Contoh Jurnal Tlc Qc 1

Journal of Chromatography A, 1112 (2006) 171180

Chromatographic fingerprint analysisquality assessment of traditional Chi

iangptorch,y ReseHom,outh Ualley,2006

Abstract

Traditional Chinese Herbal Medicine (TCHM) contain multiple botanicals, each of which contains many compounds that may be relevant to themedicines putative activity. Therefore, analytical techniques that look at a suite of compounds, including their respective ratios, provide a morerational approach to the authentication and quality assessment of TCHM. In this paper we present several examples of applying chromatographicfingerprint aauthenticatiohigh perform(TGP), to be(HPLC) fingImmature Ffinished pro(Ginkgo biloof chromato 2006 Else

Keywords: HComputer-aid

1. Introdu

Unlikemodern phtraditionalcompound,representinvidual analquality is cciples.

CorresponJiuzhou Avenfax: +86 756

E-mail ad

0021-9673/$doi:10.1016/jnalysis for determining the identity, stability, and consistency of TCHM as well as the identification of adulterants as follows: (1) speciesn of various species of ginseng (Panax ginseng, Panax quinquefolium, Panax noto-ginseng) and stability of ginseng preparations usingance thin-layer chromatography (HPTLC) fingerprint analysis; (2) batch-to-batch consistency of extracts of Total Glycosides of Peonyused as a raw material and in finished products (TGP powdered extract products), using high performance liquid chromatography

erprint analysis with a pattern recognition software interface (CASE); (3) documenting the representative HPLC fingerprints ofruits of Terminalia chebula (IFTC) through the assessment of raw material, in-process assay of the extracts, and the analysis of theduct (tablets); (4) HPLC fingerprint study demonstrating the consistent quality of total flavonoids of commercial extracts of ginkgoba) leaves (EGb) along with detection of adulterations. The experimental conditions as well as general comments on the applicationgraphic fingerprint analysis are discussed.vier B.V. All rights reserved.

igh performance liquid chromatography (HPLC); High performance thin layer chromatography (HPTLC); Fingerprint analysis; Pattern recognition;ed-similarity-evaluation (CASE); Ginseng; Peony root; Terminalia chebula; Ginkgo biloba

ction

the single chemical entity that forms the basis ofarmacology and drug development, the paradigm ofChinese herbal medicine (TCHM) views the multi-multi-ingredient preparations typical of TCHM as

g the activity of the herbal drug. Selection of indi-ytical compounds for determining either efficacy orontrary to traditional Chinese medicine (TCM) prin-

ding author. Present address: B20D, Yihai Dasha Building,ue East, Zhuhai 519015, China. Tel.: +86 756 3326296;3326961.dress: [email protected] (P.S. Xie).

The common clinical use of TCHM requires the combinationof two or more herbals based on recipes and formulae derivedfrom historical references and empirical evidence of TCM prac-titioners. Herbal drugs, singularly and in combinations, containa myriad of compounds in complex matrices in which no sin-gle active constituent is responsible for the overall efficacy. Thiscreates a challenge in establishing quality control standards forraw materials and the standardization of finished herbal drugs.This difficulty has been acknowledged in the draft of a Strate-gic Plan for Regional Traditional Medicine of the World HealthOrganization (WHO) [1].

Currently, there is a common practice among natural prod-ucts analysts to select one or more compounds as either activeor markers for purposes of identification and quality assess-ment. As many substances used in TCHM contain the same

see front matter 2006 Elsevier B.V. All rights reserved..chroma.2005.12.091Peishan Xie a,, Sibao Chen b, Yi-zeng LRuntao Tian a, Roy U

a Zhuhai Chromap Institute of Herbal Medicine Reseab State Key Laboratory of Pharmacy and Molecular Pharmacolog

The Hong Kong Polytechnic University, Hungc Research Center of Modernization of Chinese Medicine, Central S

d American Herbal Pharmacopoeia, Scotts VAvailable online 3 Februarya rational approach fornese herbal medicine

c, Xianghong Wang a,

n dZhuhai 519085, PR Chinaarch of Traditional Chinese Medicine,Hong Kong, Chinaniversity, Changsha 410083, PR ChinaCA 95067, USA

172 P.S. Xie et al. / J. Chromatogr. A 1112 (2006) 171180

compounds such an approach fails to be able to even confirmthe identity of a specific plant, let alone make any determinationregarding its quality. This problem is compounded when onesubstance tbined withcompoundsassessmentistics of TCmultiple cocan be idenassessmentCE, GC, Hrecognitionpattern ofonly the abthe complechromatoga compreheauthenticattency andSeveral exachromatogof TCHM.

