全 文 ：广西民族学院学报(自然科学版)
第 10卷第 1 期 JOURNAL OF GUANGXI UNIVERSITY FOR NATIONALITIES Vol.10 No.1
2004年 2 月 (Natural Science Edition) Feb.2004
Solid phase extraction and GC/MS
determination of free phenolic acids
in Echinacea species
YAO Xing-dong1 ,WANG Hai-ping2 ,
NIE Yuan-mei1 ,Nirmalendu Datta-Gupta3
(1.College of Chemistry and Ecological Engineering , Guangxi University for Nationalities , Nanning
530006 , China;2.The Quality Determination Institute of Beihai , Beihai536000 , China;3.Depart-
ment of Physical Sciences , South Carolina State University , Orangeburg , SC 29117 , USA)
Abstract:A new method is described combining solid phase extraction , trimethylsilyl derivatization
and GC/MS for derivatization , purification and quantitative determination of 7 free phenolic
acids(caffeic acid , chlorogenic acid , 4-coumaric acid , protocatechuic acid , syringic
acid , vanillic acid and 4-hydroxybenzoic acid)in three different Echinacea species.
Key Words:sample preparation;Echinacea;Phenolic acids
中图分类号:O6-332 文献标识码:A 文章编号:1007-0311(2004)01-0100-04
1 Introduction
Echinacea is one of the most widely applied herbs in
US.It has been used for immunostimulation and wound
healing for a long time.The beneficial effects have been
attributed partly to the phenolic compounds and its associ-
ated antioxidant activity
[ 1 , 2].A lot of methods , including some
standardization method , have been developed for the deter-
mination of phenolic compounds in Echinacea species.
Most of these methods are based on HPLC separation and
UV detection
[ 2-6].Solid-Phase Extraction(SPE)technique
was also applied for sample pretreatment
[ 7].Colorimetric
measurement was developed for total phenolic determination
with Folin-Denis reagent [ 6].HPLC is a very convenient
and powerful method.However , due to the structural simi-
larity of phenolic compounds , and a large number of bulk
compounds with UV absorption in herb , The separation and
determination of phenolics may be difficult or interfered
from other phenolics or bulk components.On the other
hand , the polysaccharides and other polymers can also con-
taminate the LC columns.
100
收稿日期:2003-12-25.
基金项目:美国农业部研究基金资助项目(编号:SCX-420-20-00).
作者简介:姚兴东(1965-),男 , 江苏人 ,广西民族学院化学与生态工程学院副教授 ,博士 , 主要研究方向:仪器分析.
DOI :10.16177/j.cnki.gxmzzk.2004.01.022
Most phenolic compounds are nonvolatile , and could
not be analyzed with GC directly.Trimethylsilyl (TMS)
derivatization can effectively increase the volatility and sta-
bility of these phenolics
[ 8].This method has been applied for
the determination of phenolics in fruits and wine
[ 9 , 10].In this
paper , we developed a Solid Phase Extraction -GC/MS
analytical method for 7 phenolic acids and reported the de-
termination result of phenolic level in three different Echi-
nacea species based on the method.
2 Experimental
2.1 Plant Tissue
3 year old plant of E.angustifolia , E.pallida and E.
purpurea aerial tops , leaf and root were purchased from
Caribon Ginseng and Starwest Botanical.They were cut
and milled before extraction.5 gram of milled plant is ex-
tracted with 150 ml of methanol for 6 hours in a Soxhlet ap-
paratus.The solvent was evaporated to keep the volume as
25 ml for SPE separation.
2.2 GC/MSAnalysis
A Hewlett-Packard (HP)model GC6890 gas chro-
matograph with a HP 5973 mass-selective detector were
employed in this work.Helium with HP mass spectrometer
gas purifier was used as the carrier gas.Chromatographic
conditions were:Injector , 250℃, Detector , 280℃, Col-
umn , HP-5MS 30.0 m x 250 um x 0.25 um , Oven tem-
perature was programmed as:Initial oven temperature
50℃, temperature was raised from 50 to 250℃ at the
speed of 15℃/min.For the MS detector , the electron
multiplier voltage (EMV)was 1294V.
