Sie sind auf Seite 1von 11

CHAPTER

27

Chromatographic
Development, Validation
and Data Handling of Tea
Fingerprints
Bieke Dejaegher, Yvan Vander Heyden
Department Analytical Chemistry and Pharmaceutical Technology (FABI), Center for
Pharmaceutical Research (CePhaR), Vrije Universiteit Brussel (VUB), Brussels, Belgium

Abbreviations
CART classification and regression trees
CC column centering
CE capillary electrophoresis
CEC capillary electrochromatography
CLC capillary liquid chromatography
COW correlation optimized warping
DAD diode array detector
DOSC direct orthogonal signal correction
ED electrochemical detector
FID flame ionization detector
GC gas chromatography
HILIC hydrophilic interaction chromatography
HPLC high-performance liquid chromatography
HPTLC high-performance thin layer chromatography
MS mass spectrometry
MSC multiplicative scatter correction
NIR near infrared
NMR nuclear magnetic resonance
OPLS orthogonal projections to latent structures
PCA principal component analysis
PCR principal component regression
PLS partial least squares
RF random forests
RMSE root mean squared error
RMSECV root mean squared error of cross-validation
RMSEP root mean squared error of prediction
rPCA robust principal component analysis
RPLC reversed-phase liquid chromatography
Tea in Health and Disease Prevention. DOI: 10.1016/B978-0-12-384937-3.00027-6
Copyright 2013 Elsevier Inc. All rights reserved.

323

SECTION 4
Compositional and Nutritional Aspects
rPLS robust partial least squares
SIMCA soft independent modeling of class analogy
SNV standard normal variate
Step-MLR stepwise multiple linear regression
SVM-C support vector machines for classification
TLC thin layer chromatography
UPLC ultra-performance liquid chromatography
UV ultraviolet
UVE-PLS uninformative variable elimination partial least squares
VIS visible

INTRODUCTION
Tea, or Camellia sinensis, belonging to the Theaceae family, can be divided into different types,
e.g. green, black, red, white, . tea, depending on the applied treatment after harvesting. For
more information on these different types, we refer to other chapters. This chapter discusses
the application of chromatographic fingerprints of teas in the context of quality control,
exploratory analysis, classification, and calibration. The idea of a chromatographic fingerprint
is to develop a chromatographic pattern of a tea extract, in which as many compounds as
possible are separated.

324

In 1991, the World Health Organization (WHO) introduced and accepted chromatographic
fingerprint technology as an identification and qualification technique for medicinal herbs
(WHO, 2000). Since 2000, the Chinese State Food and Drug Administration (SFDA) demands
the use of fingerprints for the quality control of certain Traditional Chinese Medicines in order
to standardize the herbs and their preparations (Drug Administration Bureau of China 2002).
The American Food and Drug Administration (FDA) (FDA, 2004) and the European Medicines
Agency (EMA) (EMA guidelines, 2006) have also accepted fingerprints as an alternative
approach for evaluating the quality of herbs and their preparations.
Three main steps can be distinguished when applying chromatographic fingerprints: (1) their
development, (2) validation, and (3) extraction of relevant information or data handling.
The first two steps are discussed briefly below, while the main part of this chapter will
discuss the data-handling aspects.

DEVELOPMENT OF FINGERPRINTS
In this section, we will only discuss briefly the analytical techniques used for fingerprinting.
The actual development of the chromatographic fingerprints with the sample preparation and
the fingerprint development steps are not discussed. These were considered to be outside the
scope of this chapter, which will mainly focus on data handling.
Fingerprints can be created using different analytical techniques, mainly of either spectroscopic or chromatographic origin. Spectroscopic fingerprints can be developed using ultraviolet (UV), visible (VIS), near infrared (NIR), Raman, or nuclear magnetic resonance (NMR)
spectroscopy. Mass spectrometric (MS) fingerprints can also be developed. The respective
spectra form the fingerprints and provide the data matrix X (see further) when measured for
several samples.
Chromatographic fingerprints can be developed using thin layer chromatography (TLC),
high-performance TLC (HPTLC), high-performance liquid chromatography (HPLC) (Hu
et al., 2009; Zheng et al., 2009; Lee and Ong, 2000; Novak et al., 2010; van Nederkassel et al.,
2005; Daszykowski et al., 2007; Dumarey et al., 2008), ultra-performance LC (UPLC)
(Pongsuwan et al., 2008; Zhao et al., 2011), capillary LC (CLC) (Dalluge et al., 1997), or gas
chromatography (GC) (Jumtee et al., 2009; Pongsuwan et al., 2007). Detection is performed
by UV absorption (Zheng et al., 2009; van Nederkassel et al., 2005; Daszykowski et al., 2007;

