Fingerprint Analysis of Eucommia Bark by LC-DAD and LC-MS with the Aid of Chemometrics

2008, 67, 211217

Yongnian Ni1,&, Yunyan Peng1, Serge Kokot2

1 2

Department of Chemistry, Nanchang University, Nanchang, Jiangxi 330047, Peoples Republic of China; E-Mail: ynni@ncu.edu.cn Inorganic Materials Program, School of Physical and Chemical Sciences, Queensland University of Technology, Brisbane, QLD 4001, Australia

Received: 20 August 2007 / Revised: 14 October 2007 / Accepted: 26 November 2007 Online publication: 4 January 2008

Abstract
Chromatographic ngerprints of 46 Eucommia Bark samples were obtained by liquid chromatography-diode array detector (LC-DAD). These samples were collected from eight provinces in China, with different geographical locations, and climates. Seven common LC peaks that could be used for ngerprinting this common popular traditional Chinese medicine were found, and six were identied as substituted resinols (4 compounds), geniposidic acid and chlorogenic acid by LC-MS. Principal components analysis (PCA) indicated that samples from the Sichuan, Hubei, Shanxi and Anhuithe SHSA provinces, clustered together. The other objects from the four provinces, Guizhou, Jiangxi, Gansu and Henan, were discriminated and widely scattered on the biplot in four province clusters. The SHSA provinces are geographically close together while the others are spread out. Thus, such results suggested that the composition of the Eucommia Bark samples was dependent on their geographic location and environment. In general, the basis for discrimination on the PCA biplot from the original 46 objects 7 variables data matrix was the same as that for the SHSA subset (36 7 matrix). The seven marker compound loading vectors grouped into three sets: (1) three closely correlating substituted resinol compounds and chlorogenic acid; (2) the fourth resinol compound identied by the OCH3 substituent in the R4 position, and an unknown compound; and (3) the geniposidic acid, which was independent of the set 1 variables, and which negatively correlated with the set 2 ones above. These observations from the PCA biplot were supported by hierarchical cluster analysis, and indicated that Eucommia Bark preparations may be successfully compared with the use of the HPLC responses from the seven marker compounds and chemometric methods such as PCA and the complementary hierarchical cluster analysis (HCA).

Keywords
Column liquid chromatography LC-DAD LC-MS Fingerprinting Principal component analysis Eucommia Bark

Introduction
Many plant extracts and medical preparations are complex mixtures for which conventional analysis is very dicult even with the use of Certied Reference Materials and pharmacologically active or bioactive markers [14]. In such cases, the World Health Organisation (WHO) has accepted chromatographic and mass spectrometric ngerprinting [58] for quality assurance purposes [9]. In general, this approach is feasible provided

Original DOI: 10.1365/s10337-007-0500-7 0009-5893/08/02

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Table 1. Geographical origin of the 46 herbal samples of the Eucommia Bark

Sample no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Origin Anhui Anhui Gansu Gansu Gansu Guizhou Guizhou Hubei Hubei Hubei Hubei Hubei Hubei Hubei Henan Henan Henan Jiangxi Jiangxi Sichuan Sichuan Sichuan Sichuan Sichuan Sichuan Sichuan Sichuan Sichuan Sichuan Sichuan Sichuan Sichuan Sichuan Sichuan Sichuan Sichuan Sichuan Sichuan Sichuan Sichuan Sichuan Sichuan Sichuan Shanxi Shanxi Shanxi Batch number 070106 0504028 060608 060826 060910 060228 061101 20060901 20060924 20061011 20061020 20061101 20061215 20070128 20060301 20060315 20060712 20060907 200511267 051218 060601 060801 061010 061208 070102 0702002 20051202 20060216 20060403 20060510 20060605 20060712 20060724 20060803 20060827 20060908 20061008 20061018 20061031 20061108 20061126 20070126 200702002 060318 061016 070103

the source material for the extract is reasonably consistent from batch to batch. However, when this is not the case, and the same mixture is apparently prepared from source materials obtained from very dierent environments, then even the conventional ngerprint comparison and identication becomes dicult. In such cases, multivariate data interpretation methods may be of assistance as was recently illustrated [10, 11]. In this paper, we investigate the multivariate data interpretation approach with the use of the well known