2.3 Standard Caffeic Acid Derivatives
Caffeic acid , chlorogenic acid , 4-coumaric acid ,
protocatechuic acid , syringic acid , vanillic acid and 4-
hydroxybenzoic acid were purchased from Sigma Chemical
Co.(St.Louis , MO).They were dissolved in methanol
and the concentration of stock solution is 1.0 mg/mL.10
-100 ? g of caffeic acid derivatives were used for the
standard curve measurement.
2.4 SPE procedure , Ryan s method [ 11]
1 ml of sample solution was mixed with 5 ml of 1 M
Na2CO3 in a separatory funnel and washed with 5 ml of
hexane to remove lipoidal material.Then the aqueous solu-
tion was acidified with 1M HCl to pH 2 and passed through
a Bakerbond SPETM Octadecyl(C18)Disposable Extrac-
tion Column (J.T.Baker , Phillipsburg , NJ)which was
preconditioned with 5 ml of methanol and 5 ml of water.
Phenolic compounds were retained on the column and were
eluted with 5 ml 50% methanol/water.The eluent was
heated to dryness for TMS derivatization.
2.5 TMS Derivatization Procedure
Soleas method [ 10]was used with few modifications.SPE
eluent was evaporated to dryness.0.5 ml of methylene
chloride was added to ensure complete removal of water.
The mixture was heated to dryness and kept at 70℃for 15
min.The residuewas mixed with 0.5ml 1:1 BSTFA/pyri-
dine(v/v)and heated at 70℃ for 30 min after vigorous
vortexing.
3 Results and Discussion
3.1 Chromatographic Resolution:
Individual phenol solution was dried and derivatized
as described above.GC/MS results at the given separations
above show good separation.The retention times and the
target ions for each phenol were listed in Table 1.Typical
GC/MS chromatogram was shown in Fig.1.All the TMS
derivatives were completely separated in the selected GC
analysis conditions.Unexpectedly the GC peak for chloro-
genic acid could not be detected.Further study is needed
for the reaction of BSTFA and chlorogenic acid.
101
2004年第 1期 ○姚兴东 ,王海萍 ,聂园梅 ,等/草药紫锥花中酚类化合物的测定
Table 1 Retention times(RT)and target ions for each pheno-
lic compound
Compound RT (min) t arget ion(m/z)
Caffeic acid 13.237 396
p-Coumaric acid 12.047 293
Ferulic acid 13.031 338
4-Hydroxybenzoic acid 9.816 267
Protocatechuic acid 11.242 193
Syringic acid 11.801 327
Vanillic acid 10.859 297
Cinnamic acid 9.140 205
Figure 1.GC/MS of TMS derivatives of phenolic compounds
1 cinnamic acid(200 ug);2 4-hydroxybenzoic acid(100 ug);3
vani llic acid(100 ug);4 protocatechuic acid (100 ug);5 syringic acid
(100 ug);6 p-coumaric acid(100ug);7 ferulic acid(100 ug);8 caf-
feic acid(100 ug)
3.2 Method Validation.
The 7 phenolic acids were analyzed in a mixture in
methanol.The amplitude of the ranges varied from 0 to 100
μg.Linear regression was performed using the formula y =
mx + b.The linear regression coefficients(R2)of stan-
dard curves for all the phenolic compounds are over 0.97.
Recovery test for 4-hydroxybenzoic acid showed good re-
sult (recovery > 90%).Standard derivation(RSD)is
1.7% for 6 measurement.
3.3 Sample Analysis
The contents of 7 phenolic compounds in E.species
were determined based on the method.The results are list-
ed in Table 2.Figure 2 shows typical chromatogram of
sample analysis.Comparing with the direct GC/MS analy-
sis , SPE technique effectively removes the bulk substances
in herb sample and improves the analytical results.
Table 2 Free phenolic acid concentration in different E.Species
Phenolic acid 1 2 3
3 year old E.purpurea root 105.2 250.6 63.6 -＊
3 year old E.purpurea aerial top 76.4 596.4 131.5 - 18.06 49.5 17.6
3 year old E.pallida root 128.7 82.7 71.1 - 17.61 63.5 15.0
E.angustifolia leaf 42.3 195.6
E.angustifolia root 371.9 124.6 160.0 - - 96.7 18.5
1 caffeic acid ;2 p-coumeric acid ;3 ferulic acid;4 4-hydroxybenzoic acid ;5
protocatechuic acid ;6 sy ringic acid ;7 vanillic acid
＊Can not be detected
Figure 2 Typical GC/MS chromatogram of Echinacea sample analy-
sis(E.Angustifolia leaf)
Up:GC/MS analysis without SPE separation
Down:GC/MS analysis with SPE separation
The results for p-coumeric acid , ferulic acid , proto-
catechuic acid , syringic acid and vanillic acid in Table 2
are similar with Glowniak s result [ 7], but the result of caffe-
ic acid in Glowniak s paper is much lower than the result
in this paper.Our results are close to Perry s result [ 6].It
can be regarded as evidence that the growing environment
(place , temperature etc.)has great effect on the concen-
tration of phenolic acids in E.species (Glowniak -
Poland;Perry -New Zealand;Ours -North America).