CHAPTER 27
Chromatographic Development, Validation and Data Handling of Tea Fingerprints

Dumarey et al., 2008), diode array detection (DAD) (Hu et al., 2009; Zhao et al., 2011; Lee and
Ong, 2000), mass spectrometry (MS) (Zheng et al., 2009; Pongsuwan et al., 2008; Zhao et al.,
2011; Dalluge et al., 1997; Pongsuwan et al., 2007), electrochemical detection (ED) (Novak
et al., 2010) or flame ionization detection (FID) (Jumtee et al., 2009). The chromatograms of
different samples form the data matrix X.
Finally, fingerprints also can be developed using electro-driven techniques, such as capillary
electrophoresis (CE) (Lee and Ong, 2000) or capillary electrochromatography (CEC), coupled
to UV, DAD, or MS detection. Then the data matrix X consists of the electropherograms. For
more information about the development of the fingerprints, we refer to the above mentioned
references.

VALIDATION OF FINGERPRINTS
After an analytical method is developed, it should be validated to ensure its suitability for
the intended purpose (Eurachem, 1998; ICH, 2005). Three golden rules exist in method
validation, i.e. validate the whole method, validate over the whole concentration range, and
validate all sample matrix types. Several performance criteria of the method are evaluated.
Examined criteria are the linearity, the detection limit, the quantification limit, the precision,
the accuracy or trueness, the range, the specificity, and the robustness. However, method
validation applies on so-called univariate methods, where a signal of the measurement technique is related to the concentration of a substance. Here, the entire fingerprint is linked to
a sample property (which mostly is not the concentration of a compound). As a consequence,
the performance criteria defined for the univariate approach do not apply to fingerprints.
Consequently, very often the chromatographic fingerprints are not validated. In cases where
some validation is performed, it is done on individual compounds (univariately) and not on
the entire fingerprint (Hu et al., 2009; Lee and Ong, 2000).
For instance, linearity is the ability of a method to obtain results (signals) proportional to the
concentration of a given compound. For individual peaks that need to be quantified,
a univariate calibration line (Hu et al., 2009; Lee et al., 2000) is measured, depending on the
absence or presence of matrix interferences.
The limit of detection is the lowest concentration of a given compound that produces a signal
that can be distinguished from the background noise. The limit of quantification is the lowest
concentration of a given compound that can be quantified properly, i.e. with an adequate
precision and bias. The limit of detection for the quantified components in Hu et al., 2009,
and Lee and Ong, 2000, was determined as the concentration resulting in a signal-to-noise
ratio of 3:1. For all other validation parameters, their estimation is equal to the quantification
of the individual compounds and not related to the global evaluation of the fingerprint.
Precision is related to random errors. It refers to the distribution of replicated results and can
be expressed as a standard deviation, s, a variance, s2, or a percentage relative standard
deviation, %RSD. The repeatability, time-different intermediate precision, and the injection
precision are precision estimates measured under different replication conditions. The precision responses which are evaluated are the retention time, peak area and/or peak height of the
most important peaks (Lee and Ong, 2000; Alaerts et al., 2007; Hu et al., 2009), e.g.
biomarkers. As well as evaluating the precision of retention time, Lee and Ong (2000) also
evaluated the repeatability and the time-different intermediate precision of the assay results of
eight commercial tea samples. The estimation of the above precision values reflects the quality
and replication similarity of the fingerprints.
The trueness is the closeness of agreement between an average value from a number of test
results and the accepted reference value, and is only related to systematic errors. Most often,
trueness is evaluated and incorrectly called accuracy. To evaluate accuracy and/or trueness,

325

SECTION 4
Compositional and Nutritional Aspects

parameters, such as bias, % bias, and % recovery can be used. The trueness, expressed as
percentage recovery, was determined as the average of the recoveries at three spiked levels for
the 14 quantified components (Hu et al., 2009).
The range is the concentration interval over which the method possesses acceptable linearity,
precision and trueness. and was evaluated for the 14 quantified components in Hu et al., 2009.