many-component traditional Chinese medicine (TCM), Eucommia Bark as an example. This popular medicine is available from many parts of China, and while it is derived from the same plant source, the plants from the dierent geographical areas are grown under very dierent environmental conditions. Thus, the ngerprints vary considerably. Eucommia Bark is the bark of Eucommia ulmoides Oliv., plant, and was noted in the ancient Chinese codex, Shen Nong Ben Cao Jing [12] and Ben Cao Gan Mu (Materia Medica, a dictionary of Chinese herbs) [13]. It is well known for its low toxicity and multiple pharmacological activities. It has been used to alleviate many diverse health problems e.g., complaints concerning liver, kidneys, bones, hypertension as well as cancer [1419]. This wide ranging apparent eectiveness of the herbal medicine has been attributed to many compounds present in the Eucommia Bark including, for example, lignin glycosides, phenols, avonoids, and amino acids. Thus, Eucommia Bark has been found as a component of typical products such as pharmaceuticals, health care treatments, cosmetics and even glue. In general, several techniques can characterize the nature and composition of substances such as Eucommia Bark. These include high performance liquid chromatography (HPLC) [6, 10, 11], gas chromatography (GC) [20], thin layer chromatography (TLC) [21], high-speed counter-current chromatography (HSCCC) [22] and capillary electrophoresis (CE) [23]. In this study, we applied LC-DAD analysis to collect the chromatographic ngerprints of the dierent Eucommia Bark samples, which were obtained from dierent provinces in China. LC-MS was used as the technique for the identication of the common chemical constituents of the Eucommia Bark samples. These data were then variously submitted to multivariate analysis pattern recognition methods such as PCA and HCA. Information extracted by these methods was then analysed for any similarities or dierences of the herbal samples derived from the dierent provinces.

Chemometric Methods
Similarity
The two ngerprints can be compared by evaluating their similarity. This can be estimated from a calculation, which uses the Euclidean distance, the correlation coecient and the cosine function [24, 25]. In this work, an estimate of the cosine value between two data vectors was used to calculate the similarities:

cosaij
P X k1

v ! ! u P P X u X t 2 2 xik xjk = xik xjk

k1 k1

1
where xik and xjk represent the kth elements of the kth peak of the two chromatographic ngerprints i.e., peak areas; P is the number of peaks. The larger values of cos aij (range 0 < cos aij 1) indicate better similarity between the two ngerprints. When cos aij is 1, they are identical.

Principal Component Analysis (PCA)

Principal component analysis is a well known unsupervised multivariate data analysis approach [26, 27]. Generally, PCA compresses the original data, and a new set of variables called principal components (PCs) is obtained. These PCs are linear combinations of the original variables, and are chosen to be orthogonal to each other. Each object is identied by a score value on each PC, and every variable is likewise associated with a loading on each PC. PC score score plots, loading variable plots and biplots are common two dimensional display methods for exploring the data structure.

Hierarchical Cluster Analysis (HCA)

The hierarchical cluster analysis method is also well known, and has been applied for ngerprint analysis [28, 29]. The Original

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method starts with n number of observations. The distances between observations are calculated. Dierent functions are available for this, including the Euclidean, Mahalanobis, and Minkowski distances [30]. Initially, the HCA method considers each sample as an independent group, i.e., there are n groups. Then, the two closest points are merged into a new group. The distance between the new group and the other n 2 groups (samples) is then calculated as previously; the closest two groups are merged into another new group. The process continues until all observations are clustered into one group. Finally, the results are displayed as a dendrogram. Then, a decision rule is used to determine the number of clusters. There are several methods for hierarchical clustering [28], such as the single and complete linkage methods. In this paper, the Euclidean distance was chosen as the measure of similarity, and the Ward method was applied for the clustering algorithm [30].

Fig. 1. Mean chromatograms of the Eucommia Bark extracts from samples collected in dierent provinces: Anhui (AH), Hubei (HB), Sichuan (SC), Shanxi (SX), Gansu (GS), Guizhou (GZ), Henan (HN) and Jiangxi (JX). The seven common peaks (17) are labeled

Tianjin, China). The water used was freshly distilled.

