Determination of Anomalin and Deltoin

in Seseli resinosum by LC Combined

with Chemometric Methods
2007, 66, 677683

zlem Bahadr1, Erdal Dinc2,&

Alev Tosun1, O
1
2

an, Ankara, Turkey

Department of Pharmacognosy, Faculty of Pharmacy, Ankara University, 06100 Tandog
an, Ankara, Turkey;
Department of Analytical Chemistry, Faculty of Pharmacy, Ankara University, 06100 Tandog
E-Mail: dinc@pharmacy.ankara.edu.tr

Received: 10 May 2007 / Revised: 20 July 2007 / Accepted: 7 August 2007

Online publication: 21 September 2007

Abstract
Liquid chromatographychemometric methods [LC-Partial least squares (LC-PLS), LC-principle
component regression (LC-PCR) and LC-artificial neural network (LC-ANN)] were developed for
the determination of ( )-anomalin (ANO) and deltoin (DEL) in the root and aerial part of Seseli
resinosum Freyn et Sint. (Umbelliferae). Firstly, chemometric conditions were optimized by testing
different mobile phases at various proportions of solvents with various flow rates in different
wavelengths by using a normal phase column to obtain the best separation and recovery results.
As a result, a mobile phase consisting of n-hexane and ethyl acetate (75:25 v/v) at a constant flow
rate of 0.8 mL min 1 on the above column system at ambient temperature were found to be the
optimal chromatographic condition for good separation and determination of ANO and DEL in
samples. Multichromatograms for the concentration set containing ANO and DEL compounds in
the concentration range of 50400 ng mL 1 were obtained by using a diode array detector
(DAD) system at selected wavelength sets, 300 (A), 310 (B), 320 (C), 330 (D) and 340 (E). Three
LC-chemometric approaches were applied to the multichromatographic data to construct chemometric calibrations. As an alternative method, traditional LC at single wavelength was used for
the analysis of the related compounds in the plant extracts. All of the methods were validated by
analyzing various synthetic ANODEL mixtures. After the above step, traditional and chemometric LC methods were applied to the real samples consisting of extracts from roots and aerial
parts of S. resinosum. The results obtained by LC-chemometric approaches were compared to
each other and with those obtained by traditional LC.

Keywords
Column liquid chromatography
Multichromatographic data
Chemometric-LC calibration
( )-Anomalin and deltoin

Introduction
The genus Seseli L. is represented by 12
taxa (11 species and one subspecies) in

Original
DOI: 10.1365/s10337-007-0392-6
0009-5893/07/11

the Flora of Turkey. Seseli resinosum

(Umbelliferae) is a perennial and endemic
species growing in northern Anatolia,
Turkey [14]. Coumarin derivatives

found in Seseli genus have some pharmacological eects. Their most important
activities are anticoagulant, antitumoral,
immunomodulating, and antiinammatory eects [5]. ( )-Anomalin (ANO)
and deltoin (DEL) in our study are
categorized into coumarin derivatives
isolated from the n-hexane extract of
the root of S. resinosum [6]. The molecular structure of ANO and DEL corresponding to (I) and (II), are shown in
Fig. 1.
No determination of ANO and DEL
in the same mixture was observed in the
literature until now. For the above reasons, the quantitative analysis of the
investigated ANO and DEL is a very
important task for future studies.
Generally, a traditional LC method
has been used for the quality control of
commercial products and their routine
analysis in laboratory, and for the determination of some active compounds in
plant extract samples. However, new
combined analytical methods called
hyphenated instrumentations such as LC
DAD and LCMS, etc., have been used
for the quantitative analysis of two or
more active compounds in complex mixtures. In some cases, the above modern
analytical methods may not give desirable
results for the multicomponent determination of compounds in samples due to
interference and matrix eects or other
environmental factors on the analysis.
Taking into account all above arguments,
the analysis of complex mixtures needs
new analytical approaches based on the

Chromatographia 2007, 66, November (No. 9/10)

