Journal of Pharmaceutical Analysis 2014;4(4):223–233

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Journal of Pharmaceutical Analysis

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REVIEW PAPER

Chemometrics: A new scenario in herbal

drug standardization
Ankit Bansal, Vikas Chhabra, Ravindra K. Rawal, Simant Sharman

Indo-Soviet Friendship (ISF) College of Pharmacy, Moga, Punjab 142001, India

Received 16 July 2013; accepted 4 December 2013

Available online 1 January 2014

KEY WORDS Abstract Chromatography and spectroscopy techniques are the most commonly used methods in
Chemometrics;
standardization of herbal medicines but the herbal system is not easy to analyze because of their complexity
HELP; of chemical composition. Many cutting-edge analytical technologies have been introduced to evaluate the
Herbal drugs; quality of medicinal plants and significant amount of measurement data has been produced. Chemometric
PCA; techniques provide a good opportunity for mining more useful chemical information from the original data.
OPA Then, the application of chemometrics in the field of medicinal plants is spontaneous and necessary.
Comprehensive methods and hyphenated techniques associated with chemometrics used for extracting
useful information and supplying various methods of data processing are now more and more widely used in
medicinal plants, among which chemometrics resolution methods and principal component analysis (PCA)
are most commonly used techniques. This review focuses on the recent various important analytical
techniques, important chemometrics tools and interpretation of results by PCA, and applications of
chemometrics in quality evaluation of medicinal plants in the authenticity, efficacy and consistency.
& 2014 Xi’an Jiaotong University. Production and hosting by Elsevier B.V.
Open access under CC BY-NC-ND license.

1. Introduction clinical therapy in many oriental countries for thousands of years.

During the past two decades, they attracted attention to a greater
Herbal medicines (HMs) including Chinese medicine (CM) and other extent in western countries because of their high pharmacological
folk medicines are getting more and more popular nowadays globally activities with low toxicity and rare complications. However, HMs
for improving health condition of human beings as well as preventing have not been officially recognized worldwide because the quantity
and curing diseases. They have played an important role in the and quality of the safety and efficacy data on traditional medicine are
far from sufficient to meet the criteria needed to support their use
n
Corresponding author. Tel.: þ91 9872931654. everywhere. Therefore, the World Health Organization (WHO)
E-mail address: [email protected] (S. Sharma). provides guidelines for the assessment of the quality of HMs [1].
Peer review under responsibility of Xi0 an Jiaotong University. Now, one or two markers or pharmacologically active compo-
nents in herbs or herbal mixtures are currently employed for
evaluating the quality and authenticity of herbal medicines.
However, this kind of determination does not give a complete
overview of an herbal product, because multiple constituents are

2095-1779 & 2014 Xi’an Jiaotong University. Production and hosting by Elsevier B.V. Open access under CC BY-NC-ND license.
http://dx.doi.org/10.1016/j.jpha.2013.12.001
224 A. Bansal et al.