DATA HANDLING OF FINGERPRINTS


After the development and validation of the chromatographic fingerprints, the desired
information needs to be extracted. The specific information that is desired will depend on the
goal of the study concerned. Regardless of the goal, chemometric techniques are usually
mandatory to extract the information. These techniques can be roughly divided into unsupervised and supervised data analysis. The difference is represented in Figure 27.1. Unsupervised methods will only use the information contained in the m x n data matrix X of the
fingerprints, with m being the number of samples, and n the number of variables. It is
used, for instance, to gather information regarding the structure of the data. Supervised
techniques, on the other hand, will try to link the information contained in the data matrix X
to an m x 1 response vector y. Prior to either unsupervised or supervised techniques, the
data often is pretreated, to ensure adequate data analysis. In the pretreatment, undesired
variability is minimized, while variability due to factors of interest is maximized.

326

FIGURE 27.1
(A) Unsupervised, and (B) Supervised Data Analysis, with Alignment of the Corresponding Chromatographic Peaks as
Data Pretreatment.
m x n data matrix X and m x 1 response vector y.

CHAPTER 27
Chromatographic Development, Validation and Data Handling of Tea Fingerprints

Data Pretreatment
Many preprocessing techniques can be used for chromatographic fingerprints (Alaerts et al.,
2010a; Tistaert et al., 2011). The most important is the alignment of the fingerprints. In
chromatography, retention time shifts (see Figure 27.1) occur because of, for instance, column
ageing, small variations in mobile phase composition, in flow rate or in temperature. It is often
of the utmost importance to align the corresponding peaks in the fingerprints, because then
the information about a given peak is contained in the same column of the matrix X for all
samples (Figure 27.1), which is needed to obtain improved results with the data analysis
techniques. Again, different techniques can be used, e.g. parametric time warping, semiparametric time warping, correlation optimized warping (COW) (Zheng et al., 2009; van
Nederkassel et al., 2005), and others.
Besides aligning, other often-applied preprocessing methods include column centering (CC),
normalization, standard normal variate (SNV), multiplicative scatter correction (MSC), and
direct orthogonal signal correction (DOSC). CC subtracts the column average from each
corresponding column element in X. Normalization, also called row scaling, divides each row
element by its corresponding row standard deviation. SNV preprocessing corresponds to row
centering, i.e. subtracting the row average from each corresponding row element, followed by
row scaling. MSC aims to put the responses of the different fingerprints into a same average
zero-level, i.e. the baselines should be the same. DOSC removes from the data matrix X that
variation orthogonal to, i.e. not-related to, the response y.

Unsupervised Data Analysis


Unsupervised data analysis (Figure 27.1A) can be used in the context of identification and
quality control, of exploratory data analysis, and of curve resolution (Alaerts et al., 2010a;
Tistaert et al., 2011). Below, the first two contexts are discussed briefly and illustrated with some
examples.

IDENTIFICATION AND QUALITY CONTROL


Hu et al. (2009) prepared infusions of green tea (non-fermented), Oolong tea (partially
fermented), black tea (fully fermented), and Pu-erh tea (microbially fermented) using two
approaches. The tea infusions were subjected to HPLC-DAD analysis. The HPLC method
allowed simultaneous identification and quantification of ten catechins, three purine alkaloids, and gallic acid. Identification was obtained by comparing the retention times and the
DAD spectra of the peaks to those of the 14 standard compounds, while regular calibration
lines were used for quantification. It was found that one of the two extraction methods was to
be preferred and that green tea contained higher catechin levels than the other three tea types.
In fermented teas, most catechins are oxidized and polymerized by enzymes during the
manufacturing process.
Similar research was described (Lee and Ong, 2000), in which in different teas catechins and
theaflavins were identified and quantified from HPLC-DAD fingerprints. Again it was
confirmed that green tea contained more catechins than fermented teas, and that fermented
teas contained more theaflavins.
Novak et al. (2010) also performed similar research. They identified and quantified catechins
and gallic acid from HPLC-ED fingerprints. However, their study showed that not all
fermented teas had lower catechin contents than green teas, which they attributed to better
quality, i.e. a higher catechin content of the tea leaves used to prepare the black teas.
For identification or similarity analysis, parameters such as the correlation coefficient, r, or the
congruence coefficient, c, can be used to evaluate the (dis)similarity of chromatographic
fingerprints. This was, for instance, done in Alaerts et al. (2010b), where a similarity analysis of
different batches of rhizoma Chuanxiong and rhizoma Ligusticum was carried out, and the

