0-00-0







5349 215 644497 C23H32O 2557 0-00-0











Table 1 Continued

















Table 1 Continued



(81) 120574-Elemene 5168 151 7427373 C15H24 1514 29873-99-2
















Table 1 Continued















(156) 120574-Pyronene 2307 127 603834 C10H16 533 514-95-4


(158) (-)-Carvone 378 235 9098454 C10H14O 3276 6485-40-1


Table 1 Continued




4138

3104

2069

1035

0

43711 49746 55781 61816 67851 73887

p-Mentha-1(7) 8(10)-dien-9-ol (138)

Dodecanal (104)

120573-Elemene (77)

2-di

men

siona

l ret

entio

n tim

e (s)


(a)

150

150

100

100

50

50

0

40 60 80 100 120 140 160 180

Inte

nsity

p-Mentha-1(7) 8(10)-dien-9-0l (138)

Peak true

119898119911

Library hit

55068946706999 7907414

9410049

1061051211912087

5567

79 93

119

134

152

45

55

57 65

67

77

79

84

91

106

119

134

41

(b)

Figure 4 Continued


55068946706999

7907414 9612316

4551

57

67

79

82

8596

105 110 119 133 140 147 161

55

7181

95

109121 135

150

150

100

100

50

50

0

10040 80 120 140 160 180

Inte

nsity

Dodecanal (104)

Library hit

Peak true

119898119911

41

60

(c)

2000

2000

1500

1500

500

500

0

40 60 80 100 120 160 180 200 220

1000

1000

Inte

nsity

120573-Elemene (77)

Peak true

119898119911

Library hit

53058426908714

8109600 930916310509300 11912087 13312993 14715757

53

67

8193

105

119133 147

161

204

189

140

55

68

8193

107121

133

147161

175

189

41

(d)


4 Conclusions




Acknowledgments




References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Processing

Identification


Extraction

Analyzing


Steam distillation


6208

5173

4138

3104

2069

1035

0

0 6035 18105 24141 30176 36211 42246 48281 54316 60351 66387 72422

2-di

men

siona

l ret

entio

n tim

e (s)


(a)

Column II

Column I

(b)



A B

6208

5173

4138

3104

2069

1035

0

2-di

men

siona

l ret

entio

n tim

e (s)

75 13535 1957 25605 31641 43711 49746 55781 61816 67851 73887 79922


TIC image

37676












664 223 151311 C28H34N2O 4404 0-00-0







5349 215 644497 C23H32O 2557 0-00-0











Table 1 Continued

















Table 1 Continued



(81) 120574-Elemene 5168 151 7427373 C15H24 1514 29873-99-2
















Table 1 Continued















(156) 120574-Pyronene 2307 127 603834 C10H16 533 514-95-4


(158) (-)-Carvone 378 235 9098454 C10H14O 3276 6485-40-1


Table 1 Continued




4138

3104

2069

1035

0

43711 49746 55781 61816 67851 73887

p-Mentha-1(7) 8(10)-dien-9-ol (138)

Dodecanal (104)

120573-Elemene (77)

2-di

men

siona

l ret

entio

n tim

e (s)


(a)

150

150

100

100

50

50

0

40 60 80 100 120 140 160 180

Inte

nsity

p-Mentha-1(7) 8(10)-dien-9-0l (138)

Peak true

119898119911

Library hit

55068946706999 7907414

9410049

1061051211912087

5567

79 93

119

134

152

45

55

57 65

67

77

79

84

91

106

119

134

41

(b)

Figure 4 Continued


55068946706999

7907414 9612316

4551

57

67

79

82

8596

105 110 119 133 140 147 161

55

7181

95

109121 135

150

150

100

100

50

50

0

10040 80 120 140 160 180

Inte

nsity

Dodecanal (104)

Library hit

Peak true

119898119911

41

60

(c)