usually accountable for its therapeutic actions and effectiveness. In this review, we have discussed the origin and development of
These multiple constituents may work ‘synergistically’ and could chemometrics in the first part and further stressed various
hardly be separated into active parts [2,3]. important chromatographic techniques for HDs fingerprint devel-
There are various techniques that are used for this sort of opment. Furthermore, multivariate methods that are used to extract
determination like high-performance liquid chromatography (HPLC), the information from the chromatographic data like pretreatment of
gas chromatography (GC), ultra-high performance liquid chromato- chromatogram, multivariate resolution methods and various clas-
graphy (UHPLC) and thin layer chromatography (TLC). Moreover, the sification and discrimination methods applied in the chemical
recent approaches including hyphenated chromatography and spectro- analysis of herbal drugs have been discussed. In the final part, we
scopy like high-performance liquid chromatography-diode array detec- have discussed its application in herbal drug standardization with
tion (HPLC-DAD), gas chromatography–mass spectrum analysis (GC– respect to authenticity and determination of chemical components
MS), capillary electrophoresis-diode array detection (CE-DAD), by analytical techniques, especially on the use of chemometrics in
HPLC–MS and HPLC–nuclear magnetic resonance (NMR) might combination with chromatographic fingerprint for quality evalua-
give the additional spectral information which may be helpful for the tion based on active ingredients.
qualitative and quantitative analysis and even for the structural
elucidation. With spectral information, the hyphenated instruments
show greatly improved performances in terms of the elimination of the 2. Origin and development of chemometrics
instrumental interferences, retention time shift correction, selectivity,
chromatographic separation abilities, measurement and precision. A Swedish scientist Svante Wold in 1971 coined term “kemometri”
Since complicated data from these techniques cannot be eval- in Swedish form and its English equivalent is “chemometrics”
uated easily, we need data processing techniques for this purpose. [11]. In year 1986 and 1987 two journals were started named as
These techniques provide a good opportunity for mining more “Chemometrics and Intelligent Laboratory Systems” and “Journal
useful chemical information from original information-rich data. of Chemometrics”, which promoted equipment intellectualization
Chemometrics is the application of mathematical and statistical and offered new methods for the construction of new and high-
techniques to retrieve more information from the chromatographic dimensional hyphenated equipment. These hyphenated equipments
data [4–6]. The International Chemometrics Society (ICS) defines have opened many new options for data analytical method improve-
chemometrics as the science of relating measurements made on a ment. Now, chemometrics has emerged to have a major role within
chemical system or process to the state of the system via application the analytical chemistry [12].
of mathematical or statistical methods [7].
It promotes equipment intellectualization and offers new ideas
and methods for the construction of new and high-dimensional and 3. Herbal drugs chromatographic fingerprinting
hyphenated equipments. Furthermore, with the rapid development
of the microcomputer and retrieval of the analysis of high- Herbal drugs may contain a large number of compounds even
dimensional data, artificial neural networks, research of artificial though many of them are present in low concentrations but they
intelligence of chemistry and expert systems have made great may be important in terms of quality, safety and efficacy of the
progress. With the development of analytical chemistry, chemo- herbal medicines because they show synergetic effects and their
metrics is also being developed vigorously [8,9]. therapeutic actions are based on interaction between numerous
With the advance of computer technology, chemometrics constituents. In order to evaluate the complete pattern of herbal
methods have become a leading tool among the scientific com- medicine, chromatography offers a powerful tool for separating the
munities towards faster analysis results and shorter product individual components and developing a characteristic profile of
development time. Among others, an unsupervised pattern recog- the sample, called a fingerprint. This section basically deals with
nition technique such as principal component analysis (PCA) is the different chromatographic techniques used for herbal fingerprint-
most often used method for handling multivariate data without ing and their advantages and disadvantages.
prior knowledge about the studied samples. While the supervised
classification procedure using soft independent modeling of class 3.1. Thin layer chromatography (TLC)
analogy (SIMCA) based on making a PCA model to assign
unknown samples into the predefine class model has also been TLC is a technique used for fast screening of samples to identify
applied to the analysis. Thus, the application of chemometrics herbal products and to differentiate between herbal species [13–16].
techniques will greatly improve the quality of the fingerprint One of the major advantages of TLC is its tractability to optimize
obtained (Fig. 1) [4,9,10]. operational parameters, such as the sample application, plate