Instrumentation and Chromatographic Procedures

The LC analysis was carried out with an Agilent 1100 series LC-DAD system (Agilent Technologies, Palo Alto, CA, USA) equipped with a G1379A vacuum degasser, a G1311A quaternary pump, a G1313A autosampler, an injector with a 100 lL loop, and a G1315B diode array detector. For chromatographic analysis, an Agilent Zorbax Eclipse XDB-C18 column (4.6 250 mm, 5 lm) with an Agilent Zorbax high-pressure reliance cartridge guard column (C18, 12.5 4.6 mm, 5 lm) was used. The injection volume was 20 lL and the column temperature was maintained at ambient. The DAD detector was set at 360 nm for acquiring chromatograms. UV spectra were acquired from 190 to 400 nm every 2 nm. The ow rate was 0.8 mL min1. The mobile phase consisted of (A) methanol, and (B) 0.1% acetate acid, and the gradient program was 10% A and 90% B at the beginning, then reached 45% A and 55% B at 100 min. The system was then restored to initial conditions (*10 min). LC-MS was performed with an Agilent 1100 Series LC and PESCIEX QSTAR MASS. The LC conditions were the same as above. The mass spectra were recorded with the use of electronspray ionization (ESI) in the negative mode with ion spray voltage at 3,300 eV, source temperature at 400 C, gas spray

1 at 60 psi, gas spray 2 at 40 psi, current gas at 40 psi, desolvent voltage 1 at 40 eV, desolvent voltage 2 at 15 eV, focus voltage at 215 eV, and the scanning range was set from 100 to 1,500 amu.

Sample Preparation
The samples were cut into small pieces, ground to powder and passed through a 20-mesh sieve. Each sample powder (3.0 g) was weighed accurately, and soaked in 25 mL of distilled water for 12 h. The mixture was then boiled for 3 h, ltered and the ltrate was diluted to 25 mL with water. This solution was ltered through a 0.45 lm lter, and a 20 lL aliquot of the ltrate was injected for LC analysis.

Experimental
Plant Materials and Reagents
Commercial Eucommia Bark samples (46) were purchased from dierent sources and chosen from dierent batches. The pharmaceutical providers were selected at random from eight dierent provinces: Anhui, Gansu, Guizhou, Hubei, Henan, Jiangxi, Sichuan and Shanxi provinces during November 2006March 2007 (Table 1). About half of the samples came from dierent parts of the Sichuan province, which is one of the largest in China, and which is also one of the main producers of the Chinese traditional medicine (CTM). The other samples were collected from the other seven provinces in approximately the same numbers from each one. Each sample was analysed in triplicate. Acetic acid was an A.R grade reagent (The Second Reagent Factory, Shanghai, China). The LC grade methanol (Damao Chemical Reagent Factory, Tianjin, China) was ltered with the use of the solvent lter (Automatic Science Co.

Data Analysis
LC-DAD chromatographic data of the 46 Eucommia Bark samples were submitted for analysis by the Computer Aided Similarity Evaluation System (CASES) to extract the characteristic ngerprints [24]. Prior to submitting the ngerprint data to chemometric analysis, the data matrix was autoscaled. The similarities of the entire set of chromatographic results were calculated with the use of CASES, and PCA biplots were obtained. Finally, HCA was applied and dendrograms were produced. All of the ngerprint data were processed by a Pentium computer, and all the programs for data processing were coded in MATLAB 6.5 (Mathworks).

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Table 2. Similarity values as compared with the mean chromatographic ngerprint from samples in Table 1
Sample no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Similarity 0.9945 0.9959 0.8424 0.7209 0.8758 0.6534 0.6244 0.9646 0.9819 0.9907 0.9834 0.9715 0.9712 0.9821 0.5217 0.5266 Sample no. 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Similarity 0.3864 0.8261 0.7524 0.9864 0.9411 0.9947 0.9633 0.9623 0.9508 0.9791 0.9493 0.9862 0.9869 0.9571 0.9930 0.9809 Sample no. 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Similarity 0.9439 0.9842 0.9694 0.9878 0.9636 0.9960 0.9978 0.9749 0.9980 0.9622 0.9519 0.9681 0.9883 0.9755

__________________________________________________________________________________ Common RT (min) [M-H]+ (m/z) Fragments M. W. Identification peak No. [M-H]+ (m/z) __________________________________________________________________________________ 1 25.37 373 -374 Geniposidic acid 2 29.23 353 191 354 Chlorogenic acid 3 56.90 177 162 178 Unknown 4 59.95 579 417 580 Syringaresinol O- -D-glucopyranoside 5 62.20 681 519, 357 682 Pinoresinol di-O- -D-glucopyranoside 6 64.27 519 357 520 Pinoresinol O- -D-glucopyranoside 7 70.34 534 372 535 1-Hydroxypinoresinol 4-O- -Dglucopyranoside ___________________________________________________________________________________ 1
COOH