2000

2000

1500

1500

500

500

0

40 60 80 100 120 160 180 200 220

1000

1000

Inte

nsity

120573-Elemene (77)

Peak true

119898119911

Library hit

53058426908714

8109600 930916310509300 11912087 13312993 14715757

53

67

8193

105

119133 147

161

204

189

140

55

68

8193

107121

133

147161

175

189

41

(d)


4 Conclusions




Acknowledgments




References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















A B

6208

5173

4138

3104

2069

1035

0

2-di

men

siona

l ret

entio

n tim

e (s)

75 13535 1957 25605 31641 43711 49746 55781 61816 67851 73887 79922


TIC image

37676












664 223 151311 C28H34N2O 4404 0-00-0







5349 215 644497 C23H32O 2557 0-00-0











Table 1 Continued

















Table 1 Continued



(81) 120574-Elemene 5168 151 7427373 C15H24 1514 29873-99-2
















Table 1 Continued















(156) 120574-Pyronene 2307 127 603834 C10H16 533 514-95-4


(158) (-)-Carvone 378 235 9098454 C10H14O 3276 6485-40-1


Table 1 Continued




4138

3104

2069

1035

0

43711 49746 55781 61816 67851 73887

p-Mentha-1(7) 8(10)-dien-9-ol (138)

Dodecanal (104)

120573-Elemene (77)

2-di

men

siona

l ret

entio

n tim

e (s)


(a)

150

150

100

100

50

50

0

40 60 80 100 120 140 160 180

Inte

nsity

p-Mentha-1(7) 8(10)-dien-9-0l (138)

Peak true

119898119911

Library hit

55068946706999 7907414

9410049

1061051211912087

5567

79 93

119

134

152

45

55

57 65

67

77

79

84

91

106

119

134

41

(b)

Figure 4 Continued


55068946706999

7907414 9612316

4551

57

67

79

82

8596

105 110 119 133 140 147 161

55

7181

95

109121 135

150

150

100

100

50

50

0

10040 80 120 140 160 180

Inte

nsity

Dodecanal (104)

Library hit

Peak true

119898119911

41

60

(c)

2000

2000

1500

1500

500

500

0

40 60 80 100 120 160 180 200 220

1000

1000

Inte

nsity

120573-Elemene (77)

Peak true

119898119911

Library hit

53058426908714

8109600 930916310509300 11912087 13312993 14715757

53

67

8193

105

119133 147

161

204

189

140

55

68

8193

107121

133

147161

175

189

41

(d)


4 Conclusions




Acknowledgments




References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















664 223 151311 C28H34N2O 4404 0-00-0







5349 215 644497 C23H32O 2557 0-00-0











Table 1 Continued

















Table 1 Continued



(81) 120574-Elemene 5168 151 7427373 C15H24 1514 29873-99-2
















Table 1 Continued















(156) 120574-Pyronene 2307 127 603834 C10H16 533 514-95-4


(158) (-)-Carvone 378 235 9098454 C10H14O 3276 6485-40-1


Table 1 Continued




4138

3104

2069

1035

0

43711 49746 55781 61816 67851 73887

p-Mentha-1(7) 8(10)-dien-9-ol (138)

Dodecanal (104)

120573-Elemene (77)

2-di

men

siona

l ret

entio

n tim

e (s)


(a)

150

150

100

100

50

50

0

40 60 80 100 120 140 160 180

Inte

nsity

p-Mentha-1(7) 8(10)-dien-9-0l (138)

Peak true

119898119911

Library hit

55068946706999 7907414

9410049

1061051211912087

5567

79 93

119

134

152

45

55

57 65

67

77

79

84

91

106

119

134

41

(b)

Figure 4 Continued


55068946706999

7907414 9612316

4551

57

67

79

82

8596

105 110 119 133 140 147 161

55

7181

95

109121 135

150

150

100

100

50

50

0

10040 80 120 140 160 180

Inte

nsity

Dodecanal (104)