2. Experim

2.1. Sampl

2.1.1. SamGinseng

from Jilin p(roots of Pa(roots and rYunnan prcommercia(SMY) capdong (rootwei zi (frusamples wbased on tedition andZhuhai ChrChina.

Total Gpowdered e

Immatufrom commfessor XuChinese mlaboratoryResearch; tCo. Ltd., C

The stanwere obtaiginkgo extZhejiang p

2.1.2. ApparatusTLC was performed on HPTLC silica gel 60 precoated plates

(Merck, Gpmere, Dm CdeveLC asystLC cbatc

25

Chesenobyo Rben

CollhebuNante fducoyl)

-O-{nosharm

lytic

Comreatteracoppedatog

repa

Samnd ee) seflas

fluxrate

ofred ee w

utionts.

Samurateth

tratesoluhat contains a specific class of compounds is com-others containing the same or different classes of. Thus, it is necessary to develop a type of qualitysystem that adequately meets the complex character-HM. Chromatographic fingerprint analysis by whichmpounds in single herbal drugs and finished TCHMtified represents a rational approach for the qualityof TCHM. It utilizes chromatographic techniques,

PLC, HPTLC, etc. [2] to construct specific patterns offor multiple compounds in herbal drugs. The entire

compounds can then be evaluated to determine notsence or presence of desired markers or actives butte set of ratios of all detectable analytes [3]. Thus,raphic fingerprint analysis of herbal drugs representsnsive qualitative approach for the purpose of speciesion, evaluation of quality, and ensuring the consis-stability of herbal drugs and their related products.mples presented in this paper elucidate the role of

raphic fingerprint analysis in the quality assessment

ental

es, apparatus, chemicals, reagents, and software

ples collected(roots of Panax ginseng C.A. Mey) white and red,rovince China and South Korea), American ginsengnax quinquefolium L. from Canada), tienchi ginsenghizome ofPanaxnotoginseng (Burk.) F.H. Chen fromovince of China); ginseng powdered extracts and al multi-ingredient ginseng productSheng Mai Yinsules and granules, and its ingredients, mai men

s of Ophiopogon japonicus (Thunb.) Ker-Gawl, wuits of Schisandra chinensis (Turcz.) Baill. All of theere identified by Drs. Sibao Chen and Peishan Xiehe description in the Chinese Pharmacopoeia 2005

retention samples are housed in the laboratory ofomap Institute of Herbal Medicine Research, Zhuhai,

lycosides of Peony (TGP) and a standardized TGPxtract from San Jiu Pharm. Co. Ltd. China.

re fruits of Terminalia chebula (IFTC) were collectedercial herb markets in China and identified by Pro-Honghua of Guangzhou University of Traditionaledicine and the retention samples are housed in theof Zhuhai Chromap Institute of Herbal Medicineablets of IFTC were obtained from Xiangxue Pharm.hina.dardized extracts of Ginkgo biloba leaves (EGb761)ned from Schwabe in Germany and commercialract (EGb) samples were obtained from Jiangsu androvinces of China.

develosoftwa(all froware (

HPHPLC

HP5m;4 mm

2.1.3.Gin

videdTobaccflorin,TCMacid, cticals,Institucal procinnamA), 3l-rhamIpsen Pof ana

2.1.4.softwa

A pPharmdevelochrom

2.2. P

2.2.1.Gri

2 sievmetricder, rethe filt1.0 mLpowdethe sample solproduc

2.2.2.Acc

0.5% mthe filextractermany); Automatic TLC sampler 4, TLC/HPTLCnt twin-trough chamber, TLC scanner 3 with WinCatsigiStore TLC documentation device and software

AMAG, Switzerland); TLC Digiscan-Acquiring soft-loped by our own lab).nalysis was performed on an Agilent 1100 series

em with autosampler and diode array detector (DAD).olumn: Lichrospher 100 RP-18, 4 mm 125 mm,

h number: 497017 (Merck); Spherisorb ODS2 C-18,0 mm, 5m (Waters).