Table 3. LC-MS data for the identication of the seven common compounds in the Eucommia Bark LC ngerprints

Similarities relative to this object were calculated (Table 2). During similarity calculation, a total of seven common peaks were involved. For classication purposes, the literature suggests that a value > 0.90 indicates strong similarity [31]. The Eucommia Bark ngerprints of samples from Sichuan, Hubei, Shanxi and Anhui (i.e., the SHSA group) were quite similar with values of ca. 0.94, while those from Gansu, Guizhou, Henan, Jiangxi were less alike (<0.87). The ngerprints of samples from Sichuan, Hubei, Shanxi and Anhui (i.e., the SHSA group) were very similar, while those from Gansu, Guizhou, Henan and Jiangxi diered from the SHSA ones. These conclusions are reected in the actual patterns and peak shapes of the chromatograms (Fig. 1). The mean chromatograms of the SHSA samples are very similar to each other, but those from Gansu and Guizhou, have very dierent intensities as compared to the responses from the Henan and Jiangxi samples. Their mean chromatograms are also quite dierent from the SHSA ones, especially their main peaks.

2
CH CH

OH O C O HO OH OH COOH

LC-MS Analysis of Eucommia Bark Samples

The electronspray ionization technique revealed the presence of organic acids, saccharides and glycosides. The mass spectra of these compounds were compared with reference data [3235], and seven principal peaks of the Eucommia Bark samples were partialy identied. Their proposed chemical structures are in Table 3: geniposidic acid (peak 1), chlorogenic acid (3-O-caeoylquinic acid, peak 2), syringaresinol O-b-D-glucopyranoside (peak 4), pinoresinol di-O-b-Dglucopyranoside (peak 5), pinoresinol O-b-D-glucopyranoside (peak 6) and 1-hydroxypinoresinol 40 -O-b-D-glucopyranoside (peak 7), respectively. Peak 3 was not identied and is labeled Unknown.

O CH2 OH OGlc

HO

4-7
R4 R 5O OCH3 O R1

OCH3 OR3 R2

4: 5: 6: 7:

R1 = R5 = H, R2 = R4 = OCH3, R3 = Glc R1 = R2 = R4 = H, R3 = R5 = Glc R1 = R2 = R3 = R4 = H, R5 = Glc R1 = OH, R2 = R3 = R4 = H, R5 = Glc

___________________________________________________________________________________

Results and Discussion

LC Fingerprints of Eucommia Bark and Identication of Common Peaks
Seven common peaks were identied from the chromatograms of the 46 samples with the use of the CASES software. The mean chromatograms of the Eucommia Bark samples from each province (Fig. 1) reect some of the differences between the samples. The Eucommia Bark ngerprints suggest that

most of the detected compounds in the dierent samples were very similar, but the relative peak intensities were signicantly dierent.

Similarity Calculation
For similarity calculations a starting or standard sample i.e., a ngerprint, has to be selected. For this work, the mean of all the available peaks for the 46 Eucommia Bark samples was calculated and dened as the standard sample.

Principal Component Analysis

A 46 object 7 variable data matrix containing the absolute peak areas of the seven principal peaks, (see above) was Original