327

SECTION 4
Compositional and Nutritional Aspects

correlation coefficients were calculated between each pair of HPLC-DAD fingerprints. In


addition, exploratory data analysis methods, such as principal component analysis and
hierarchical cluster analysis, were used to evaluate the (dis)similarity of the samples.

EXPLORATORY DATA ANALYSIS


Exploratory data analysis techniques can be used to check the data structure in the context of
identification and quality control or prior to supervised data analysis. Regularly applied
methods are principal component analysis (PCA), robust PCA (rPCA), projection pursuit, and
cluster analysis. These methods visualize the multivariate data and allow an insight into the
structure or clustering tendency of the data (Alaerts et al., 2010a; Tistaert et al., 2011).
Zhao et al. (2011) used PCA to distinguish between three tea types, i.e. green, white, and green
Pu-erh tea, using UPLC-DAD-MS fingerprints. When using the chromatographic profile after
alignment as the X matrix, no groups were seen. However, when using the contents of 54
components as the X matrix, the three groups could be distinguished by PCA.
Zheng et al. (2009) used PCA on HPLC-UV-MS fingerprints prior to classification (supervised
methods) to distinguish six tea groups: Assam, Ceylon, Darjeeling, English breakfast, green
and decaffeinated tea. Before alignment with COW, the six groups were not clearly separated
(Figure 27.2A), while after alignment, they clearly were (Figure 27.2B).

328

PCA was also used by Pongsuwan et al. (2008) prior to multivariate calibration to explore
green tea UPLC-MS fingerprints. Along PC1, a distinction could be made according to the
green tea rank (between 0 and 60), which was based on sensory analysis by professional tea
tasters, including evaluation of leaf appearance, smell, taste and color of the brew. At one side
of PC1, the high-ranked samples are found and at the other, the low-ranked. The PC1 scores
thus can be linked to the tea quality.
van Nederkassel et al. (2005) applied rPCA on HPLC-UV fingerprints of green tea samples,
prior to multivariate calibration, in order to detect, from the score diagnostic plot, outlying
objects in the matrix X. In such a score diagnostic plot, the distance of an object/sample to
the PCA model space (orthogonal distance) is plotted as a function of the distance of an
object/sample to the data majority (robust distance) (Figure 27.3). For both distances, a cutoff value is calculated. Objects that do not exceed both cut-off values are considered ordinary

FIGURE 27.2
Principal Component Analysis: PC1-PC2 Score Plot (A) Before and (B) After Alignment of the HPLC-UV Fingerprints Used
as X Matrix. (:) Decaffeinated, (-) green, (A) Darjeeling, (>) Ceylon, ()) English breakfast and () Assam tea.
(Reproduced with permission from Zheng et al., 2009.)

CHAPTER 27
Chromatographic Development, Validation and Data Handling of Tea Fingerprints

FIGURE 27.3
Score Diagnostic Plot of Green
Tea HPLC-UV Fingerprints. The
two cut-off lines also are shown.
(Reproduced with permission from
van Nederkassel et al., 2005.)

objects (Quadrant III). Objects that exceed the cut-off value of the orthogonal distance, but
not that of the robust distance are orthogonal outliers (Quadrant IV). Objects that exceed the
cut-off value of the robust distance, but not that of the orthogonal distance are good leverage
objects (Quadrant II). Finally, objects that exceed both cut-off values are called bad leverage
objects (Quadrant I). Three different types of outlying objects thus can be detected and
occasionally removed before modeling. From Figure 27.3, prior to a multivariate calibration,
the orthogonal outliers 1 and 2 are removed to obtain a better model for the antioxidant
activity of green tea samples. Besides using rPCA to remove outliers in the matrix X,
a histogram of the antioxidant activity values was also made to discover outliers in the
response vector y. Samples with an outlying antioxidant activity, seen in the histogram, are
removed for further analysis.