micals and reagentsside Rb1, -Re, Rg1, pseudoginsenoside-F11 (pro-Dr. Hyunjoo Sohn of the Korea Ginseng andesearch Institute, Taejon, South Korea); peoni-zoyl-paeoniflorin, albiflorin (provided by Anhuiege, Hefei, Anhui, China); gallic acid, chebulagiclinic acid (from China University of Pharmaceu-

jing, China); Rutin (provided by Chinese Nationalor the Control of Pharmaceutical and Biologi-ts, Beijing, China); 3-O-{2-O-[6-O-(p-hydroxy-cis---glucose]--l-rhamnose} quercetin (heteroside2-O-[6-O-(p-hydroxy-cis-cinnamoyl)--glucose]--e} kaempferol (heteroside B) (provided by Beaufour-

. Co. Ltd., France). All chemicals and solvents wereal grade.

puter-aided-similarity-evaluation (CASE)

n recognition program recommend by the Chineseoeial committee and the complementary softwareby our lab that allows for a statistical evaluation ofraphic patterns.

ration of sample solutions

ple solution of ginseng rootsach of the roots of ginseng to a coarse powder (no.parately and place in an appropriately sized volu-k. To each, add 30 mL of methanol to 1 g of pow-in an Erlenmeyer flask for 30 min, filter, evaporateon a water bath to dryness, dissolve the residue inmethanol. Prepare the sample solution of ginsengxtracts and 0.5 g of SMY capsules and granules inay as the ginseng root solution. These are the sam-s for the various ginseng roots and finished ginseng

ple solution of TGP and TGP powdered extractely weigh 20 mg of the TGP, dissolve it in 10 mL ofanol, filter through a 0.45m filter membrane, useas sample solution; prepare the standardized TGPtion in the same manner.

P.S. Xie et al. / J. Chromatogr. A 1112 (2006) 171180 173

2.2.3. Sample solution of IFTC and IFTC tabletsGrind the IFTC to a coarse powder and place in an appro-

priately sized volumetric flask. Add 50 mL of acetone to 50 mgof the powder, reflux in an Erlenmeyer flask for 30 min, filter,evaporate tof water, refilter membAccuratelyan approprcate twicefilter, combsolve the rfilter througthe IFTC ta

2.2.4. SamDissolve

through aEGb sampl

2.3. Prepa

2.3.1. RefeDissolve

pseudoginsof methanoence soluti

2.3.2. Refeextract

Dissolveof benzoyl-respectivelas the paeoysis.

2.3.3. Refetablets

Dissolveacid referetively; use

2.3.4. RefeDissolve

substancesreference s

2.4. Metho

2.4.1. HPTStationarynumber: ORelative hber with tMobile p(15:40:22lower pha

Sample application: Apply 0.4L of sample solution as spotsor bands onto HPTLC plate).Development: Developing distance should be 8 cm from thelower edg

r 5 mol, hle.tion

HPLoma

mn: Lmn teient

min)

ratelengime:ivein; p

paroma

HPLoma

mn:ck);mn tele phnd 0thylient:

min)

ratelengime:ive rin; c

erveespe

HPLomahe filtrate to dryness, dissolve the residue in 10 mLfrigerate at 5 C for 1 h, then filter through a 0.45mrane; use the filtrate as the IFTC sample solution.weigh 5 mg of powdered IFTC tablets and place in

iately sized volumetric flask, add acetone, and soni-for 20 min each, using 20 mL of acetone each time,ine the filtrate, evaporate the filtrate to dryness, dis-

esidue in water and refrigerate at 5 C for 1 h, thenh a 0.45m filter membrane; use the final filtrate asblets sample solution.

ple solution of ginkgo extract80 mg of extract of EGb in 5 mL of methanol, filter

0.45m filter membrane; the filtrate is used as thee solution.

ration of chemical reference solutionsrence solution for analysis of ginseng

0.5 mg of ginsenoside-Rb1, Re, Rg1, Rf,neoside-F11 chemical reference substances in 1 mLl, respectively; use these as the ginsenosides refer-

ons for ginseng analysis.

rence solution for analysis of TGP and TGP

1 mg of paeoniflorin, 0.5 mg of albiflorin, 0.2 mgpaeoniflorin reference substances in 1 mL methanol,y; filter through a 0.45m filter membrane; use theseniflorin glycosides reference solution for TGP anal-

rence solution for analysis of IFTC and IFTC

0.2 mg of gallic acid, chebulagic acid, chebulinicnce substances in 1 mL of 30% methanol, respec-these as the reference solutions for IFTC analysis.

rence solution for analysis of EGb0.2 mg of rutin, heteroside A, heteroside B reference

in 1 mL of methanol, respectively; use these as theolutions for EGb analysis.