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submitted to PCA after data pretreatment (autoscaling) [36]. The PC1 versus PC2 biplot (Fig. 2) accounted for 83% data variance (PC1 = 56%, PC2 = 27%), and showed that ve clusters formed from the 46 Eucommia Bark samples from the eight provinces. The Eucommia Bark samples from the Guizhou (lled square) province had the highest positive scores on PC1, those from Gansu (lled triangle) and Henan (triangle) provinces were a somewhat less positive, and the scores associated with the Jiangxi (square) province had the lowest values. However, the remaining Eucommia Bark samples from the other four provinces formed a clearly separated cluster with mostly negative scores close to zero. In other words, the Eucommia Bark samples from the SHSA group all formed one group. Loadings vectors for compounds 27 were positive on PC1 with the vector for peak 3 being the largest and that for peak 2 the smallest. Thus, compound 2 contributed very little to data variance. Only, the loadings vector for peak 1 (geniposidic acid) had a relatively large negative vector. It is this compound that is mainly responsible for the separation of the SHSA sample group from the other objects. On PC2, most of the objects clustered near the origin. However, Guizhou (most positive scores) and Jiangxi objects were separated by loadings 1, 2, 57 with the loadings vector 2 again having only a small eect. Only the responses of the samples from the Gansu province had objects with signicant negative scores on PC2, and these were separated from the rest by loadings vectors 3 and 4. This analysis of the biplot indicates that the SHSA group were principally separated by the geniposidic acid (peak 1) loadings vector. This vector is approximately at right angles to the three closely correlated loadings vectors 57 i.e., pinoresinol di-O-b-D-glucopyranoside (peak 5), pinoresinol O-b-Dglucopyranoside (peak 6) and 1-hydroxypinoresinol 40 -O-b-D-glucopyranoside (peak 7), and consequently, the peak 1 vector is independent. This vector was also almost diametrically opposed to the loadings vectors 3 and 4, i.e., syringaresinol O-b-D-glucopyranoside (peak 4) and the unknown peak 3. This suggests Original

Fig. 2. Biplot of the 46 chromatographic responses of the Eucommia Bark samples (83% data variance explained) from Sichuan (5), Hubei (), Shanxi (x), Anhui (v), Henan (4), Gansu (m), Jiangxi (h) and Guizhou (j). The seven loadings vectors (d) are numbered 17

that samples high in the geniposidic acid (peak 1) contain little or no substances corresponding to these two peaks. The small loadings vector (peak 2) on both PCs, suggests that this compound has little eect on any sample. This vector also correlates moderately well with the already mentioned loadings vectors 57, which indicates that the related compounds may be found together. It is well known that climatic and geographical conditions can inuence plant development as reected, for example, by the amounts of their marker compounds. This is apparently well illustrated by the distribution of the Eucommia Bark objects in the above biplot because the objects are dened by their compositon of the marker compounds as reected by the LC ngerprint measurements described above. Thus, PC2 discriminates between the samples from the Guizhou and Jiangxi (positive scores) and those from the Gansu and the Henan (negative scores). These provinces are geographically scattered with the latter two lying at a higher latitude than the former pair. This necessarily implies dierences in climatic conditions such as sunlight, temperature and rainfall all of which are typical of the climatic conditions that might inuence the amounts of the marker compounds. It was clear from the above PCA biplot that the SHSA group of samples

played a minor role, and the samples from the smaller provinces diered markedly from the SHSA ones. However, because the SHSA group contained most of the experimental samples, many of which were obtained from Sichuan the largest province, this sample group was submitted for further PCA studies. The new PC1 versus PC2 biplot (85% variance described, Fig. 3) indicates that the Sichuan samples (down arrow) clearly show a trend on PC1, which is characterized by a well spread out group with positive scores (SHSA3), a group with negative scores close to zero (SHSA2) and a compact group of objects with negative scores (SHSA1). The rst group (SHSA3) is associated with loadings vectors 27, while the last (SHSA1) is discriminated by the negative loadings vector 1. Thus, the distribution of the loadings vectors is similar to that in the previous biplot based on the total data matrix (Fig. 2). This indicates that the arguments regarding the dierences in climatic and geographical conditions, which inuence the amounts of the compounds detected by the LC ngerprints, may be applicable to a smaller subgroup of samples. This could explain why some ngerprint objects from the Hubei province are closely associated with the Sichuan cluster with negative PC1 scores, and others overlay the Sichuan cluster with the positive PC1

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Fig. 3. Biplot of the 36 chromatographic responses of the Eucommia Bark samples (85% data variance explained) from Sichuan (5), Hubei (), Shanxi (x), Anhui (v). The seven loadings vectors (d) are numbered as 17. Dashed lines (- - -) indicate notional boundaries of the SHSA (13) groups of the objects

structuraly similar; they appear to be the common compounds present across the sample set, and belong to the substituted pineresinols. Compound 4 is also based on the pinoresinol molecule but importantly it has an OCH3 substituent in the R4 position. This substituent is absent from the R14 positions in compounds 57 just discussed. It is possible that this dierence in chemistry inuences the amount of this compound in the Eucommia Bark samples, and is sucient to distinguish the samples containing it from the others as discussed above. However, the loading vectors 57 are approximately at right angles to the vector for peak 4, which indicates that this vector is independent from the others i.e., the presence of compounds 57 does not inuence the amount of compound 4 in Eucommia Bark.