2007 Friedr. Vieweg & Sohn Verlag/GWV Fachverlage GmbH

677

simultaneous use of classical analytical

methods (LC, LCDAD and LCMS,
etc.) and powerful mathematical algorithms (PLS, PCR and ANN, etc.) to
obtain more accurate and precise results.
In the previous studies, the combined use
of LC and chemometric calibrations have
been used for the resolution of mixture
systems [7, 8].
In this study, the quantitative analysis of ANO and DEL in the extracts of
root and aerial part of S. resinosum was
performed by the proposed LC-PLS,
LC-PCR and LC-ANN approaches. As
a comparison method, traditional LC
was applied to the quantitative analysis
of the extracts containing ANO and
DEL compounds. The same chromatographic conditions were used for both
traditional LC and chemometric LC
methods. All of the proposed analytical
methods were subjected to the synthetic mixtures and real samples (plant
extracts) of the ANO and DEL
compounds. The determination results
provided by the traditional and chemometric LC methods were compared to
each other. It was observed that the
relative standard deviation (RSD)
values obtained by LC-chemometric approaches became smaller than those
obtained by traditional LC calculations
for recovery studies and real sample
analysis. These RSD values indicate that
the simultaneous use of chemometric
calibration and LC methods is an
appropriate
approach
to
provide
successful results.

Experimental
LC-Chemometric Methods
In this investigation, the multichromatograms of ANO and DEL at the ve
wavelengths via DAD system are plotted
and stored in a computer. The detector
responses at the multiwavelengths are
measured in terms of peak area. The recorded multichromatographic data corresponding to the concentration and
sample sets are transferred into Microsoft
EXCEL and are processed by three different chemometric calibration methods,
PLS, PCR and ANN mathematical
algorithms. The theoretical details of the
application of the chemometric approaches to the multichromatographic
data are explained below.

678

LC-PLS Method

The PLS calibration using the orthogonalized PLS algorithm developed by

Wold [9, 10] and extensively discussed by
Martens and Naes [11] involves simultaneously the independent and the dependent variables on the data compression
and decomposition operations.
In the LC data analysis, the LC-PLS
calibration is done by decomposition of
both concentration and the ratio of peak
area matrix into latent variables, R = T
PT + E and C = U QT + F. The linear
regression Cprediction = b Rsample, is
used for the estimation of the compounds
in the samples. The vector b is given as
b = W (PT W) 1 Q, where W is a
weight matrix. Application of this method was performed by means of PLS
toolbox 3.0 in Matlab 7.0 software.
LC-PCR Method

In the application of the LC-PCR, the

peak areas of the concentrations sets of
the related compounds were reprocessed
by mean-centering as Ro and Co, respectively. The covariance dispersion matrix
of the centered matrix Ro was computed.
The normalized eigenvalues and eigenvectors were calculated starting from
square covariance matrix. The number
of the optimal principal components
[eigenvectors (P)] is selected by considering only the highest values of the eigenvalues. The other eigenvalues and their
corresponding eigenvectors are eliminated. To reach this objective, the coecient b dened as b = P q is calculated,
where P is the matrix of eigenvectors and
q is the C-loadings given by q =
D TT Ro. TT is the transpose of the
score matrix T, and D is a diagonal matrix having the inverse components of the
selected eigenvalues. The content of
compounds in samples were evaluated by
using the Cprediction = b Rsample. PLS
toolbox 3.0 in Matlab 7.0 software was
used for data treatment.

LC-ANN Method

ANN serves as powerful computational

tool in a diversity of applications including classication, pattern recognition,
function approximation, and the modeling of biological neural networks. When
equipped with procedures for learning
from examples, ANN can solve problems
for which no algorithmic solution exists.

The ANN methodology is based on the

attempt to model a biological from a
classical statistical method for analysis.
Neural networks need less formal statistical training and can detect complex
non-linear relationships between dependent and independent variables as well as
possible interactions without complicated
equations, and may use multiple training
algorithms. In addition, ANN can combine and incorporate both literaturebased and experimental data to solve
problems.