d of preparing chromatographic ngerprintLC ngerprint of various ginseng species [4,5]phase: HPTLC plate (10 cm 10 cm; Merck; batchB247237).umidity: 3247% (pre-equilibrate twin-trough cham-he mobile phase for 30 min prior to analysis).hase: Chloroformethyl acetatemethanolwater:10; store at 10 C for 1 h prior to analysis; use these for analysis).

air foethanvisibDetec

2.4.2.Chr

ColuColuGrad

Time (0

15

Flowwave

Run tRelat0.9 m

Comthe chr

2.4.3.Chr

Colu(MerColuMobition a(C) eGrad

Time (05152035

Flowwave

Run tRelat1.0 m

Obstions, r

2.4.4.Chre of the plate. Dry the plate in a stream of coldin. Spray plate with 10% sulphuric acid in 70%

eat at 100 C until the spots (or bands) are clearly

: Observe in daylight and at UV 366 nm (see Fig. 1).

C ngerprint of TGP [6]tographic conditions:

ichrospher 100 RP-18, 4 mm 125 mm, 5m;mperature: 20 C;of mobile phase:

Phosphoric acid (0.1%) (%) Acetonitrile (%)90 1060 40

(mL/min): 1.0; injection volume: 5L; detectionth: 230 nm;16 min;retention time of reference substances: albiflorinaeoniflorin1.0 min; benzoylpaeoniflorin 2.2 min.

e the chromatograms of the sample solutions againsttograms of the reference solution.

C ngerprint of IFTC and IFTC tablets [7]tographic conditions:

Lichrospher 100 RP-18, 4 mm 125 mm, 5m

mperature: 20 C;ase: (A) 0.05 mol/L phosphoric acid aqueous solu-.05 mol/L KH2PO4 aqueous solution; (B) methanol;acetate

A (%) B (%) C (%)94 6 096 3 193 2 589 6 555 40 5

(mL/min): 1.0; injection volume: 5L; detectionth: 280 nm;35 min;etention time of reference substances: gallic acidhebulagic acid 6.3 min; chebulinic acid 7.2 min.

and compare the chromatograms of the sample solu-ctively, against the reference fingerprint of IFTC.

C ngerprint of EGb [8]tographic conditions:


Column: Spherisorb ODS2 C-18, 4 mm 250 mm, 5m(Waters);Column temperature: 25 C;Mobile phase: (A) wateracetonitrileisopropanolcitric acid(1000:200:30:4.92 g); (B) wateracetonitrileisopropanolcitric acid (1000:470:50:6.08 g)Gradient: 0 min: 100% A; 25 min 100% BFlow rate (mL/min): 1.0; injection volume: 5L; detectionwavelength: 250 nm, 360 nmRun time: 25 min.

Observe and compare of the chromatograms of the sam-ple solutions, respectively against the reference fingerprint ofEGb761.

3. Results and discussion

3.1. HPTLC ngerprint analysis of ginseng

3.1.1. HPTLC chromatographic differentiation for theauthentication of selected ginseng species

Ginsenosides are triterpenoid saponins that are commonto the three species of ginseng analyzed. However, the con-centration, distribution, and proportion of saponins differbetween the species, each presenting a unique fingerprint pattern(Figs. 1 and 2; Table 1) [4].

Fig. 1. HPTLAmerican ginginseng) (NGto top): ginsenPanax ginsenPanax notogivent system):10 C for 1 h;phuric acid ethunder UV 366store device (

HPTLC image and digital scanning profiles of white Panax ginsenged Panax ginseng (RG), American ginseng (AG), Panax notoginsengd ginsenosides reference substances mixture (cf. Fig. 1).

Monitoring stability of ginsenosides after processingeng extract using HPTLCd quality crude ginseng root has a characteristic finger-]. When analyzed using HPTLC the primary ginsenosidesible (see Fig. 3A) and are therefore good marker com-s for determining the stability of these compounds in thed extract. A comparison of the raw material (Fig. 3A) andd extract (Fig. 3B) shows this clearly. The chromatogramfinished extract shows that the primary ginsenosides (e.g.

e, and Rg1) originally observed in the raw materialsed substantially while some minor ginsenosides consid-increased. This indicates that the main ginsenosides, suchC images of white Panax ginseng (WG), red Panax ginseng (RG),seng (Panax quincefolius) (AG), and Tienchi ginseng (Panax noto-). Lane 1, ginsenosides reference substances mixture (from bottomoside-Rb1, -Re, -Rg1, -Rf, pseudoginsenoside-F11; lane 2, white

g root; lane 3, red Panax ginseng; lane 4, American ginseng; 5,nseng. Plate: HPTLC silica gel 60 (Merck); Mobile phase (sol-chloroformethyl acetatemethanolwater (15:40; 22; 10; store atuse lower phase for analysis). Derivatization: spraying 10% sul-anolic solution. Observation: check the fluorescent chromatogramnm. Documentaion: prepare the HPTLC image photo with Digi-

Camag).