Supervised Data Analysis


Supervised data analysis (Figure 27.1B) can be used for pattern recognition or classification,
and for multivariate calibration. The difference between the two approaches is the response
vector y. When y is categoric and thus contains classes, classification methods are applied,
while when y is continuous, multivariate calibration methods are used (Alaerts et al., 2010a;
Tistaert et al., 2011).

PATTERN RECOGNITION OR CLASSIFICATION METHODS


Well-known classification methods that can be applied are linear discriminant analysis,
quadratic discriminant analysis, classification and regression trees (CART), random forests
(RF, i.e. an ensemble of CART trees), k-nearest neighbors, partial least squares discriminant
analysis, orthogonal projections to latent structures discriminant analysis, soft independent
modeling of class analogy (SIMCA), artificial neural Networks, and support vector machines
for classification (SVM-C) (Alaerts et al., 2010a; Tistaert et al., 2011).
In Zheng et al. (2009), SIMCA, SVM-C, and RF models were built to classify six groups of teas:
Assam, Ceylon, Darjeeling, English breakfast, green and decaffeinated tea. The three techniques allowed classifying the samples correctly, using the aligned HPLC-UV fingerprints as
matrix X. PCA was applied to reduce the number of variables and the SVM-C and RF methods
were applied using only the first six PCs as matrix X. Again all samples could be correctly
classified.

329

SECTION 4
Compositional and Nutritional Aspects

MULTIVARIATE CALIBRATION METHODS


Stepwise multiple linear regression (step-MLR), principal component regression (PCR), partial
least squares (PLS), robust PLS (rPLS), uninformative variable elimination PLS (UVE-PLS),
orthogonal projections to latent structures (OPLS), and support vector machines for regression
are multivariate calibration methods which are often applied.
The data set from van Nederkassel et al. (2005), consisting of the HPLC-UV fingerprints of
green tea samples, was used to model the antioxidant activity. After outlier removal and COW,
Dumarey et al. (2008) used Step-MLR, PCR, PLS, UVE-PLS and OPLS as modeling techniques,
while Daszykowski et al. (2007) applied PLS without outlier removal, PLS with outlier
removal, and rPCA without outlier removal after COW. The results are presented in Table 27.1.
Dumarey et al. (2008) concluded that the models of the different methods have a similar
predictive ability. Daszykowski et al. (2007) found that PLS without outlier removal resulted in
very large prediction errors. However, PLS after outlier removal and rPLS without outlier
removal gave similar predictive errors, meaning that rPLS is able to provide good models even
with outliers in the data set.
PLS was used by Pongsuwan et al. (2008) to model the rank (between 0 and 60, and based on
sensory analysis by professional tea tasters) of green tea samples as a function of their UPLCMS fingerprints. In Figure 27.4, the observed rank was plotted against that predicted. In
Pongsuwan et al. (2007), PLS and OPLS were used to model the rank of green tea samples
based on their GC-MS fingerprints. The OPLS model was found to have a better predictive
ability than the PLS model.

330

Jumtee et al. (2009) used PLS and OPLS to model the quality ranking (between 0 and 60, as
judged by professional tea tasters) of green tea samples as a function of their GC-FID and GCMS fingerprints. For both types of fingerprints, the OPLS model showed a better predictive
ability than the PLS model. In Figure 27.5, the observed rank was plotted against the predicted
one for the built OPLS models. The predictions were found to be very accurate.