Instrumentation
and Chromatography
A Shimadzu UV-160 double beam UV
Vis spectrophotometer possessing a xed
slit width (2 nm) connected to a computer loaded with Shimadzu UVPC
software was used to record the absorption spectra.
Chromatography was performed with
an Agilent 1100 series HPLC system (Agilent Technologies, CA, USA) provided
with a quaternary pump, a thermostatted
autosampler, a thermostatted column
compartment, and a multiwavelength
diode array detector (DAD). Data
were acquired and processed using HP
Chem Station for LC [Rev. A0.01 (403)]
(Agilent). Waters Spherisorb S5W
(25 cm 4 mm 5 lm) column was used
for the chromatographic separation. The
ow rate was maintained at 0.8 mL min 1
and the injection volume was 10 lL. The
mobile phase was prepared daily and ltered through a 0.45 lm membrane lter.
In fact the same chromatographic
conditions were used for both, traditional
and LC chemometric analyses.

Standard Solutions
Stock solution of ANO and DEL
(50 mg 50 mL 1 for each compound)
were prepared with n-hexane and ethyl
acetate (50:50 v/v). A concentration set of
mixture solutions containing both compounds in the range of 50400 ng mL 1
was obtained from the above stock solutions. A validation set consisting of six
synthetic mixture solutions of ANO and
DEL in the above concentration range
was prepared. All the solutions were
prepared freshly and protected from
light.

Chromatographia 2007, 66, November (No. 9/10)

Original

Sample Collection
and Preparation for Analysis

O
O

O
O

(I)

(II)

Fig. 1. Molecular structures of ANO (I) and DEL (II) compounds

1.6
1.4
1.2

Abs.

Seseli resinosum Freyn et Sint. (Umbelliferae) was collected from Cakraz-Bartin

in July 2000 at an altitude of 05 m during
the owering season. The identication of
voucher specimen was performed by Prof.
H. Duman (Gazi University, Faculty of
Science and Letters) and the sample was
deposited at the Herbarium of the Faculty
of Pharmacy at Ankara University (AEF
21696).
Dried roots and aerial parts of S.
resinosum were pulverized and extracted
under reux with n-hexane. The n-hexane extract were evaporated to dryness
under vacuum to obtain crude extract.
The amount corresponding to 0.6349
and 0.2537 g crude extracts of roots (a)
and aerial parts (b) were weighed and
transferred into 25 mL calibrated asks.
After this procedure, the transferred
contents of the asks were dissolved in
n-hexane and ethyl acetate (50:50 v/v).
The solutions of (a) and (b) were diluted
to the working concentration range by
using the above mentioned solvent system. Finally these solutions were injected
into the LC column system for the
quantitative analysis of ANO and DEL
compounds.

1.0
0.8
0.6
0.4

Results and Discussion

Method Optimization
and Improvement
Absorption spectra of ANO and DEL in
the mixture of n-hexane and ethyl acetate
(50:50 v/v) are presented in Fig. 2. As can
be seen from Fig. 2, their absorption
spectra overlap in the same spectral
range. Quantitative analysis of ANO and
DEL is not possible by spectrophotometric methods due to the above reason
and the matrix eect coming from plant
extracts. For these reasons, we focused
mainly on the LC analysis in combination with chemometric calibrations for
the determination of the subjected compounds in the samples.
In the chromatographic studies, the
simultaneous registration of multiple
chromatograms at multi-wavelength sets
is obtained by LC-DAD. In addition, the
multivariate LC data corresponding to
multiple chromatograms is used to construct the LC-chemometric calibration
[7, 8]. As in traditional LC at single
Original

0.2
0.0
240

260

280

300

320

340

360

380

Wavelength (nm)
Fig. 2. Absorption spectra of ANO () and DEL () compounds in the mixture of n-hexane and
ethyl acetate (50:50 v/v)

wavelength, the optimization of chromatographic conditions such as column

temperature, ow rate and composition
and variety of mobile phase plays a very
important role to provide a good separation and determination, and to reach
the best recovery values in the application
of the LC-chemometric calibrations.
The basic aim of the application of
chemometric calibrations (PCR, PLS and
ANN) to the multivariate LC data is to
eliminate or reduce the errors coming
from sample injection, peak uctuation in
the column system and experimental
environment that aect the peak area. To
obtain calibration graphs, LC-chemometric calibration based on the measure-