Fig. 2.(WG), r(NG) an

3.1.2.of gins

Gooprint [2are vispoundfinishefinisheof theRb1, Rdecreaerably


Table 1The distribution and proportion of ginsenosides in Ginseng species

Ginsenosidesa Ra Rb1 Rb2 Rc Re NR1 Rd Rg1 Rf F11 Minor ginsenosides

Panax ginseng + +++ + + ++ N/A + +++ + N/A +Panax quequifolium N/A ++++ N/A + ++ N/A + ++ N/A + Panax notoginseng N/A +++ N/A N/A +++ + + +++ N/A N/A +++: high content, ++: medium content, +: low content, : traces, N/A: not detected.

a The distribution of the ginsenosides were tested and described in ref. [8]. NR1, notoginsenoside-R1.

Fig. 3. Tracincomparison win the extractincrease in th

as -Rb1, -Rtion duringprone to hywater, partiextractionless than op

3.1.3. Moncommercia

Eleven sration, She

collected from different manufacturers and exemplify the dif-ferences in HPTLC fingerprint patterns that can be obtained(Fig. 4). Each of the product manufacturers claimed conformityto standards established by the Pharmacopoeia of the PeoplesRepublic of China (PPRC). Study of the chromatography showssubstantial inconsistencies between the commercial SMY prepa-rations. In some of the samples none of the primary ginsenosideswere detected (Fig. 4 samples 2, 3, 6, 7 and 12). This implieseither a lack of conformity to PPRC raw material standards orinconsistencies in processing techniques between manufactur-

iminis sh

PLCASE

HPr) ofith thtency

Fig. 4. HPTLmaterial); lang the HPTLC fingerprint of a sample of Panax ginseng extract byith the fingerprint of Panax ginseng root. The primary ginsenosideshave been substantially hydrolyzed resulting in a proportionate

e minor saponins.

ers claanalys

3.2. Husing C

Thefactureison wconsise and -Rg1 have undergone significantly degrada-processing. It is well known that ginsenosides aredrolyzation when exposed to rigorous heating withcularly in a lower pH environment [5]. Therefore, theprocedures used by this particular manufacturer aretimal for preservation of ginsenosides.

itoring consistency of a multi-ingredientl ginseng compoundamples of a classical multi-ingredient ginseng prepa-ng Mai Yin (SMY) capsule and granules, were

gerprint ofpaeonifloriflorin; the oratios of thof peak 3 (to peak 3 trespectivelEvaluationof the commTGP referelarity betw

C image and digital scanning profiles of various commercial preparations of a ginsene 24, 6, 7 and 11: oral liquid; lane 5, 8, 9, 11 and 12, capsules; lane 12, Injection dog to meet the same standards. HPTLC fingerprintows these inconsistencies clearly.

ngerprint of TGP with statistical evaluationsoftware

LC fingerprints of 10 batches (from the same manu-the TGP powdered extract were evaluated in compar-e fingerprint of the TGP to determine batch-to-batch. There are approximately 8 peaks in the HPLC fin-the TGP reference standard. Peak 3 correlates to

n, peak 2 to albiflorin, and peak 8 to benzoylpaeoni-thers are unknown (Fig. 5). The complete set of the

e height of all peaks are shown in Fig. 6. The heightpaeoniflorin) was given a value of 1. In relationshiphe relative ratio of the height of all of the peaks are,y, 0.05, 0.28, 1.0, 0.03, 0.03, 0.15, 0.03 and 0.04.by CASE software showed that the authenticationercial TGP product complies with the standardized

nce sample. It also shows a high degree of simi-een the 10 batches of TGP analyzed, suggesting ag compound formulaSMY. Lane 1, Panax ginseng root (rawsage form. HPTLC experimental condition: cf. Fig. 1.


Fig. 5. 3D HP , peakLichrospher 1 queou230 nm.