TABLE 27.1 Multivariate Calibration Results from Models to Predict the Antioxidant
Activity of Green Tea. The Results for Two Types of Fingerprints
(of Different Length) are Given
Pooled Standard Deviation [ 143 (8.65%)

TEAC Assay
Multivariate
Calibration
Step-MLR after outlier
removal
PCR after outlier
removal
PLS without outlier
removal
PLS after outlier
removal
rPLS without outlier
removal
UVE-PLS after outlier
removal
OPLS after outlier
removal

Short (2 min) Fingerprints


RMSECV

RMSE

RMSEP

Long (11 min) Fingerprints


RMSECV

RMSE

RMSEP

214

162

186

182

140

86

216

189

192

227

194

227

721

350

206

177

177

159

80

174

172

186

215

195

208

158

105

198

209

177

176

166

80

168

Root mean squared error of cross-validation (RMSECV), root mean squared error for calibration set (RMSE), and root mean
squared error of prediction for test set (RMSEP). [line break] / = not specified [line break]
(Adapted from Daszykowski et al., 2007, and Dumarey et al., 2008)

CHAPTER 27
Chromatographic Development, Validation and Data Handling of Tea Fingerprints

FIGURE 27.4
Observed Versus Predicted Rank
Results. Prediction was from a PLS
model to model the rank (between 0 and
60) of green tea samples as a function of
their UPLC-MS fingerprints: both samples
from the calibration and the test
(encircled) set are shown. (Reproduced
with permission from Pongsuwan et al.,
2008.)

331

FIGURE 27.5
Observed Versus Predicted Rank Results. Prediction was from an OPLS model to model the rank (between 0 and 60) of
green tea samples as a Function of their (A) GC-FID and (B) GC-MS fingerprints: both samples from the calibration (:) and
the test (D) are shown. (Reproduced with permission from Jumtee et al., 2009.)

CONCLUDING SUMMARY
In this chapter, a short overview was given concerning the development, validation, and data
handling of tea fingerprints; the main focus was on data handling. This was divided into a data
pretreatment, an unsupervised data analysis, and a supervised data analysis section. A number
of possible methods and approaches were discussed and illustrated with some examples from
the literature.

SUMMARY POINTS
l

l
l
l

A chromatographic fingerprint is a chromatographic profile of a complex sample, in which


as many peaks as possible are separated.
Fingerprints can be developed using different analytical techniques.
Fingerprint validation is rarely performed.
Data pretreatment is often needed to be able to extract the desired information from the
fingerprints.

SECTION 4
Compositional and Nutritional Aspects

Unsupervised data analysis uses only the information contained in the fingerprint data
matrix X to extract desired information.
Supervised classification methods try to link the information contained in the fingerprint
data matrix X to a response vector y containing classes.
Supervised multivariate calibration methods try to link the information contained in the
fingerprint data matrix X to a response vector y containing a continuous response.

Acknowledgments
Bieke Dejaegher is a postdoctoral fellow of the Fund for Scientific Research (FWO) e Vlaanderen, Belgium.

References
Alaerts, G., Dejaegher, B., Smeyers-Verbeke, J., Vander Heyden, Y., 2010a. Recent developments in chromatographic
fingerprints from herbal products: Set-up and data analysis. Comb. Chem. High Throughput Screen. 13,
900e922.
Alaerts, G., Matthijs, N., Smeyers-Verbeke, J., Vander Heyden, Y., 2007. Chromatographic fingerprint development
for herbal extracts: A screening and optimization methodology on monolithic columns. J. Chromatogr. A 1172,
1e8.
Alaerts, G., Merino-Arevalo, M., Dumarey, et al., 2010b. Exploratory analysis of chromatographic fingerprints to
distinguish rhizoma Chuanxiong and rhizoma Ligustici. J. Chromatogr. A 1217, 7706e7716.
Dalluge, J.J., Nelson, B.C., Thomas, J.B., et al., 1997. Capillary liquid chromatography/electrospray mass spectrometry for the separation and Ddetection of catechins in green tea and human plasma. Rapid Commun. Mass
Spectrom. 11, 1753e1756.
Daszykowski, M., Vander Heyden, Y., Walczak, B., 2007. Robust partial least squares model for prediction of green
tea antioxidant capacity from chromatograms. J. Chromatogr. A 1176, 12e18.
Drug Administration Bureau of China, 2002. Requirements for Studying Fingerprints of Traditional Chinese
Medicine Injection. Drug Administration Bureau of China, Beijing, China.