ment at multi-wavelength sets does not

require the selection of a specic wavelength while the traditional LC method
needs the selection of a specic single
wavelength.
In our case, various chromatographic
conditions were tested to nd the optimal
separation conditions to obtain desirable
determination results. As a result, a good
chromatographic separation of ANO and
DEL was carried out at ambient temperature on a Waters Spherisorb S5W
(25 cm 4 mm 5 lm) by using a mobile phase containing n-hexane and ethyl
acetate (75:25 v/v) at a ow rate of
0.8 mL min 1; 10 lL were used as injection sample volume during the chro-

Chromatographia 2007, 66, November (No. 9/10)

679

mAU
200

A, 1 =300 nm
B, 2 =31 0 nm
C, 3 =320 nm
D, 4 =330 nm
E, 5 =340 nm

175
150

mAU

A, 1 =300 nm

125

B, 2 =310 nm

100

75

50

25

C, 3 =320 nm

1600

D, 4 =330 nm

B
A

0
0

10

15

Concentration set 1

E, 5 =340 nm

25

20

Time (min)
mAU

A, 1 =300 nm
B, 2 =31 0 nm
C, 3 =320 nm
D, 4 =330 nm
E, 5 =340 nm

600
500
400

1400

300

E
D

200

1200

C
100

B
A

0
0

10

15

Concentration set 2

20

25

Time (min)

mAU

A, 1 =300 nm
B, 2 =31 0 nm
C, 3 =320 nm
D, 4 =330 nm
E, 5 =340 nm

1200
1000

1000
a

800
600

E
D

400

800

200

B
A

0
0

10

15

Concentration set 3

20

25

Time (min)
mAU

600

A, 1 =300 nm
B, 2 =31 0 nm
C, 3 =320 nm
D, 4 =330 nm
E, 5 =340 nm

1600
1400
1200

1000

800

600

400

400

C
B

200

0
0

10

15

Concentration set 4

20

25

Time (min)

200

A, 1 =300 nm
B, 2 =31 0 nm
C, 3 =320 nm
D, 4 =330 nm
E, 5 =340 nm

mAU
2000
1750
1500

1250
1000

750

500

250

Concentration set 4

B
A

0
0

10

15
Time (min)

Concentration set 5

20

25

10

15

20

25

Time (min)

Fig. 3. Multiple chromatograms of 300 ng mL

concentration set 1 5

matographic analysis. In fact the same

chromatographic conditions were used
for both traditional LC and LC-chemometric analyses.
We observed that sensitivity, accuracy
and precision of LC-chemometric calibrations increased when LC-PCR, LCPLS and LC-ANN approaches were
compared to the traditional LC method.

Multichromatographic Data
Processing
A concentration set of ve dierent mixture solutions of ANO and DEL in the

680

ANO a and DEL b in the concentration set 4. Small multiple chromatograms correspond to the

linear concentration range of 50400 ng

mL 1 was prepared for the chemometric
calibrations. The multiple chromatograms and their peak area at a vewavelength sets [300 (A), 310 (B), 320 (C),
330 (D) and 340 nm (E)] were plotted and
stored in a computer to be processed
chromatographically with traditional and
chemometric LC calibrations. In the
chromatographic separation, the retention times of ANO and DEL were observed as of 7.83 and 11.93 min,
respectively (see Fig. 3). Multiple chromatograms corresponding to the concentration sets given in Table 1 are presented
in Fig. 3. Multichromatographic data at a

ve-wavelength set were transferred into

Microsoft Oce Excel 2003 and then the
data of the peak area of the ANO and
DEL for both calibration and determination were normalized as 0 >  for
the construction of traditional and chemometric LC calibrations (see Table 1).
In the above procedure, x-values correspond to the peak area of compounds.
Normalized chromatographic peak area
was processed by using PCR, PLS and
ANN algorithms to construct LC-PCR,
LC-PLS and LC-ANN calibrations. The
determination of ANO and DEL in plant
extracts were carried out by the following
LC-chemometric calibrations.