Fig. 1 Schematic interpretation of complicated data comes from various hyphenated instruments by various chemometrics techniques.
Chemometrics in herbal drug standardization 225

development and derivatization. The main disadvantages of TLC by using solid phase particles of less than 2 mm in diameter to
in the analysis and quality control of HDs are its low reproduci- achieve superior sensitivity and resolution. Smaller particle size
bility and resolution [16–18], need for high compound concentra- leads to higher separation efficiency and shorter columns size leads
tion for detection [16] and the semi-quantitative nature of the to shorter analysis time with little solvent consumption [35].
technique. The main factors that are hard to control in TLC and Within a period of last few years, UHPLC fingerprints of herbal
affect its precision include sample spotting, saturation of the products were developed instead of conventional HPLC approach
developing chamber and the instability of color appearance when [36,37]. In comparison to HPLC, UHPLC analyses reported a
coloring reagents are used for detection [16]. Nevertheless, TLC is decreased analysis time by a factor up to eight without loss of
still being used as it is a promptly available, easy to use and information [38,39]. The results obtained not only showed
economical technique and recent developments and modifications decreased analysis time but also proved a great enhancement in
of the technique remarkably improved its reproducibility, resolu- selectivity compared to conventional HPLC analysis.
tion and sensitivity.
Digital scanning and documentation software have improved 3.4. Hydrophilic interaction chromatography (HILIC)
the applicability of TLC for the comprehensive identification and
assessment of HDs resulting in a more adequate extraction of the HILIC has gained attention in herbal fingerprinting because of
information. In addition, high performance thin layer chromato- good separation quality of hydrophilic compounds. Many of polar
graphy (HPTLC) by using a smaller particle size range (5–20 mm) compounds of herbal medicines are extracted by using aqueous
and automation of different steps increase the reproducibility and solution which might be better separated by means of HILIC [40].
resolution [13,16,17,19]. Micro-emulsion TLC (ME-TLC) devel- HILIC was introduced as an alternative for normal-phase liquid
opment leads to increased separation efficiency and signal chromatography (NPLC); HILIC enables the separation of polar
enhancement and resulting in an improved sensitivity [16]. Other compounds on polar stationary phases with aqueous mobile
accepted modifications in herbal analysis include TLC densito- phases. It is based on the principle of partitioning between a
metry [20], two-dimensional TLC (2D-TLC) [21,22] and coupled- water-enriched layer in the hydrophilic stationary phase and a
layer planar chromatography (graft-TLC) [22]. relatively hydrophobic mobile phase usually containing 5–40%
water in organic solvent. This technique is more eco-friendly as
3.2. High performance liquid chromatography (HPLC) compared to NPLC because of the use of water and polar organic
solvents as mobile phase. In addition, the polar compounds are
HPLC is the most popular analytical technique used in HDS more soluble in the mobile phase of HILIC [40,41].
[23–25]. HPLC is easy to operate and a fully automatable technique As HILIC is a relatively recent technique, few papers analyzing
with high rates of precision, resolution, selectivity and sensitivity. herbal products have been published yet. Most papers usually
One of the main advantages of HPLC is the possibility to make describe a methodology exploiting the orthogonal character of the
hyphenation with different detectors such as ultra violet (UV) for UV HILIC and reversed-phase liquid chromatography (RPLC) methods
absorbing compounds [25–27], and diode array detector (DAD) for for quality control [41,42].
herbal fingerprinting [24,28,29], evaporative light scattering detector
(ELSD) [27] and chemo luminescence detectors (CL) for non-UV 3.5. Gas chromatography (GC)
absorbing compounds, NMR for metabolomic profiling [30] and
mass spectrometry (MS) for identification of the separated com- GC is a well established analytical technique commonly used for the
pounds [28,31,32]. characterization, quantization and identification of volatile com-
However, the main drawback in HPLC is the requirement of pounds. The powerful separation efficiency and sensitive detection
expensive machinery, large volumes of environmentally unfriendly make GC a useful tool for the analysis of essential oils [43]. Despite
liquids, the undetected co-elution of compounds and the susceptibility its advantages, GC analysis of herbal products is usually limited to
of conventional (silica-based) columns to relatively basic (pH49) or the essentials oils because of possible degradation of thermo-labile
acidic (pHo2) mobile phases and high temperatures. An important compounds and the requirement of volatile compounds makes GC
factor in fingerprint development is the selection of stationary phase. unsuitable for many herbal compounds [44].
Commonly used stationary phases for herbal fingerprinting are particle- The hyphenation of GC–MS leads to reducing analysis times of
based C-8, C-12 and C18 columns, and efficiency of these columns is essential oils (40–100 s) as well as decreased detection limits. GC–MS
dependent on their particle size (currently 3–5 mm). Smaller particle analysis of essentials oils, showing faster analysis and high efficiency,
size drastically increases the backpressure: when the particle size is made use of micro-bore capillary columns [45] with reduced stationary
halved, the pressure quadruples. As the current HPLC instrumentation phase film thickness (10 m  100 mm I.D. and 5 m  50 mm I.D.) with
can withstand up to 6000 psi, higher efficiencies and shorter separation rapid temperature programming (20 1C/s), fast data acquisition by FID
times by decreasing particle size and increasing the flow rates are and high split ratio. Finally, low-pressure GC–MS using mega-bore
limited [33,34]. analytical columns (10 m  530 mm with 0.25–1 mm film thickness)
was investigated on the essential oils and led to a slightly reduced
3.3. Ultra-high performance liquid chromatography (UHPLC) efficiency but manifold decreased analysis times [46,47].