Hierarchical Cluster Analysis (HCA)

It is generally good practice to test the conclusions drawn on the basis of one method of chemometrics analysis by submitting the data to analysis by another method. In such cases, it is preferable to apply a method that is based on a completely dierent algorithm. Hierarchical cluster analysis is a non-parametric data interpretation method, and is suitable for ngerprint analysis in practice because it is simple to use and ready available. In this work it was applied to the original data matrix of the 46 Eucommia Bark objects, and then also to the SHSA subset. The dendrogram (Fig. 4) indicates that objects from the original data matrix may be divided into ve main clusters. Eucommia Bark samples from Sichuan (2043), Hubei (814), Shanxi (4446), Anhui (12) were classied into one group (1) i.e., the SHSA cohort, and Gansu (35), Guizhou (67), Henan (15 17) and Jiangxi (1819) samples were classied into their own separate groups (25). The dendrogram for SHSA shows that it can be clearly divided into three groups (labeled as 1, 2 and 3), and they correspond to SHSA1-3 as described above. The results shown by the two Original

Fig. 4. HCA dendrogram for the 46 Eucommia Bark samples. The samples were the same as listed in Table 1 and the abbreviations are the same as in Fig. 1

scores. For example, they could come from similar climatic regions such as those close to their border or a common river valley. Approximately half the ngerprint objects have negative scores on PC2 (Fig. 3), and they are strongly discriminated by the loadings vector 3 and to a lesser extent, vector 4, i.e., peak 3 (the unknown compound) and to a lesser extent peak 4 (syringaresinol O-b-Dglucopyranoside). The other half of the ngerprint objects are associated with the compounds: geniposidic acid (peak

1), chlorogenic acid (peak 2), pinoresinol di-O-b-D-glucopyranoside (peak 5), pinoresinol O-b-D-glucopyranoside (peak 6) and 1-hydroxypinoresinol 40 -Ob-D-glucopyranoside (peak 7). If the two biplots (Figs. 2, 3) are compared, then it becomes apparent that three dierent chemical structures underpin the three general groupings discriminated in the analysis of the total and SHSA-only data matrices. These compounds are represented by: (1) peaks 57, (2) peak 4, and (3) peak 1. The rst three compounds, 57, are

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HCA support the main subdivision of the ngerprint samples by the PCA.

Acknowledgments
The authors gratefully acknowledge the nancial support of this study by the National Natural Science Foundation of China (NSFC20365002 and NSFC20562009), the State Key Laboratories of Electroanalytical Chemistry of Changchun Applied Chemical Institute (SKLEAC2004-3) and the Chemo/ Biosensing and Chemometrics of Hunan University (SKLCBC2005-22), the Jiangxi Provincial Natural Science Foundation (JXNSF062004), and the program for Changjiang Scholars and Innovative Research Team in Universities (IRT0540).

Conclusion
In this work, 46 Eucommia Bark samples were collected from eight Chinese provinces, and LC-DAD chromatographic analysis yielded seven common peaks that could be used for ngerprinting this common popular traditional medicine. Six of these compounds were identied by mass spectrometry as substituted resinols (4 compounds), geniposidic acid and chlorogenic acid. PCA of the 46 7 data matrix discriminated the objects on the basis of the provinces from which the samples were obtained. Samples sourced from the SHSA provinces were very similar and clustered together. They were further investigated by PCA. This analysis showed that in general, the basis for discrimination of the original data and that from the SHSA subset was the same. The loadings vectors of the seven marker compounds grouped into three sets: (1) three closely correlating substituted resinol compounds and chlorogenic acid; (2) the fourth resinol compound identied by the OCH3 substituent in the R4 position, and the unknown compound; and (3) the geniposidic acid, which was independent of set 1 variables, and which negatively correlated with the set 2 ones above. The discrimination of objects according to their provinces was supported by the classication results with the use of the complementary non-parametric HCA. Thus, this work indicated that Eucommia Bark preparations may be successfully compared with the use of the LC responses from the seven marker compounds and chemometrics methods such as PCA and the complementary HCA.

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