Chromatographia 2007, 66, November (No. 9/10)

Original

Table 1. Concentration set and its corresponding normalized peak area data
Concentration set
1

Normalized peak area set of ANO

1

Normalized peak area set of DEL

ANO (ng mL )

DEL (ng mL )

300 nm

310 nm

320 nm

330 nm

340 nm

300 nm

310 nm

320 nm

330 nm

340 nm

50
100
200
300
400

50
100
200
300
400

0.1591
0.2873
0.4832
0.6991
1.0000

0.1618
0.3135
0.5260
0.8031
0.9694

0.1739
0.3109
0.5217
0.7957
0.9647

0.1775
0.3160
0.5296
0.8069
0.9779

0.1648
0.3069
0.5139
0.7824
0.9447

0.1661
0.2773
0.5832
0.7476
0.9799

0.1680
0.2860
0.6006
0.7683
0.9953

0.1725
0.2873
0.6029
0.7707
0.9974

0.1708
0.2841
0.5956
0.7602
0.9809

0.1751
0.2900
0.6073
0.7747
1.0000

Table 2. Linear regression analysis and its corresponding statistical results

Parameter

300 nm

310 nm

ANO
m
n
r
SE(n)
SE(m)
SE(r)
LOD (ng mL 1)
LOQ (ng mL 1)

DEL

2.33 10
3.65 10
0.9966
2.73 10
1.11 10
3.18 10
5.66
18.86

3
2

2
4
2

2.32 10
6.28 10
0.9956
3.09 10
1.26 10
3.60 10
4.07
13.57

320 nm

ANO
3
2

2
4
2

DEL

2.33 10
6.61 10
0.9967
2.69 10
1.09 10
3.13 10
7.84
26.14

3
2

2
4
2

2.36 10
6.71 10
0.9950
3.39 10
1.38 10
3.94 10
5.04
16.79

330 nm

ANO
3
2

2
4
2

DEL

2.29 10
7.28 10
0.9974
2.33 10
9.49 10
2.72 10
8.36
27.85

3
2

2
5
2

2.36 10
7.03 10
0.9950
3.37 10
1.37 10
3.93 10
11.31
37.69

340 nm

ANO
3
2

2
4
2

DEL

2.32 10
7.51 10
0.9974
2.36 10
9.59 10
2.75 10
9.02
30.06

3
2

2
5
2

ANO

2.32 10
7.11 10
0.9948
3.39 10
1.38 10
3.94 10
9.71
32.38

3
2

2
4
2

2.25 10
6.97 10
0.9970
2.49 10
1.01 10
2.90 10
6.43
21.45

DEL
3
2

2
4
2

2.36 10
7.33 10
0.9948
3.44 10
1.40 10
4.01 10
10.17
33.89

3
2

2
4
2

Table 3. Recovery data obtained by application of the proposed methods to the synthetic mixtures
Mixture
(ng mL 1)

Traditional HPLC
300 nm

ANO

50
200
400
200
200
200

DEL

200
200
200
50
200
400

ANO
Found
45.6
187.8
387.7
189.4
193.1
189.2

310 nm

DEL
(ng mL
191.1
192.9
193.2
46.4
194.7
403.3

Recovery (%)
91.1
95.6
93.9
96.5
96.9
96.6
94.7
92.8
96.6
97.4
94.6 100.8
Mean
SD
RSD (%)