In recent years, UHPLC has been emerging as a feasible technique 3.6. Two-dimensional (2D) chromatography
for the quality control of herbal products. UHPLC can withstand a
pressure of at most 8000 psi and it brings liquid chromatographic Before going to 2D chromatography, firstly one has to under-
analysis to another level by hardware modifications of the stand the difference between multi-dimensional fingerprint gener-
conventional HPLC machinery [35]. UHPLC makes it possible ated by hyphenated detection techniques and 2D chromatography.
to perform high resolution separations superior to HPLC analysis In multi-dimensional fingerprinting, the hyphenated detector
226 A. Bansal et al.

collects information from the eluting compounds while a finger- resolution and extensive applicability. LC–MS, GC–MS, and LC–
print is recorded. In 2D chromatography, fractions eluting from a NMR have been increasingly used in complex chemical identifica-
first chromatographic system are chromatographed on a second tion of HDs [52]. This advancement in instrumentation is able to
system having different separation properties resulting in increased generate enormous amounts of data which record small differences
the peak capacity of entire separation (Fig. 2). between samples and this enables us to provide large implications
To reveal all characteristics of complex HDs, 2D chromatogra- for the discrimination of herbal plants.
phy was proposed. With the development of 2D chromatographic So we discuss here application of chemometrics in the analysis of
systems, a new era in herbal fingerprinting has been clicked. The herbal drugs. But before analyzing data, pretreatment of data is
main advantage of 2D chromatography over conventional one- essential because unknown components or unclear interferences cause
dimensional chromatography is to obtain high peak capacity which overlapped peaks and shifted baseline. Thus, we discuss here various
theoretically equals to the product of the peak capacities of two commonly used chemometric techniques in herbal drug standardiza-
individual dimensions. However, a major limitation is very long tion (HDS) such as PCA, linear discriminate analysis (LDA), spectral
time span needed to reach this maximum capacity [48]. correlative chromatography (SCC), information theory (IT), local
Literature review reveals that 2D chromatography has been used least square (LLS), heuristic evolving latent projections (HELP) and
with different techniques such as 2D TLC [21], 2D HPLC [49], 2D orthogonal projection analysis (OPA).
GC [50] and 2D chromatography combining size-exclusion and
RPLC [51]. 4.1. Tools for preprocessing data

To eliminate or reduce unwanted sources of variations due to

4. Common chemometric tools used in chemical analysis instrumental responses from hyphenated techniques and to obtain
of herbal drugs more efficient results, data preprocessing techniques are applied.
Commonly applied preprocessing techniques include LLS and
As is known, HPLC is extensively applied for quality control normalization. To obtain accurate quantitative results from the
of herbal drugs due to its sensitivity, superior precision, high fingerprint, pretreatment of chromatographic data is necessary.

