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C for 1 h, then
lter through a 0.45 m lter membrane; use the nal ltrate as
the IFTC tablets sample solution.
2.2.4. Sample solution of ginkgo extract
Dissolve 80 mg of extract of EGb in 5 mL of methanol, lter
through a 0.45 m lter membrane; the ltrate is used as the
EGb sample solution.
2.3. Preparation of chemical reference solutions
2.3.1. Reference solution for analysis of ginseng
Dissolve 0.5 mg of ginsenoside-Rb1, Re, Rg1, Rf,
pseudoginsneoside-F11 chemical reference substances in 1 mL
of methanol, respectively; use these as the ginsenosides refer-
ence solutions for ginseng analysis.
2.3.2. Reference solution for analysis of TGP and TGP
extract
Dissolve 1 mg of paeoniorin, 0.5 mg of albiorin, 0.2 mg
of benzoyl-paeoniorin reference substances in 1 mL methanol,
respectively; lter through a 0.45 mlter membrane; use these
as the paeoniorin glycosides reference solution for TGP anal-
ysis.
2.3.3. Reference solution for analysis of IFTC and IFTC
tablets
Dissolve 0.2 mg of gallic acid, chebulagic acid, chebulinic
acid reference substances in 1 mL of 30% methanol, respec-
tively; use these as the reference solutions for IFTC analysis.
2.3.4. Reference solution for analysis of EGb
Dissolve 0.2 mg of rutin, heteroside A, heteroside Breference
substances in 1 mL of methanol, respectively; use these as the
reference solutions for EGb analysis.
2.4. Method of preparing chromatographic ngerprint
2.4.1. HPTLC ngerprint of various ginseng species [4,5]
Stationary phase: HPTLC plate (10 cm10 cm; Merck; batch
number: OB247237).
Relative humidity: 3247%(pre-equilibrate twin-trough cham-
ber with the mobile phase for 30 min prior to analysis).
Mobile phase: Chloroformethyl acetatemethanolwater
(15:40:22:10; store at 10
C;
Gradient of mobile phase:
Time (min) Phosphoric acid (0.1%) (%) Acetonitrile (%)
0 90 10
15 60 40
Flow rate (mL/min): 1.0; injection volume: 5 L; detection
wavelength: 230 nm;
Run time: 16 min;
Relative retention time of reference substances: albiorin
0.9 min; paeoniorin1.0 min; benzoylpaeoniorin 2.2 min.
Compare the chromatograms of the sample solutions against
the chromatograms of the reference solution.
2.4.3. HPLC ngerprint of IFTC and IFTC tablets [7]
Chromatographic conditions:
Column: Lichrospher 100 RP-18, 4 mm125 mm, 5 m
(Merck);
Column temperature: 20
C;
Mobile phase: (A) 0.05 mol/L phosphoric acid aqueous solu-
tion and 0.05 mol/L KH
2
PO
4
aqueous solution; (B) methanol;
(C) ethyl acetate
Gradient:
Time (min) A (%) B (%) C (%)
0 94 6 0
5 96 3 1
15 93 2 5
20 89 6 5
35 55 40 5
Flow rate (mL/min): 1.0; injection volume: 5 L; detection
wavelength: 280 nm;
Run time: 35 min;
Relative retention time of reference substances: gallic acid
1.0 min; chebulagic acid 6.3 min; chebulinic acid 7.2 min.
Observe and compare the chromatograms of the sample solu-
tions, respectively, against the reference ngerprint of IFTC.
2.4.4. HPLC ngerprint of EGb [8]
Chromatographic conditions:
174 P.S. Xie et al. / J. Chromatogr. A 1112 (2006) 171180
Column: Spherisorb ODS2 C-18, 4 mm250 mm, 5 m
(Waters);
Column temperature: 25
C;
Mobile phase: (A) wateracetonitrileisopropanolcitric acid
(1000:200:30:4.92 g); (B) wateracetonitrileisopropanol
citric acid (1000:470:50:6.08 g)
Gradient: 0 min: 100% A; 25 min 100% B
Flow rate (mL/min): 1.0; injection volume: 5 L; detection
wavelength: 250 nm, 360 nm
Run time: 25 min.
Observe and compare of the chromatograms of the sam-
ple solutions, respectively against the reference ngerprint of
EGb761.