Library hit

Peak true

119898119911

41

60

(c)

2000

2000

1500

1500

500

500

0

40 60 80 100 120 160 180 200 220

1000

1000

Inte

nsity

120573-Elemene (77)

Peak true

119898119911

Library hit

53058426908714

8109600 930916310509300 11912087 13312993 14715757

53

67

8193

105

119133 147

161

204

189

140

55

68

8193

107121

133

147161

175

189

41

(d)


4 Conclusions




Acknowledgments




References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 1 Continued

















Table 1 Continued



(81) 120574-Elemene 5168 151 7427373 C15H24 1514 29873-99-2
















Table 1 Continued















(156) 120574-Pyronene 2307 127 603834 C10H16 533 514-95-4


(158) (-)-Carvone 378 235 9098454 C10H14O 3276 6485-40-1


Table 1 Continued




4138

3104

2069

1035

0

43711 49746 55781 61816 67851 73887

p-Mentha-1(7) 8(10)-dien-9-ol (138)

Dodecanal (104)

120573-Elemene (77)

2-di

men

siona

l ret

entio

n tim

e (s)


(a)

150

150

100

100

50

50

0

40 60 80 100 120 140 160 180

Inte

nsity

p-Mentha-1(7) 8(10)-dien-9-0l (138)

Peak true

119898119911

Library hit

55068946706999 7907414

9410049

1061051211912087

5567

79 93

119

134

152

45

55

57 65

67

77

79

84

91

106

119

134

41

(b)

Figure 4 Continued


55068946706999

7907414 9612316

4551

57

67

79

82

8596

105 110 119 133 140 147 161

55

7181

95

109121 135

150

150

100

100

50

50

0

10040 80 120 140 160 180

Inte

nsity

Dodecanal (104)

Library hit

Peak true

119898119911

41

60

(c)

2000

2000

1500

1500

500

500

0

40 60 80 100 120 160 180 200 220

1000

1000

Inte

nsity

120573-Elemene (77)

Peak true

119898119911

Library hit

53058426908714

8109600 930916310509300 11912087 13312993 14715757

53

67

8193

105

119133 147

161

204

189

140

55

68

8193

107121

133

147161

175

189

41

(d)


4 Conclusions




Acknowledgments




References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 1 Continued



(81) 120574-Elemene 5168 151 7427373 C15H24 1514 29873-99-2
















Table 1 Continued















(156) 120574-Pyronene 2307 127 603834 C10H16 533 514-95-4


(158) (-)-Carvone 378 235 9098454 C10H14O 3276 6485-40-1


Table 1 Continued




4138

3104

2069

1035

0

43711 49746 55781 61816 67851 73887

p-Mentha-1(7) 8(10)-dien-9-ol (138)

Dodecanal (104)

120573-Elemene (77)

2-di

men

siona

l ret

entio

n tim

e (s)


(a)

150

150

100

100

50

50

0

40 60 80 100 120 140 160 180

Inte

nsity

p-Mentha-1(7) 8(10)-dien-9-0l (138)

Peak true

119898119911

Library hit

55068946706999 7907414

9410049

1061051211912087

5567

79 93

119

134

152

45

55

57 65

67

77

79

84

91

106

119

134

41

(b)

Figure 4 Continued


55068946706999

7907414 9612316

4551

57

67

79

82

8596

105 110 119 133 140 147 161

55

7181

95

109121 135

150

150

100

100

50

50

0

10040 80 120 140 160 180

Inte

nsity

Dodecanal (104)

Library hit

Peak true

119898119911

41

60

(c)

2000

2000

1500

1500

500

500

0

40 60 80 100 120 160 180 200 220

1000

1000

Inte

nsity

120573-Elemene (77)

Peak true

119898119911

Library hit

53058426908714

8109600 930916310509300 11912087 13312993 14715757

53

67

8193

105

119133 147

161

204

189

140

55

68

8193

107121

133

147161

175

189

41

(d)