Fig. 2 3D data obtained from 2D LC/MS (HPLC–HPLC–TOF/MS) and resolution of data by employing PCA and HELP.
Chemometrics in herbal drug standardization 227

4.1.1. Normalization chromatogram. The more they separate with uniform concentrations,
Variation in sample concentration might affect the multivariate the higher the value of information contents, i.e., more chemical
analysis of the entire chromatographic profile. So normalization of information can be obtained from this chromatogram. Information
data is examined before carrying out multivariate analysis in the content Φ of a chromatogram is calculated using following equation:
following way: each chromatogram consists of N peaks with each Z
px log ðpx Þ
peak area ci of the ith component being utilized. The total area of Φ¼  dx ð3Þ
sumðpx Þ sumðpx Þ
all peaks can be calculated by
N
where px is the real chromatographic response of all chemical
C ¼ ∑ ci ð1Þ components involved in the chromatogram. By comparing the
i¼1 magnitude of information content, maximal chemical information
So the peak area after normalization is as follows: under certain chromatographic conditions that include all extrac-
ci tion and detection parameters can be found out [56].
ci ¼ ð2Þ
C
4.3. Tools for resolution of mixtures
Then, each peak is expressed as percentage of the sum of peak
areas [53].
The main goal in the analysis of any multicomponent system is to
get useful information from the raw experimental data and
4.1.2. Local least square (LLS) method
knowledge of the number of chemical components in the analyzed
Major problem in analyzing HDs is occurrence of signal shift.
sample, especially in herbal drugs (Fig. 2). The common purpose
It causes a significant influence in chromatographic profile. To
of all resolution methods is to provide the linear model of
eliminate the chromatographic shift, the LLS method is used to
individual component contributions using solely the raw experi-
correct retention time shift. When we are using pattern approach,
mental measurements. HELP and OPA are two important and
then it is necessary to correct retention time in all chromatograms
commonly used multivariate tools.
unless it causes serious problem. To match all chromatographic
profiles with their retention times, all common constituents will be
selected and utilized for chromatographic alignment [54]. 4.3.1. HELP
HELP is a multivariate resolution method used to resolve two-way
bilinear data into spectra and chromatograms of the pure consti-
4.2. Tools for extracting chemical information
tuents [57,58]. This method employs the feature of visual interface
from latent variable projection graph and also provides information
The aim of these methods is to detect similarities or extract useful of the local rank of the data matrix.
information from the data obtained from analytical instrument. This method employs the following steps:
These methods are important for finding out the interested analyte
in complex mixture of plant fingerprint and similarity analysis 1. To find out the total number of components by employing
(SA), SCC and IT are commonly used tools. singular value decomposition (SVD).
2. Further, eigenvalue plot is constructed which is plotted
4.2.1. SCC between logarithms of eigenvalues against retention time.
It is a technique used to identify the chemical component present 3. Then in the next step zero concentration regions and selective
in different chromatograms as acquired from hyphenated instru- concentration region of the compound are determined from
ment. It is based on the fact that the same chemical components eigenvalue plot. Selective concentration region is defined as
should have the same spectra no matter what or how they are the region in which only a single component elutes out i.e.,
eluted through diverse chromatographic columns. The spectral having rank equal to one and zero concentration region in
information is utilized to pick up the targeted component from the which no component elutes out.
other two-way chromatograms. The procedure for carrying out 4. Then evolving latent projection (ELP) graph gives us
SCC is given in the following steps: information about the selective region of the individual
component in wavelength as well as retention time space.
1. Assess peak purity of targeted component and obtain its UV Such region can be identified by passing straight lines in ELP
or MS spectrum from the chromatogram. graphs.
2. Identify this component in the chromatogram of interest 5. In the end, spectra and chromatogram of individual compo-
through comparison by a series of spectrum at each scan nents can be resolved through simple mathematic computa-
point of other chromatogram by their correlation coefficients. tion
3. Get a curve of the correlation coefficient versus scan point in
S ¼ ðC T CÞ  1 CT X ð4Þ
the direction of retention time and further validate step 2 with
consideration of the information in the local chromatographic where X represents data matrix generated by hyphenated
cluster where the target exists. system with total rows representing spectral profile S and
columns showing chromatograms C.
The result obtained by SCC is highly accurate in the case of MS
because the mass spectrum provides uniqueness compared to the
UV spectrum [55]. 4.3.2. OPA
OPA is a stepwise process and selects one key variable in each
4.2.2. Information theory (IT) step [59,60]. This method calculates dissimilarity based on the
The value of information content depends on the separation degree mathematical concept of orthogonalisation [61,62]. The method is
and concentration distribution of each chemical component in a based on the fact that pure spectra are extreme spectra and will
228 A. Bansal et al.