3. Results and discussion
3.1. HPTLC ngerprint analysis of ginseng
3.1.1. HPTLC chromatographic differentiation for the
authentication of selected ginseng species
Ginsenosides are triterpenoid saponins that are common
to the three species of ginseng analyzed. However, the con-
centration, distribution, and proportion of saponins differ
between the species, each presenting a unique ngerprint pattern
(Figs. 1 and 2; Table 1) [4].
Fig. 1. HPTLC images of white Panax ginseng (WG), red Panax ginseng (RG),
American ginseng (Panax quincefolius) (AG), and Tienchi ginseng (Panax noto-
ginseng) (NG). Lane 1, ginsenosides reference substances mixture (frombottom
to top): ginsenoside-Rb1, -Re, -Rg1, -Rf, pseudoginsenoside-F11; lane 2, white
Panax ginseng root; lane 3, red Panax ginseng; lane 4, American ginseng; 5,
Panax notoginseng. Plate: HPTLC silica gel 60 (Merck); Mobile phase (sol-
vent system): chloroformethyl acetatemethanolwater (15:40; 22; 10; store at
10
C for 1 h; use lower phase for analysis). Derivatization: spraying 10% sul-
phuric acid ethanolic solution. Observation: check the uorescent chromatogram
under UV 366 nm. Documentaion: prepare the HPTLC image photo with Digi-
store device (Camag).
Fig. 2. HPTLC image and digital scanning proles of white Panax ginseng
(WG), red Panax ginseng (RG), American ginseng (AG), Panax notoginseng
(NG) and ginsenosides reference substances mixture (cf. Fig. 1).
3.1.2. Monitoring stability of ginsenosides after processing
of ginseng extract using HPTLC
Good quality crude ginseng root has a characteristic nger-
print [2]. WhenanalyzedusingHPTLCthe primaryginsenosides
are visible (see Fig. 3A) and are therefore good marker com-
pounds for determining the stability of these compounds in the
nished extract. Acomparison of the rawmaterial (Fig. 3A) and
nished extract (Fig. 3B) shows this clearly. The chromatogram
of the nished extract shows that the primary ginsenosides (e.g.
Rb1, Re, and Rg1) originally observed in the raw material
decreased substantially while some minor ginsenosides consid-
erably increased. This indicates that the main ginsenosides, such
P.S. Xie et al. / J. Chromatogr. A 1112 (2006) 171180 175
Table 1
The distribution and proportion of ginsenosides in Ginseng species
Ginsenosides
a
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. Tracing the HPTLC ngerprint of a sample of Panax ginseng extract by
comparison with the ngerprint of Panax ginseng root. The primary ginsenosides
in the extract have been substantially hydrolyzed resulting in a proportionate
increase in the minor saponins.
as -Rb1, -Re and -Rg1 have undergone signicantly degrada-
tion during processing. It is well known that ginsenosides are
prone to hydrolyzation when exposed to rigorous heating with
water, particularly in a lower pHenvironment [5]. Therefore, the
extraction procedures used by this particular manufacturer are
less than optimal for preservation of ginsenosides.
3.1.3. Monitoring consistency of a multi-ingredient
commercial ginseng compound
Eleven samples of a classical multi-ingredient ginseng prepa-
ration, Sheng Mai Yin (SMY) capsule and granules, were
collected from different manufacturers and exemplify the dif-
ferences in HPTLC ngerprint patterns that can be obtained
(Fig. 4). Each of the product manufacturers claimed conformity
to standards established by the Pharmacopoeia of the Peoples
Republic of China (PPRC). Study of the chromatography shows
substantial inconsistencies betweenthe commercial SMYprepa-
rations. In some of the samples none of the primary ginsenosides
were detected (Fig. 4 samples 2, 3, 6, 7 and 12). This implies
either a lack of conformity to PPRC raw material standards or
inconsistencies in processing techniques between manufactur-
ers claiming to meet the same standards. HPTLC ngerprint
analysis shows these inconsistencies clearly.
3.2. HPLC ngerprint of TGP with statistical evaluation
using CASE software
The HPLC ngerprints of 10 batches (from the same manu-
facturer) of the TGPpowdered extract were evaluated in compar-
ison with the ngerprint of the TGP to determine batch-to-batch
consistency. There are approximately 8 peaks in the HPLC n-
gerprint of the TGP reference standard. Peak 3 correlates to
paeoniorin, peak 2 to albiorin, and peak 8 to benzoylpaeoni-
orin; the others are unknown (Fig. 5). The complete set of the
ratios of the height of all peaks are shown in Fig. 6. The height
of peak 3 (paeoniorin) was given a value of 1. In relationship
to peak 3 the relative ratio of the height of all of the peaks are,
respectively, 0.05, 0.28, 1.0, 0.03, 0.03, 0.15, 0.03 and 0.04.