94.6
2.10
2.21

ANN

96.6
2.61
2.70

ANO

320 nm

330 nm

PCR

PLS

340 nm

DEL

ANO

DEL

ANO

DEL

ANO

DEL

ANO

DEL

ANO

DEL

ANO

DEL

)
45.7
198.8
401.3
195.2
195.0
203.6

197.4
188.1
194.0
48.2
184.7
394.5

46.5
197.7
405.1
183.4
193.4
188.0

183.0
186.1
195.5
48.6
184.6
393.7

44.5
198.5
399.2
190.7
193.6
196.2

198.2
194.2
201.8
45.7
203.2
400.3

45.0
202.5
413.2
186.5
194.7
198.2

191.6
189.7
189.2
51.2
182.8
376.8

46.6
198.3
393.8
198.5
200.3
199.8

195.0
193.9
199.6
51.7
196.7
400.0

47.9
197.0
400.7
189.2
194.0
195.1

192.4
190.4
194.9
49.6
190.2
391.9

48.0
197.0
400.7
189.2
194.0
195.1

192.4
190.9
195.1
49.7
190.2
392.0

91.3
99.4
100.3
97.6
97.5
101.8

98.7
94.1
97.0
96.3
92.3
98.6

93.0
98.9
101.3
91.7
96.7
94.0

91.5
93.0
97.8
97.1
92.3
98.4

89.1
99.2
99.8
95.3
96.8
98.1

99.1
97.1
100.9
91.4
101.6
100.1

89.9
101.2
103.3
93.2
97.4
99.1

95.8
94.9
94.6
102.5
91.4
94.2

93.3
99.2
98.5
99.2
100.1
99.9

97.5
97.0
99.8
103.3
98.3
100.0

95.7
98.5
100.2
94.6
97.0
97.5

96.2
95.2
97.4
99.2
95.1
98.0

96.0
98.5
100.2
94.6
97.0
97.5

96.2
95.5
97.5
99.4
95.1
98.0

98.0
3.66
3.73

96.2
2.54
2.64

95.9
3.69
3.85

95.0
3.07
3.23

96.4
3.93
4.08

98.4
3.75
3.81

97.4
5.00
5.14

95.6
3.70
3.88

98.4
2.56
2.60

99.3
2.31
2.33

97.3
1.99
2.04

96.9
1.64
1.69

97.3
1.95
2.00

97.0
1.65
1.70

SD Standard deviation, RSD relative standard deviation

LC-PLS Method

In the LC-PCR method, the concentration set and normalized chromatographic data sets given in Table 1 were
considered for LC-PLS calibration. In
all of the chemometric and traditional
calibration models, normalized chromatographic peak area values were used.
According to the PLS algorithm, LCPLS calibration were computed by using
the linear combination based on the
Original

decomposed values of the concentration

data set and corresponding decomposed
chromatographic data. The computed
LC-PLS calibrations for ANO and DEL
compounds were used to determine two
compounds in the synthetic mixtures,
root and aerial part samples, and successful results were obtained. The
mathematical treatments were performed
by means of the PLS toolbox 3.5 in
Matlab 7.0 software.

LC-PCR Method

The PCR algorithm was applied to the

normalized chromatographic data and
concentration sets given in Table 1. In
this chemometric model, LC-PCR calibration for each ANO and DEL compound were obtained by using the
relationship between concentration sets
and its corresponding normalized chromatographic peak area. LC-PCR calibrations obtained in the above step were

Chromatographia 2007, 66, November (No. 9/10)

681

Table 4. Assay results obtained by applying the proposed analytical approaches to the root
samples
Method

k (nm) Compound Repetition experiments

Root samples
Traditional 300
HPLC
310
320
330
340
ANN
PCR
PLS

ANO
DEL
ANO
DEL
ANO
DEL
ANO
DEL
ANO
DEL
ANO
DEL
ANO
DEL
ANO
DEL

Mean
SD
(lg mg 1,
%)