encompass the mixture spectra. OPA compares each spectrum with where X is a given data matrix, ui and pi are score and loading
one or more than one reference spectra and searches for the least vector, respectively and orthogonal to each other. Generally score
correlated spectrum. The first dissimilar plot represents a compar- matrix (U) gives the relationship between samples while loading
ison of each spectrum with the average spectrum. matrix (P) shows the importance of each variable (Fig. 3). When a
The procedure of OPA consists of three steps fingerprint with unexpected features that differ from those of
majority fingerprint is encountered, it will be analyzed differently
1. Comparison of each spectrum of data matrix X with spectra
and displayed as outlier. Their difference in score plot can be
used in equation
expressed by a measure of distance like euclidean distance (EDA).
d i ¼ detðY Ti Y i Þ i ¼ 1; 2………m ð5Þ qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
where dissimilarity of the ith spectrum, di, is defined as the EDA ¼ ðxtest xA Þðxtest xA ÞT ð7Þ
determinant of the dispersion matrix of Yi. In general,
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
matrices Yi consist of one or more reference spectra and the
EDB ¼ ðxtest xB Þðxtest xB ÞT ð8Þ
spectrum measured at the ith analysis time.
2. Plotting of the dissimilarity plot between dissimilarity values xA and xB are the mean of training set for herb A and herb B,
and analysis time. respectively [64,66].
3. Then in the last step selection of the spectrum with the
highest dissimilarity value by including it as reference in
matrix Yi. 4.4.2. LDA
LDA is a supervised pattern recognition method. It seeks a linear
4.4. Tools for displaying data function of the variable in multivariate space which maximizes the
ratio between both variances compared to the within-group
These tools are useful to resolve net analytical signal by variance. Since multi-dimensional data arise when the number of
discovering the dominant factors while excluding the relevant variables larger than the number of observations, then we cannot
interference, thus giving a more accurate estimate. These include use LDA directly. In that case PCA is employed to compress data
various methods such as LDA, hierarchical clustering analysis and to transform the original dataset comprising of a large number
(HCA), SIMCA and PCA, but among these PCA and LDA are of inter-correlated variables into a reduced new set of variables.
found to be most commonly used. LDA makes a discriminate function for each group according to
this equation:
4.4.1. PCA n
The central idea of PCA is to reduce the dimensionality of a data set FðGi Þ ¼ k i þ ∑ wij pij ð9Þ
j¼1
consisting of a large number of interrelated variables, while keeping
maximum variation in the data set. This is accomplished by where i is number of groups (G), ki is the constant of each group, n
transforming to latent variables (Fig. 3), the principal components is number of parameters used to classify a set of data into a given
(PCs) that are uncorrelated and ordered so that the first few PCs group and wj is weight coefficient, assigned by LDA to a selected
retain most of the variation in all of the original variables [63,64]. parameter (pi) [67,68].
PCA [63,65], a multivariate tool, is used to find out the main
source of variability present in the data sets. It is used to detect
cluster formatting and to establish relationship between object and
variable. The sample variation in data is given in terms of latent 5. Application of chemometrics in HDS
variable as given below:
A
We will describe the application of chemometrics in HDS by
X ¼ UPt ¼ ∑ ui pti ð6Þ giving detailed examples regarding authenticity, efficacy and
i¼1 chemical analysis of HDs.