Evaluation by CASE software showed that the authentication
of the commercial TGP product complies with the standardized
TGP reference sample. It also shows a high degree of simi-
larity between the 10 batches of TGP analyzed, suggesting a
Fig. 4. HPTLC image and digital scanning proles of various commercial preparations of a ginseng compound formulaSMY. Lane 1, Panax ginseng root (raw
material); lane 24, 6, 7 and 11: oral liquid; lane 5, 8, 9, 11 and 12, capsules; lane 12, Injection dosage form. HPTLC experimental condition: cf. Fig. 1.
176 P.S. Xie et al. / J. Chromatogr. A 1112 (2006) 171180
Fig. 5. 3D HPLC-DAD ngerprint of the Total Glycosides of Peony (TGP). Peak 2: abiorin, peak 3: paeoniorin and peak 8: benzoylpaeoniorin. HPLC column:
Lichrospher 100 RP-18, column temperature: 20
C; mobile phase: A, 0.1%phosphoric acid aqueous solution; B, acetonitrile, gradient elution; detection wavelength:
230 nm.
Fig. 6. Peak height of the HPLC ngerprints of 10 batches of TGP (shows the
quality consistency among the samples).
standardized consistency in rawmaterial quality, manufacturing
practices, or both, represented by a correlation coefcient (r) of
higher than 0.98 (r ranges between 0, completely dissimilar and
1, completely identical).
3.3. HPLC ngerprint of IFTC and IFTC tablets
Immature fruits of T. chebula (IFTC) contains a relatively
high content of polygalloyl glucose esters such as chebulinic
acid, chebulagic acid, and the monomer gallic acid (Fig. 7).
Gallic acid is often used as a qualitative and quantitative
marker.
However, content of gallic acid alone is not sufcient for the
quality assessment of IFTC products. Improperly made IFTC
extracts can yield higher concentrations of gallic acid than what
originally existed in the crude drug due to hydrolyzation of
polygalloyl glucose esters togallic acid. Therefore, simplydeter-
mining the content of gallic acid is not sufcient for evaluation
of the quality of IFTC and its products. The HPLC ngerprint
provided (Fig. 8) was established under optimized experimental
conditions to construct an overall pattern that is specic for the
identication and quality assessment of IFTC. For convenience
of recognition, the total ngerprint was divided into three sec-
tions; section I contains peaks 15, peak 3 corresponds to gallic
acid (retention time region from 2 to 10.6 min); section II con-
tains peak 611 (retention time region from 11 to 22.5 min);
section III contains peak 1215, peak 13 corresponds to chebu-
lagic acid and peak 15 to chebulinic acid (retention time region
from 23 to 40 min). Reviewing the line chart generated from the
original HPLCngerprint (the bottomof Fig. 8) it is very easy to
recognize the chromatographic patterns of the various sections.
This can be considered as a characteristic HPLC ngerprint for
IFTC.
When comparing the ngerprint of raw material IFTC with
the ngerprint of the extract some changes in the extract n-
Fig. 7. Chemical structures of chebulagic acid, chebulinic acid, and gallic acid.
P.S. Xie et al. / J. Chromatogr. A 1112 (2006) 171180 177
Fig. 8. HPLC ngerprint 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%/LKH
2
PO
4
aqueous solution; (B) methanol; (C) ethyl acetate, non-linear gradient
elution; detection wavelength: 280 nm.
gerprint can be seen, as shown in Fig. 9. The intensity of peak
3 (gallic acid) in section I is dramatically increased, while the
intensity of peak 13 (chebulagic acid) and peak 15 (chebulinic
acid) in section III is proportionately decreased. In this case, the
ngerprint pattern of the extracts is considerably different when
compared with that of the rawmaterial. The results of ngerprint
analysis may enable the producer to understand the reason that
degradation of ingredients occurs and rene the extracting pro-
Fig. 9. HPLC ngerprint and the line chart of extracts of IFTC and the tablets comparing original (A; sub-optimal) extraction method with improved extraction (B).
178 P.S. Xie et al. / J. Chromatogr. A 1112 (2006) 171180
Fig. 10. HPLC ngerprint of standardized extract of Ginkgo biloba
leaves (EGb761). HPLC column: Spherisorosorb ODS C18; column tem-
perature: 25