2.06
2.95
2.01
2.88
2.15
3.07
2.09
2.96
2.16
3.03
2.16
3.01
2.10
2.98
2.09
2.99

2.08
2.96
2.04
2.92
2.10
2.95
2.08
2.95
2.10
3.10
2.17
3.00
2.10
2.97
2.09
2.98

2.07
2.98
2.07
3.04
2.09
3.08
2.18
3.08
2.07
3.07
2.20
3.07
2.12
3.02
2.11
3.01

2.12
2.95
2.07
2.88
2.10
2.93
2.06
2.91
2.07
3.06
2.19
3.04
2.13
2.97
2.09
3.01

2.04
3.01
2.07
2.95
2.12
2.99
2.11
2.99
2.13
2.98
2.17
3.00
2.10
2.99
2.11
3.00

2.04
2.98
2.06
2.89
2.08
3.05
2.08
2.94
2.04
3.09
2.14
3.01
2.07
2.99
2.08
3.00

2.04
3.03
2.01
2.87
2.15
2.91
2.15
3.04
2.10
3.00
2.19
3.00
2.10
2.97
2.09
2.98

2.06
2.98
2.05
2.92
2.11
3.00
2.11
2.98
2.10
3.05
2.17
3.02
2.10
2.98
2.09
3.00

0.03
0.03
0.03
0.06
0.03
0.07
0.04
0.06
0.04
0.05
0.02
0.03
0.02
0.02
0.01
0.01

RSD

1.56
1.02
1.40
2.07
1.36
2.30
2.11
1.97
1.92
1.50
0.96
0.93
0.89
0.63
0.55
0.39

Condence limit (p = 0.05)

were used to calculate the LC-ANN calibration. In this case, normalized chromatographic data for the concentration
data set (see Table 1) were utilized as an
input data. To reach the optimal HPLCANN calibration model with dierent
neuron size, various topological networks
were assayed and a training network
having ve neurons in the input layer
with one hidden layer and one output for
the prediction of each ANO and DEL in
samples were found to be suitable for the
construction of the LC-ANN calibrations. In this chemometric ANN approach, logarithmic signal transfer
function in three layers was used for
obtaining the LC-ANN calibrations for
each compound.
During a back propagation ANN
algorithm application, the relationship
between the mean square error (MSE) of
the networks and epochs for the training
set was performed. A dierence between
the calculated MSE and predened MSE
(1.0 10 10) values was not observed for
the calculated ANN calibration. The obtained LC-ANN calibration was used for
the prediction of ANO and DEL in
samples mentioned above. ANN application gives us a good agreement with
PCR and PLS calibrations.

Traditional LC Method

Fig. 4. Multiple chromatograms of root extract from S. resinosum

used for the determination of both ANO

and DEL in the synthetic mixtures and
real samples. The calculation of calibration and data treatment were performed
by the PLS toolbox 3.5 in Matlab 7.0
software.
According to the cross validation
procedure one factor for calibrations was

682

used for both LC-PCR and LC-PLS

calibrations.
LC-ANN Method

The concentration set and multivariate

chromatographic data in the application
of the LC-PCR and LC-PLS calibrations

The multiple chromatograms of ANO

and DEL in the concentration range of
50400 ng mL 1 were recorded by using
LC-DAD at the ve-wavelength set as
shown in Fig. 3. The detector responses
were measured in terms of peak area.
Chromatographic separation of ANO
and DEL was performed under the
above-mentioned chromatographic conditions. In the application of the traditional LC method, calibration graphs for
each compound at each wavelength in the
wavelength set (300, 310, 320, 330 and
340 nm) were obtained by using the
relationships between concentrations and
corresponding normalized peak area.
Linear regression analysis and its statistical results were summarized in Table 2.
In this computation, correlation coecients for calibration equations were
found higher than 0.99.
Calibration equations at single wavelengths from 300 to 340 were applied to
the quantitative evaluation of ANO and
DEL in synthetic samples and plant extracts.

Chromatographia 2007, 66, November (No. 9/10)

Original

Method Validation
The validity of the LC-chemometric and
traditional LC methods was performed
for obtaining reliable results of analysis.
This procedure was carried out by using
the following parameters. In the traditional LC method, linearity for ANO and
DEL was observed in the concentration
range of 50400 ng mL 1 because the
values of correlation coecients being
close to 1.0 as depicted in Table 2. In case
of the traditional LC method, the limit of
detection (LOD) and the limit of quantitation (LOQ) were calculated by using
100 ng mL 1 for ANO and DEL for six
replicates and their numeric values were
presented in Table 2.
The percent of mean recoveries and
their relative standards deviations for two
compounds were obtained by application
of all of the proposed methods to six
dierent mixtures of ANO and DEL in
dierent concentration levels. Accuracy
and precision corresponding to the percent of mean recoveries and relative
standards are shown in Table 3. We observed that the proposed methods are
available for the quantitative analysis of
the subjected compounds due to the
above-mentioned accuracy and precision
values.