4 Conclusions




Acknowledgments




References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 1 Continued















(156) 120574-Pyronene 2307 127 603834 C10H16 533 514-95-4


(158) (-)-Carvone 378 235 9098454 C10H14O 3276 6485-40-1


Table 1 Continued




4138

3104

2069

1035

0

43711 49746 55781 61816 67851 73887

p-Mentha-1(7) 8(10)-dien-9-ol (138)

Dodecanal (104)

120573-Elemene (77)

2-di

men

siona

l ret

entio

n tim

e (s)


(a)

150

150

100

100

50

50

0

40 60 80 100 120 140 160 180

Inte

nsity

p-Mentha-1(7) 8(10)-dien-9-0l (138)

Peak true

119898119911

Library hit

55068946706999 7907414

9410049

1061051211912087

5567

79 93

119

134

152

45

55

57 65

67

77

79

84

91

106

119

134

41

(b)

Figure 4 Continued


55068946706999

7907414 9612316

4551

57

67

79

82

8596

105 110 119 133 140 147 161

55

7181

95

109121 135

150

150

100

100

50

50

0

10040 80 120 140 160 180

Inte

nsity

Dodecanal (104)

Library hit

Peak true

119898119911

41

60

(c)

2000

2000

1500

1500

500

500

0

40 60 80 100 120 160 180 200 220

1000

1000

Inte

nsity

120573-Elemene (77)

Peak true

119898119911

Library hit

53058426908714

8109600 930916310509300 11912087 13312993 14715757

53

67

8193

105

119133 147

161

204

189

140

55

68

8193

107121

133

147161

175

189

41

(d)


4 Conclusions




Acknowledgments




References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 1 Continued




4138

3104

2069

1035

0

43711 49746 55781 61816 67851 73887

p-Mentha-1(7) 8(10)-dien-9-ol (138)

Dodecanal (104)

120573-Elemene (77)

2-di

men

siona

l ret

entio

n tim

e (s)


(a)

150

150

100

100

50

50

0

40 60 80 100 120 140 160 180

Inte

nsity

p-Mentha-1(7) 8(10)-dien-9-0l (138)

Peak true

119898119911

Library hit

55068946706999 7907414

9410049

1061051211912087

5567

79 93

119

134

152

45

55

57 65

67

77

79

84

91

106

119

134

41

(b)

Figure 4 Continued


55068946706999

7907414 9612316

4551

57

67

79

82

8596

105 110 119 133 140 147 161

55

7181

95

109121 135

150

150

100

100

50

50

0

10040 80 120 140 160 180

Inte

nsity

Dodecanal (104)

Library hit

Peak true

119898119911

41

60

(c)

2000

2000

1500

1500

500

500

0

40 60 80 100 120 160 180 200 220

1000

1000

Inte

nsity

120573-Elemene (77)

Peak true

119898119911

Library hit

53058426908714

8109600 930916310509300 11912087 13312993 14715757

53

67

8193

105

119133 147

161

204

189

140

55

68

8193

107121

133

147161

175

189

41

(d)


4 Conclusions




Acknowledgments




References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















55068946706999

7907414 9612316

4551

57

67

79

82

8596

105 110 119 133 140 147 161

55

7181

95

109121 135

150

150

100

100

50

50

0

10040 80 120 140 160 180

Inte

nsity

Dodecanal (104)

Library hit

Peak true

119898119911

41

60

(c)

2000

2000

1500

1500

500

500

0

40 60 80 100 120 160 180 200 220

1000

1000

Inte

nsity

120573-Elemene (77)

Peak true

119898119911

Library hit

53058426908714

8109600 930916310509300 11912087 13312993 14715757

53

67

8193

105

119133 147

161

204

189

140

55

68

8193

107121

133

147161

175

189

41

(d)


4 Conclusions




Acknowledgments




References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article characterization of chemical composition of ...pericarpium citri reticulatae...

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