332

Dumarey, M., van Nederkassel, A.M., Deconinck, E., Vander Heyden, Y., 2008. Exploration of linear multivariate
calibration techniques to predict the total antioxidant capacity of green tea from chromatographic fingerprints.
J. Chromatogr. A 1192, 81e88.
EMA, 2006. Guideline on Quality of Herbal Medicinal Products/Traditional Herbal Medicinal Products. Committee
for medicinal products for human use (CHMP), European Medicines Agency Inspections, October 1st, 2006,
CPMP/QWP/2819/00 Rev 1. EMEA/CVMP/814/00 Rev 1. http://www.ema.europa.eu/home.htm (accessed
25.5.11).
Eurachem, 1998. The Fitness for Purpose of Analytical Methods: A Laboratory Guide to Method Validation and
Related Topics, pp. 1e75.
Food and Drug Administration, 2004. Guidance for Industry: Botanical Drug Products, Food and Drug Administration, June 2004, p. 10. http://www.fda.gov/cder/guidance/4592fnl.pdf (accessed 23.05.09).
Hu, B., Wang, L., Zhou, B., et al., 2009. Efficient procedure for isolating methylated catechins from green tea and
effective simultaneous analysis of ten catechins, three purine alkaloids, and gallic acid in tea by highperformance liquid chromatography with diode array detection. J. Chromatogr. A 1216, 3223e3231.
ICH, 2005. Guidelines prepared within the International Conference on Harmonisation of Technical Requirements
for the Registration of Pharmaceuticals for Human Use (ICH), Validation of Analytical Procedures: Text and
Methodology, Q2(R1), pp. 1e13, http://www.ich.org/.
Jumtee, K., Bamba, T., Fukusaki, E., 2009. Fast GC-FID based metabolic fingerprinting of Japanese green tea leaf for
its quality ranking prediction. J. Sep. Sci. 32, 2296e2304.
Lee, B.-L., Ong, C.-N., 2000. Comparative analysis of tea catechins and theaflavins by high-performance liquid
chromatography and capillary electrophoresis. J. Chromatogr. A 881, 439e447.

Novak, I., Seruga,
M., Komorsky-Lovric, S., 2010. Characterisation of catechins in green and black teas using squarewave voltammetry and RP-HPLC-ECD. Food Chem. 122, 1283e1289.
Pongsuwan, W., Bamba, T., Harada, K., et al., 2008. High-throughput technique for comprehensive analysis of
Japanese green tea quality assessment using ultra-performance liquid chromatography with time-of-flight mass
spectrometry (UPLC/TOF MS). J. Agric. Food Chem. 56, 10705e10708.
Pongsuwan, W., Fukusaki, E., Bamba, T., et al., 2007. Prediction of Japanese green tea ranking by gas chromatography/mass spectrometry-based hydrophilic metabolite fingerprinting. J. Agric. Food Chem. 55, 231e236.
Tistaert, C., Dejaegher, B., Vander Heyden, Y., 2011. Chromatographic separation techniques and data handling
methods for herbal fingerprints: A review. Anal. Chim. Acta 690, 148e161.

CHAPTER 27
Chromatographic Development, Validation and Data Handling of Tea Fingerprints

van Nederkassel, A.M., Daszykowski, M., Massart, D.L., Vander Heyden, Y., 2005. Prediction of total green tea
antioxidant capacity from chromatograms by multivariate modeling. J. Chromatogr. A 1096, 177e186.
WHO, 2000. General Guidelines for Methodologies on Research and Evaluation of Traditional Medicines, Geneva.
Available from: http://www.who.int/ (accessed on 26.06.08).
Zhao, Y., Chen, P., Lin, L., et al., 2011. Tentative identification, quantitation, and principal component analysis of
green pu-erh, green, and white teas using UPLC/DAD/MS. Food Chem. 126, 1269e1277.
Zheng, L., Watson, D.G., Johnston, B.F., et al., 2009. A chemometric study of chromatograms of tea extracts by
correlation optimization warping in conjunction with PCA, support vector machines and random forest data
modeling. Anal. Chim. Acta 642, 257e265.

333