Fig. 3 Interpretation of 3 dimensional data by using first 2 principal components where score vector ui gives grouping in data sets and loading
vector pi direction of variability in data sets.
Chemometrics in herbal drug standardization 229

5.1. Authenticity classification and discrimination of these fruit samples than the
UV/vis spectra [83]. Herbs (mint, thyme and rosemary) and spices
Ascertaining authenticity is the important step to assess the quality of (black pepper, chili pepper, cinnamon, cumin, sweet red pepper
plant medicine. Each medicinal plant contains certain characteristic and turmeric) were analyzed using atomic spectrometry. Principle
constituents. Therefore, constituents including their respective che- component analysis and HCA classified the samples into five
mical ratios can be analyzed to identify medicinal plants and groups and LDA was used to show how these group members
distinguish the fakes. were correctly classified with regard to the original group [84].
The analysis of fingerprint of 46 Cassia seeds samples was Ultra-performance liquid chromatography-quadrupole time-of-
carried out by applying chemometric methods at two wavelengths flight mass spectrometry (UPLC–QTOF/MS) and multivariate
of HPLC–UV. Samples were clustered into four groups according statistical analysis were used to investigate the processing technol-
to the plant sources and preparation procedures. Chemometric ogy of Loquat (Eriobotrya japonica) leaf (pipaye, PPY). The
tools were effectively applied to predict the category of the four differences in samples processed under different methods were
different samples in the test set [69]. Several studies concerning revealed by unsupervised PCA [85].
the quality control of Camellia sinensis have been undertaken. HPLC-DAD–MS method has been developed and compared
The total antioxidant capacity of C. sinensis extracts is considered with the fingerprints of another non-official species Cistanche
as an important quality criterion. Different regression models such salsa and Cistanche sinensis with the official one Cistanche
as PCA and OPA were applied for the prediction of total deserticola. It is found that C. salsa has high similarity with the
antioxidant capacity using chromatographic fingerprints [70–72]. standard while C. sinensis is suggested that C. salsa may be used
Ultra-high performance liquid chromatography–electrospray as an alternative species [86]. Notopter giumincium having
ionization-mass spectrometry (UHPLC–ESI-MS) was utilized for complex fingerprint obtained by GC–MS is resolved by using
metabolite profiling of saponins to discriminate Panax notogin- OPA [87]. A similar study was carried out for Artemisia
seng parts in order to classify them according to their phytochem- capillaries with the aid of a fixed size moving window evolving
ical diversity and saponins accounting for such variations were factor analysis (FSMWEFA) [88]. Several studies were conducted
identified through the loading plots of PCA [73,74]. for quality control of the herb Houttuynia cordat utilizing
The high performance liquid chromatography-diode array FSMWEFA, PCA and subwindow factor analysis (SFA) [89–91].
detector (HPLC-DAD) method was used to determine 10 triterpe-
noid acids simultaneously. The present HPLC-DAD method,
combined with chemometrics, was demonstrated to be very helpful
in searching Ziziphus jujuba resources and possibly useful in 5.2. Efficacy and consistency
chemotaxonomic characterization [75]. Data of HPLC analyses
of Ocimum americanum, O. basilicum, O. Citriodorum and The efficacy of plant medicines is closely related to their chemical
O. Minimum were utilized for authentication purposes to determine constituents and their concentrations. Consistency may slightly
the surface flavonoid of different basil cultivar by applying PCA vary according to differences in climate, cultivating and harvest
[76,77]. Atractylis chinensis is a medicinal plant used for establish- time, possessing procedure and storage.
ment of HPLC fingerprints together with the metal profile which Evaluating the recorded fingerprints, minor differences in