Fig. 6. Peak hquality consis

standardizepractices, ohigher than1, complete

3.3. HPLC

Immatuhigh conteacid, chebu

acidr.

eve

ass

s canLC-DAD fingerprint of the Total Glycosides of Peony (TGP). Peak 2: abiflorin00 RP-18, column temperature: 20 C; mobile phase: A, 0.1% phosphoric acid a

Gallicmarke

Howqualityextracteight of the HPLC fingerprints of 10 batches of TGP (shows thetency among the samples).

d consistency in raw material quality, manufacturingr both, represented by a correlation coefficient (r) of0.98 (r ranges between 0, completely dissimilar andly identical).

ngerprint of IFTC and IFTC tablets

re fruits of T. chebula (IFTC) contains a relativelynt of polygalloyl glucose esters such as chebuliniclagic acid, and the monomer gallic acid (Fig. 7).

originallypolygalloymining theof the qualprovided (Fconditionsidentificatiof recognittions; sectiacid (retentains peaksection IIIlagic acid afrom 23 tooriginal HPrecognize tThis can beIFTC.

When cthe fingerp

Fig. 7. Chemical structures of chebulagic acid, chebulin3: paeoniflorin and peak 8: benzoylpaeoniflorin. HPLC column:s solution; B, acetonitrile, gradient elution; detection wavelength:

is often used as a qualitative and quantitative

r, content of gallic acid alone is not sufficient for theessment of IFTC products. Improperly made IFTC

yield higher concentrations of gallic acid than whatexisted in the crude drug due to hydrolyzation ofl glucose esters to gallic acid. Therefore, simply deter-content of gallic acid is not sufficient for evaluationity of IFTC and its products. The HPLC fingerprintig. 8) was established under optimized experimentalto construct an overall pattern that is specific for theon and quality assessment of IFTC. For convenienceion, the total fingerprint was divided into three sec-on I contains peaks 15, peak 3 corresponds to galliction time region from 2 to 10.6 min); section II con-611 (retention time region from 11 to 22.5 min);contains peak 1215, peak 13 corresponds to chebu-nd peak 15 to chebulinic acid (retention time region40 min). Reviewing the line chart generated from theLC fingerprint (the bottom of Fig. 8) it is very easy to

he chromatographic patterns of the various sections.considered as a characteristic HPLC fingerprint for

omparing the fingerprint of raw material IFTC withrint of the extract some changes in the extract fin-

ic acid, and gallic acid.


Fig. 8. HPLC fingerprint of Immature Fruits of Terminalia chebula (IFTC) and the line chart. HPLC column: Lichrospher 100 RP-18, column temperature: 20 C;mobile phase: (A) 0.05% mol/L phosphoric acid aqueous solution and 0.05%/LKH2PO4 aqueous solution; (B) methanol; (C) ethyl acetate, non-linear gradientelution; detection wavelength: 280 nm.

gerprint can be seen, as shown in Fig. 9. The intensity of peak3 (gallic acid) in section I is dramatically increased, while theintensity ofacid) in sec

fingerprint pattern of the extracts is considerably different whencompared with that of the raw material. The results of fingerprint

is mation

Fig. 9. HPLCpeak 13 (chebulagic acid) and peak 15 (chebuliniction III is proportionately decreased. In this case, the

analysdegradfingerprint and the line chart of extracts of IFTC and the tablets comparing original (ay enable the producer to understand the reason thatof ingredients occurs and refine the extracting pro-A; sub-optimal) extraction method with improved extraction (B).


Fig. 10. HPLC fingerprint of standardized extract of Ginkgo bilobaleaves (EGb761). HPLC column: Spherisorosorb ODS C18; column tem-perature: 25 C; mobile phase: (A) wateracetonitrileisopropanolcitricacid (1000:200:30:4.92 g); (B) wateracetonitrileisopropanolcitric acid(1000:470:50:6.08 g); gradient elution; detection wavelength: 250 nm.

cedure accordingly. In this case, specific changes in processingwere made and new extracts showed recognition patterns thatwere satisfactorily similar to the chromatographic fingerprint tothe raw material. This verified the changes in the manufacturingprocesses were successful in creating greater conformity withthe reference standard (Fig. 9).

