Research Article Characterization of Chemical Composition of ...Pericarpium Citri Reticulatae Extraction Analyzing Zoex GC × GC-TOF-MS analysis Steam distillation F : Flow chart of
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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
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
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
Evidence-Based Complementary and Alternative Medicine 5
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
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)
Evidence-Based Complementary and Alternative Medicine 11
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
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
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
Evidence-Based Complementary and Alternative Medicine 5
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
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)
Evidence-Based Complementary and Alternative Medicine 11
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
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
Evidence-Based Complementary and Alternative Medicine 5
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
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)
Evidence-Based Complementary and Alternative Medicine 11
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
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
Evidence-Based Complementary and Alternative Medicine 5
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
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)
Evidence-Based Complementary and Alternative Medicine 11
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
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)
Evidence-Based Complementary and Alternative Medicine 11
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
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)
Evidence-Based Complementary and Alternative Medicine 11
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
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)
Evidence-Based Complementary and Alternative Medicine 11
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
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)
Evidence-Based Complementary and Alternative Medicine 11
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
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)
Evidence-Based Complementary and Alternative Medicine 11
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
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)
Evidence-Based Complementary and Alternative Medicine 11
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
Evidence-Based Complementary and Alternative Medicine 11
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