was used to assess the quality procedures and data were evaluated concentrations might influence the quality of the herbal plant
by using PCA and LDA. The results obtained showed that the while small differences between the fingerprints can discriminate
samples were discriminated on the basis of the processing methods among species. PCA, SSC and LDA can classify and discriminate
[78]. HPLC-DAD method was developed to evaluate the quality of medicinal plants fingerprints effectively. Fingerprint analysis of
Receptaculum Nelumbinis (dried receptacle of Nelumbo nucifera) Chrysanthemum morifolium was developed by combining chemo-
through establishing chromatographic fingerprint and simultaneous metric methods such as SA, HCA, and PCA with ultra-performance
determination of five flavonol glycosides, including hyperoside, liquid chromatography [92].
isoquercitrin, quercetin-3-O-β-D-glucuronide, isorhamnetin-3-O-β- Ultra-performance liquid chromatography with photodiode array
D-galactoside and syringetin-3-O-β-D-glucoside [79]. To evaluate detector (UPLC-PAD) method for quantitative analysis of five active
the quality consistency of commercial medicinal herbs, a simple alkaloids of Rhizoma coptidis has been developed. For the classifica-
and reliable HPLC method with a UV/vis detector was developed, tion of samples, chromatographic data were analyzed by SA, PCA
both for fingerprint analysis and quantitation of some pharmaco- and HCA. This method, in combination with chemometrics, demon-
logically active constituents. Melissa officinalis L. (lemon balm) strated to be very helpful in the quality control and evaluation of
was chosen for this study because it is widely used as an aromatic, R. coptidis [12]. PCA, SIMCA and HCA were applied to HPLC
culinary and medicine remedy [80]. fingerprint of Epimedium wushanenseas in order to identify and
Fourier transform infrared (FTIR) and chemometrics have distinguish their secondary metabolites [93,94]. Differentiation of
been used to analyze adulteration in cod liver oil (CLO). This Teucrium flavum was carried out by GC–MS and then investigation
method successfully allowed one to make a classification of pure of their chemical and genetic profiles was carried out by utilizing
CLO and adulterated CLOs with vegetable oils [81]. HPLC application of PCA and cluster analysis (CA) as classification
fingerprint analysis followed by PCA and discriminate analysis methods. The obtained results showed that the genetic background
(DA) provided good discrimination of Cinnamomum cassia bark was solely responsible for differences in the volatile oil composition
and twings samples and cinnamaldehyde was found to be the most [95]. In another study, differentiation and prediction of cultivation age
abundant marker component [82]. A simultaneous fingerprinting using ginseng samples cultivated under standardised protocols or
analysis of some kiwi and pomelo fruits has been performed by guidelines were investigated by NMR-based metabolomics techni-
employing HPLC and UV/vis spectroscopy, followed by applica- ques using various solvents to develop a differentiation method for
tion of chemometric tools such as cluster analysis, PCA and LDA ginseng cultivation age. The PLS-DA method has successfully
for data analysis. The digitized chromatograms provided a better differentiated the 1–6 years old ginseng root samples [96]. A method
230 A. Bansal et al.

for chemical fingerprint analysis of Hibiscus mutabilis leaves was [4] K. Wiberg. Multivariate Spectroscopic Methods for the Analysis of
developed by using UPLC-PAD combined with SA and HCA [97]. Solutions, Akademitryck AB, Edsbruk, Stockholm, 2004.
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sensitive determination of parabens and p-hydroxybenzoic acid in
Ankit Bansal is grateful to AICTE, New Delhi and PSCST, Punjab waters by non-aqueous capillary electrophoresis, Anal. Chim. Acta
(Project PSO/POS/80/9334) for financial support. Authors are also 647 (2009) 104–111.
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