3.4. HPLC ngerprint of EGb (extract of Ginkgo bilobaleaves)

The ginkgo extract most widely tested for clinical efficacy(EGb761; Schwabe, Germany) is chemically characterized tocontain approximately 24% total flavonoids and 6% triterpenelactones. The four primary terpenes (ginkolides AC and bilob-alide) can be assayed using already existing published testingmethodologies (HPLC/ELSD) [9]. The chromatographic condi-tions of the HPLC fingerprint method of the total flavonoidsas described in Section 2.4.4 was optimized for full patternrecognition. This methodology is a complementary approachfor assessing the quality of Ginkgo biloba products. For conve-nience of evaluation, the chromatogram was divided into fivesections and characterized as follows: seven peaks in section I(tR region from 2 to 7 min), three peaks in section II (tR regionfrom 7.5 to 9 min), four peaks in section III (tR region from9 to 12 min), three peaks in section IV (tR region from 12 to18.5 min), and several minor peaks in section V (tR region from

Fig. 11. The similarity of HPLC fingerprints of 19 commercial samples of Ginkgo biloba extrasimilarity-evaluation (CASE) software.cts (EGb) from different sources derived from computer-aided-


19 to 23 min). These minor peaks are of less significance tothe overall characteristics of the pattern. A simple review of thechromatogram allows the analyst to quickly and reliably identifya characteristic pattern, including peak height and peak-to-peakratios before reading peak area values. In the chromatogrampresented (Fig. 10), the highest peak (8) in the fingerprint isattributed to rutin (quercetin-3-rutinoside); peak 16 to heterosideA (quercetin cinnamoyl-glycoside), and peak 17 to heterosideB (kaempferol cinnamoyl glycoside); the approximate peak topeak ratios of peaks 3, 16, and 17 are approximately 1:0.5:0.45.

Using the HPLC fingerprint of EGb761 as the standard pat-tern against which to compare other preparations, 19 samples ofEGb from different sources were comparatively analyzed. Cal-culating the raw signal points set of all samples by using the

Fig. 12. The Hation with thefingerprint in

Fig. 13. The ssamples of EGaway from the

CASE softof the samplation coeffidegree of sless than 0distributionbiloba leavthe fingerprno. 1, 2, aflavonoid rtotal flavonno. 8 wasis suggestiPrincipal Cproducers ddance withadulterated ginkgo extracts only been analyzed by quantitationof total flavonoids by conventional HPLC test, rather than bypattern recognition, this adulteration would not be evident.

4. Conclusion

omatographic fingerprint analysis is a rational and practi-lytical strategy to assess the authenticity, quality consis-

and stability of TCHM, as well as other herbal medicines.formation gathered from the fingerprint is more compre-e than that provided from the typical approach of onlyg on the quantitation of individual markers or active con-

ts for identity and quantitative assay. In China, chromato-c fingerprinting is gradually being applied in the quality

ent of TCHM preparations. It is currently required, byinese State Food and Drug Administration, to ensure

ality control of injectable herbal preparations and is pro-for use in the manufacture of oral preparations. This isaging TCHM manufacturers to introduce more stringentPLC fingerprint of EGb samples nos. 1, 2, and 4 showing adulter-flavonoid rutin; note abnormally increased peak dominating the

comparison with the fingerprint of standardized EGb761.

Chrcal anatency,The inhensivfocusinstituengraphiassessm

the Chthe qumotedencourcore plot obtained by principal components analysis (PCA) of 19b. The projection points of the samples nos. 1, 2, and 4 are farmain body in the graph (cf. Fig. 12).

ware, the results showed a high degree of similarityles collected to the EGb761 represented by a corre-cient of more than 0.94; five batches showed a lowerimilarity represented by a correlation coefficient of.87 (Fig. 11). This indicates that the proportion and

of the total flavonoids in most extracts of Ginkgoes possess a high level of consistency. Additionally,int analysis shows that three of the products (samples

nd 4) were adulterated, likely with the inexpensiveutin, which can be used to artificially increase theoid content. In this fingerprint chromatogram, peakuncharacteristically predominating (Fig. 12), whichve of adulteration. This was further confirmed byomponent Analysis (PCA) (Fig. 13), although theeclared the quality of their products to be in accor-the standardized EGb specification. Had the three


management over the collection of raw materials, increasingcompliance with good manufacturing practices (GMP), and oth-erwise ensuring the overall quality control of TCHM.

Provided are only a few examples of proposed chromato-graphic fingerprint analyses. More methodological validationwork is required on more botanicals and botanical products. Thefundamental requirement for developing a chromatographic fin-gerprint is specificity, reproducibility, and applicability. Oncean official chromatographic fingerprint is established and anacceptable allowance range is given, all manufacturers shouldbe required to meet these specifications. Eventually, the pro-cess for accepting or developing an herbal drug should requirethe combination of a specific chromatographic fingerprint withpharmacological and clinical evidence of efficacy and safety.