Sample Analysis
In the application of the proposed
methods to the real samples, LC-ANN,
LC-PCR, LC-PLS and traditional LC
approaches were successfully applied to
the quantitative analysis of ANO and
DEL in the root and aerial part of S.
resinosum. As an example for the chromatographic separation obtained in the
method application to the sample, a
multiple chromatogram of the root extract is presented in Fig. 4.
Assay results (n = 7) obtained in the
application of the proposed traditional
LC and LC-chemometric approaches to
the root and aerial part samples are given
in Tables 4 and 5, respectively. Generally
successful results provided by the LCchemometric and traditional LC methods
were obtained for the analysis of related
compounds. Therefore, we observed that
the ANO and DEL % RSD values

Original

Table 5. Assay results obtained by applying the proposed analytical approaches to the aerial part
samples
Method

SD RSD
Mean
(lg mg 1,
%)

4.92
11.06
4.48
10.94
4.36
10.77
4.37
10.65
4.44
10.52
4.58
10.11
4.43
10.76
4.51
10.79

4.78
10.90
4.14
10.26
4.14
10.08
4.16
9.95
4.23
10.23
4.38
10.21
4.50
10.46
4.51
10.49

4.71
11.23
4.54
11.11
4.41
10.85
4.43
10.78
4.49
10.65
4.50
10.19
4.53
10.80
4.53
10.72

4.73
11.23
4.52
11.08
4.39
10.88
4.41
10.75
4.47
10.62
4.50
10.51
4.52
10.88
4.51
10.90

4.69
10.72
4.28
10.57
4.15
10.74
4.15
10.25
4.23
10.13
4.36
10.48
4.41
10.56
4.43
10.58

4.61
11.03
4.31
10.72
4.19
11.04
4.22
10.14
4.31
10.07
4.47
10.28
4.44
10.58
4.51
10.61

4.61
10.91
4.64
10.68
4.39
11.03
4.38
10.50
4.46
10.41
4.45
10.40
4.41
10.68
4.50
10.69

4.72
11.01
4.42
10.77
4.29
10.77
4.30
10.43
4.38
10.38
4.46
10.31
4.46
10.68
4.50
10.68

k (nm) Compound Repetition experiments

Aerial part samples

Traditional 300
ANO
HPLC
DEL
310
ANO
DEL
320
ANO
DEL
330
ANO
DEL
340
ANO
DEL
ANN
ANO
DEL
PCR
ANO
DEL
PLS
ANO
DEL

0.11
0.19
0.18
0.30
0.12
0.33
0.12
0.32
0.11
0.23
0.07
0.15
0.05
0.15
0.03
0.14

2.28
1.69
3.98
2.81
2.88
3.04
2.84
3.10
2.60
2.25
1.64
1.49
1.16
1.40
0.70
1.31

obtained by application of the LC-chemometric approaches to plant extracts

were in the range of 0.391.64 with mean
values (lg mg 1, %) being close to 2 for
ANO and 3 for DEL. In the above cases,
the traditional LC method gives relatively
poor results.

the LC-chemometric calibration in comparison to the traditional LC method.

We believe that the LC-chemometric
approaches create alternative and dierent perspectives of the application of LC
to the mixture analysis containing ANO
and DEL in the root and aerial part.

Conclusions

References

In this study, LC-chemometric and traditional LC approaches were applied to

the simultaneous quantitative analysis of
the mixture containing ANO and DEL in
the root and aerial part.
As can be seen from Tables 3, 4, 5, the
traditional LC corresponding to single
wavelength did not give better RSD
values than that of the LC-chemometric
methods. It was shown that LC-chemometric approaches allow the elimination
or reduction of the disadvantages of traditional LC. In other words, we suppose
that the eect of the selection of wavelength, peak uctuation in column system, noise coming from instrumentation
and experimental condition caused this
unwanted situation in the application of
the traditional LC method. Chemometric
calibrations were obtained by considering
the multi-wavelength set (300, 310, 320
330 and 340 nm) without the selection of
a single wavelength as well as traditional
LC application. This is an advantage of

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