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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 classication 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 ame 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
323
Tea in Health and Disease Prevention. DOI: 10.1016/B978-0-12-384937-3.00027-6
Copyright 2013 Elsevier Inc. All rights reserved.
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 classication
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 ngerprints of teas in the context of quality control,
exploratory analysis, classication, and calibration. The idea of a chromatographic ngerprint
is to develop a chromatographic pattern of a tea extract, in which as many compounds as
possible are separated.
In 1991, the World Health Organization (WHO) introduced and accepted chromatographic
ngerprint technology as an identication and qualication technique for medicinal herbs
(WHO, 2000). Since 2000, the Chinese State Food and Drug Administration (SFDA) demands
the use of ngerprints 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 ngerprints as an alternative
approach for evaluating the quality of herbs and their preparations.
Three main steps can be distinguished when applying chromatographic ngerprints: (1) their
development, (2) validation, and (3) extraction of relevant information or data handling.
The rst two steps are discussed briey below, while the main part of this chapter will
discuss the data-handling aspects.
DEVELOPMENT OF FINGERPRINTS
In this section, we will only discuss briey the analytical techniques used for ngerprinting.
The actual development of the chromatographic ngerprints with the sample preparation and
the ngerprint 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 spectro-
scopic or chromatographic origin. Spectroscopic ngerprints can be developed using ultra-
violet (UV), visible (VIS), near infrared (NIR), Raman, or nuclear magnetic resonance (NMR)
spectroscopy. Mass spectrometric (MS) ngerprints can also be developed. The respective
spectra form the ngerprints and provide the data matrix X (see further) when measured for
several samples.
Chromatographic ngerprints 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;
324
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Compositional and Nutri tional Aspects
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 ame ionization detection (FID) (Jumtee et al., 2009). The chromatograms of
different samples form the data matrix X.
Finally, ngerprints 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 ngerprints, 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 quantication limit, the precision,
the accuracy or trueness, the range, the specicity, and the robustness. However, method
validation applies on so-called univariate methods, where a signal of the measurement tech-
nique is related to the concentration of a substance. Here, the entire ngerprint is linked to
a sample property (which mostly is not the concentration of a compound). As a consequence,
the performance criteria dened for the univariate approach do not apply to ngerprints.
Consequently, very often the chromatographic ngerprints are not validated. In cases where
some validation is performed, it is done on individual compounds (univariately) and not on
the entire ngerprint (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 quantied,
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 quantication is the lowest
concentration of a given compound that can be quantied properly, i.e. with an adequate
precision and bias. The limit of detection for the quantied 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 quantication
of the individual compounds and not related to the global evaluation of the ngerprint.
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, s
2
, 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 preci-
sion 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 reects the quality
and replication similarity of the ngerprints.
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,
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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 quantied 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 quantied components in Hu et al., 2009.
DATA HANDLING OF FINGERPRINTS
After the development and validation of the chromatographic ngerprints, the desired
information needs to be extracted. The specic 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 unsu-
pervised and supervised data analysis. The difference is represented in Figure 27.1. Unsu-
pervised methods will only use the information contained in the m x n data matrix X of the
ngerprints, 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.
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.
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Compositional and Nutri tional Aspects
326
Data Pretreatment
Many preprocessing techniques can be used for chromatographic ngerprints (Alaerts et al.,
2010a; Tistaert et al., 2011). The most important is the alignment of the ngerprints. In
chromatography, retention time shifts (see Figure 27.1) occur because of, for instance, column
ageing, small variations in mobile phase composition, in owrate or in temperature. It is often
of the utmost importance to align the corresponding peaks in the ngerprints, 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, semi-
parametric 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 ngerprints 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 identication and
quality control, of exploratory data analysis, and of curve resolution (Alaerts et al., 2010a;
Tistaert et al., 2011). Below, the rst two contexts are discussed briey 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 identication and quantication of ten catechins, three purine alka-
loids, and gallic acid. Identication 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 quantication. 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
theaavins were identied and quantied from HPLC-DAD ngerprints. Again it was
conrmed that green tea contained more catechins than fermented teas, and that fermented
teas contained more theaavins.
Novak et al. (2010) also performed similar research. They identied and quantied catechins
and gallic acid from HPLC-ED ngerprints. 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 identication or similarity analysis, parameters such as the correlation coefcient, r, or the
congruence coefcient, c, can be used to evaluate the (dis)similarity of chromatographic
ngerprints. 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
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327
correlation coefcients were calculated between each pair of HPLC-DAD ngerprints. 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
identication 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 ngerprints. When using the chromatographic prole 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 ngerprints prior to classication (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).
PCA was also used by Pongsuwan et al. (2008) prior to multivariate calibration to explore
green tea UPLC-MS ngerprints. 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 ngerprints 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 cut-
off 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.)
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Compositional and Nutri tional Aspects
328
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 classication,
and for multivariate calibration. The difference between the two approaches is the response
vector y. When y is categoric and thus contains classes, classication 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 classication methods that can be applied are linear discriminant analysis,
quadratic discriminant analysis, classication 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), articial neural Networks, and support vector machines
for classication (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 tech-
niques allowed classifying the samples correctly, using the aligned HPLC-UV ngerprints as
matrix X. PCA was applied to reduce the number of variables and the SVM-C and RF methods
were applied using only the rst six PCs as matrix X. Again all samples could be correctly
classied.
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.)
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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 ngerprints 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 UPLC-
MS ngerprints. 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 ngerprints. The OPLS model was found to have a better predictive
ability than the PLS model.
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 GC-
MS ngerprints. For both types of ngerprints, 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
TEAC Assay Pooled Standard Deviation [143 (8.65%)
Multivariate
Calibration
Short (2 min) Fingerprints Long (11 min) Fingerprints
RMSECV RMSE RMSEP RMSECV RMSE RMSEP
Step-MLR after outlier
removal
214 162 186 182 140 86
PCR after outlier
removal
216 189 192 227 194 227
PLS without outlier
removal
/ 721 350 / / /
PLS after outlier
removal
206 177 177 159 80 174
rPLS without outlier
removal
/ 172 186 / / /
UVE-PLS after outlier
removal
215 195 208 158 105 198
OPLS after outlier
removal
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 specied [line break]
(Adapted from Daszykowski et al., 2007, and Dumarey et al., 2008)
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330
CONCLUDING SUMMARY
In this chapter, a short overview was given concerning the development, validation, and data
handling of tea ngerprints; 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 A chromatographic ngerprint is a chromatographic prole of a complex sample, in which
as many peaks as possible are separated.
l Fingerprints can be developed using different analytical techniques.
l Fingerprint validation is rarely performed.
l Data pretreatment is often needed to be able to extract the desired information from the
ngerprints.
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 ngerprints: both samples
from the calibration and the test
(encircled) set are shown. (Reproduced
with permission from Pongsuwan et al.,
2008.)
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 ngerprints: both samples from the calibration (:) and
the test (D) are shown. (Reproduced with permission from Jumtee et al., 2009.)
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l Unsupervised data analysis uses only the information contained in the ngerprint data
matrix X to extract desired information.
l Supervised classication methods try to link the information contained in the ngerprint
data matrix X to a response vector y containing classes.
l Supervised multivariate calibration methods try to link the information contained in the
ngerprint data matrix X to a response vector y containing a continuous response.
Acknowledgments
Bieke Dejaegher is a postdoctoral fellow of the Fund for Scientic Research (FWO) e Vlaanderen, Belgium.
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