102
广西民族学院学报(自然科学版) 2004年 2月 第 10卷
4 Acknowledgement
This work was funded by US Department of Agricul-
ture(USDA)-1890 Research Program at South Carolina
State University , Grant #SCX-420-20-00.
[ References]
[ 1] R.Bauer , Chemistry , analysis and immunological investigations of Echi-
nacea phytopharmaceuticals[ J] .In Immunomodulatory Agents from plants;
Edit , Wagner , Birkhauser Verlag:Basel , 1999.H., 41-88
[ 2] Hu C.;Kitts D.D.Studies on the Antioxidant Activity of Echinacea Root
Extract , J.Agric.Food Chem.[ J] , 2000 , 48, 1466-1472.
[ 3] Kim H.O.;Durance , T.D.;Scaman , C.H.etc.Retention of caffeic
acid derivatives in dried Echinacea purpurea , J.Agric.Food Chem[ J] .
2000 , 48 , 4182-4186.
[ 4] Schenk , R.;Franke , R.Content of echinacoside in Echinacea root s of
different origin.Beit r[ J] .Zuechtungforsch.1996 , 2.
[ 5] Stuart , D.L.;Wills , R.B.H.Alkylamide and cichoric acid levels in
Echinacea purpurea tissues during plant growth.J.Herbs Spices Med[ J] .
Plants 2000, 7 , 91-101.
[ 6] Perry , N.B.;Burgess, E.J.;Glennie , V.Echinacea Standardization:
Analytical Methods for Phenolic Compounds and Typical Levels in Medicinal
Species , J.Agric.Food Chem[ J] .2001, 49 , 1702-1706 .
[ 7] Glowniak , K.;Zgorka , G.;Kozyra , M.Solid-phase extraction and re-
versed-phase high-performance liquid chromatography of free phenolic
acids in some Echinacea species , J.Chromatography A[ J] .1996 , 730 ,
25-29.
[ 8] Little , J.I.Artifacts in Trimethylsilyl Derivatization Reactions and Ways
to Avoid Them J.Chromatogr[ J] .A , 1999, 844 , 1-22.
[ 9] Antolovich , M.;Prenzler P.;Robards , K.etc.Sample preparation in the
determination of phenolic compounds in fruits Analyst , 2000 , 125, 989-
1009.
[ 10] Soleas , G.J.;Diamandis , E.P.;Karumanchiri , A.etc.A Mul-
tiresidue Derivatization Gas Chromatographic Assay for Fifteen Phenolic
Constituents withMass Selective Detection Anal.Chem[ J] .1997 , 69 ,
4405-4409.
[ 11] Ryan D.;Robards K.And Lavee S.Determination of phenolic com-
pounds in olives by reversed-phase chromatography and mass spectrome-
t ry J.Chromatogr[ J] .A , 1999 , 832, 87-96.
[责任编辑 黄招扬]
[责任校对 苏 琴]
草药紫锥花中酚类化合物的测定
姚兴东1 ,王海萍2 ,聂园梅1 , Nirmalendu Datta-Gupta3
(1.广西民族学院化学与生态工程学院 , 广西 南宁 530006;
2.北海质检所 ,广西 北海 536000 ;
3.南卡罗莱纳州立大学 物理科学系 ,南卡州 奥林治堡 , 29117)
摘 要:报道了一种固相萃取-三甲基硅烷衍生化-气相色谱/质谱联用法测定草药紫锥花的不同
品种中 7种游离酚类化合物的新方法.
关键词:试样制备;紫锥花;酚类化合物
103
2004年第 1期 ○姚兴东 ,王海萍 ,聂园梅 ,等/草药紫锥花中酚类化合物的测定