An optimized chromatographic fingerprint should provide asdetailed information regarding quality assessment as possible.Because of the complexity of ingredients in herbal drugs, morethan one fingerprint may be needed for adequately assessingquality. Practically, it may be impossible, perhaps even unneces-sary, to establish an all-embracing chemical fingerprint. Rather,as a first step, it may be more acceptable to create a representativefingerprint, as presented here, and develop further fingerprintsas new evidence demands.

Yet another challenge is how to make a qualitative finger-print quantifiable. The primary parameters of chromatographicfingerprints in column chromatography are retention time (tR)and the integration of data regarding peak area and height; inplanar chroRf value asTLC imagebecause ofoccurs in d

tion of chemical profiles of individual herbs also may influencethe patterns of chromatographic fingerprint analysis. Therefore,a computer recognition system and similarity evaluation soft-ware, such as the one used in this study (CASE), should beestablished to create patterns of recognition that are represen-tative of materials that have been found to be both safe andefficacious [2].

Acknowledgements

The authors thank Ms. Yuzhen Yan, Ms. Pinghua Lu, and Mr.Haoquan Qian for their participation in the experiment cited inthis paper.

References

[1] World Health Organization (WHO), WPR/RC52/7: A Draft RegionalStrategy for Traditional Medicine in Western Pacific. WHO RegionalCommittee, 52nd Session Brunei Darussalam, 1014 September 2001.

[2] Y.Z. Liang, P.S. Xie, K. Chan, J. Chromatogr. B 812 (2004) 53.[3] P.S. Xie, in: The Proceedings of the International Symposium of Chro-

matogr. Fingerprint of Chinese Herbal Medicine, Guangzhou, 2001, pp.4056.

[4] P.S. Xie, Y.Z. Yan, High Res. Chromatogr. Chromatogr. Comm. (HRCC)10 (1987) 607.

[5] P.S. Xie, Y.Z. Yan, J. Planar Chromatogr.-Modern TLC 1 (1988) 29.[6] P.S. Xie (Chief ed.), The Chromatographic fingerprint of Traditional Chi-

nese Medicine (in Chinese), Peoples Health Publishing House, Beijing,2005, p. 292.

[7] P.S. Xie (Chief ed.), The Chromatographic fingerprint of Traditional Chi-Med, p. 2

. Qianvan

pter 9matography the primary parameters are the relativewell as the color and intensity of spots/bands of the. However, these data are all relative, not absolute,the unavoidable differences in chromatograms that

ifferent laboratories. Additionally, the natural devia-

nese

2005[8] H.Q[9] T.A.

Chaicine (in Chinese), Peoples Health Publishing House, Beijing,68., P.S. Xie, J. Instrum. Anal. (in Chinese) 23 (2004) 7.Beek, Ginkgo biloba, Harwood Academic Publishers, 2000,, pp. 151178.

Chromatographic fingerprint analysis-a rational approach for quality assessment of traditional Chinese herbal medicineIntroductionExperimentalSamples, apparatus, chemicals, reagents, and softwareSamples collectedApparatusChemicals and reagentsComputer-aided-similarity-evaluation (CASE) software

Preparation of sample solutionsSample solution of ginseng rootsSample solution of TGP and TGP powdered extractSample solution of IFTC and IFTC tabletsSample solution of ginkgo extract

Preparation of chemical reference solutionsReference solution for analysis of ginsengReference solution for analysis of TGP and TGP extractReference solution for analysis of IFTC and IFTC tabletsReference solution for analysis of EGb

Method of preparing chromatographic fingerprintHPTLC fingerprint of various ginseng species [4,5]HPLC fingerprint of TGP [6]HPLC fingerprint of IFTC and IFTC tablets [7]HPLC fingerprint of EGb [8]

Results and discussionHPTLC fingerprint analysis of ginsengHPTLC chromatographic differentiation for the authentication of selected ginseng speciesMonitoring stability of ginsenosides after processing of ginseng extract using HPTLCMonitoring consistency of a multi-ingredient commercial ginseng compound

HPLC fingerprint of TGP with statistical evaluation using CASE softwareHPLC fingerprint of IFTC and IFTC tabletsHPLC fingerprint of EGb (extract of Ginkgo biloba leaves)

ConclusionAcknowledgementsReferences

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