New HPLC-chemometric approaches to the analysis of isoflavones in Trifolium lucanicum Gasp

Research Article

New HPLC-chemometric approaches to theanalysis of isoflavones in Trifoliumlucanicum Gasp.

New HPLC-chemometric approaches were proposed for the simultaneous chromato-

graphic quantification of daidzein, genistein, formononetin, and biochanin A in the

samples consisting of the aerial parts of Trifolium lucanicum Gasp. (Leguminosae). Partial

least squares and principal component regression algorithms were applied to the multiple

chromatographic data set obtained by measuring at 240, 248, 256, and 264 nm to

construct HPLC-partial least squares and HPLC-principal component regression calibra-

tions. Chromatographic separation was carried out by using a mobile phase containing

methanol, acetate buffer (pH 5 4.75) and acetonitrile (21:58:21, v/v/v) on the reversed

phase column, SupelcosilTM LC-18 (15 cm� 4.6 mm id). In addition, conventional HPLC

based on the detection at a single wavelength was used for the determination of each

compound in the extracts of T. lucanicum. The validity and applicability of the proposed

HPLC-chemometric and conventional HPLC methods were performed by analyzing

various synthetic plant samples. A good agreement was observed in the application of the

proposed HPLC-chemometric tools to the synthetic and extracted samples of T. lucanicum.

Keywords: HPLC-chemometric methods / Isoflavones / Leguminosae / Quanti-tative analysis / Trifolium lucanicumDOI 10.1002/jssc.201000273

1 Introduction

Trifolium lucanicum Gasp. (Leguminosae) is an annual plant

with flowering calyx with subulate, hirsute teeth, and corolla

somewhat longer than the calyx, leaflets thickened, with

prominent lateral nerves arcuate-recurved at the margin.

The natural habitat of T. lucanicum is dry places and rocky

ground and its distribution is Northwest, West and South

Anatolia. Besides, it is also widely distributed in France,

Italy, and Balkans. The mentioned plant is a Mediterranean

element [1].

Nowadays, red clover (T. pratense L.) has a very interest

importance for many human health benefits, particularly in

the area of women’s health. However, red clover is the main

source of phytoestrogen isoflavones. Phytoestrogens, poly-

phenolic non-steroidal plant compounds, may have protec-

tive effects on estrogen-related conditions such as

menopausal symptoms and estrogen-related diseases, such

as prostate, breast cancers, osteoporosis, and cardiovascular

diseases. In red clover, the fundamental isoflavones are

formononetin (FNT) and biochanin A (BC), with low

concentration levels of daidzein (DZ) and genistein (GT) [2].

Genus Trifolium L. is represented by 128 taxons, in

which 11 are endemic in flora of Turkey [1, 3–6]. Up to date,

not any analytical study has been performed on Trifoliumspecies growing in Turkey. A series of isoflavones consisting

of DZ, GT, FNT and BC were subjected to our investigation.

According to the substituents in the main isoflavone struc-

ture, the molecular structures of DZ, GT, FNT and BC are

shown in Fig. 1. In the previous studies, some potential

investigations on the analysis of isoflavones in plant mate-

rials and biological samples were reported by chromato-

graphic methods [7–9].

Nowadays, chemometric methods, particularly partial

least squares (PLS) and principal component regression

(PCR), have been used as chemometric powerful tools to

quantify two or more active substances in their mixtures

using spectrophotometric data. However, some application

of the chemometric calibration techniques in the chroma-

tographic analysis of complex samples were reported

[10–12].

The first main aim of this study is to develop and apply

new HPLC-chemometric methods for the simultaneous

quantification of DZ, GT, FNT, and BC compounds in the

extracted samples of T. lucanicum. The second aim is to

Nurgun Kuc- ukboyacı1

Ays-egul Guvenc-2

Erdal Dinc-3

Nezaket Adıguzel4

Barıs- Bani4

1Department of Pharmacognosy,Faculty of Pharmacy, GaziUniversity, Ankara, Turkey

2Department of PharmaceuticalBotany, Faculty of Pharmacy,Ankara University, Ankara,Turkey

3Department of AnalyticalChemistry, Faculty of Pharmacy,Ankara University, Ankara,Turkey

4Department of Biology, Facultyof Arts and Science, GaziUniversity, Ankara, Turkey

Received April 21, 2010Revised June 30, 2010Accepted July 1, 2010

Abbreviations: BC, biochanin A; DAD, diode array detector;DZ, daidzein; FNT, formononetin; GT, genistein; IS, internalstandard; PCR, principal component regression; PLS, partialleast squares

Correspondence: Professor Nurgun Kuc- ukboyacı, Department ofPharmacognosy, Faculty of Pharmacy, Gazi University, 06330Etiler, Ankara, TurkeyE-mail: [email protected]: 190-312-2235018

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2010, 33, 2558–25672558

compare the results obtained by applying HPLC-chemo-

metric approaches with those obtained by conventional

single-wavelength HPLC. After the optimization of the

chromatographic settings and conditions, a mobile phase

consisting of methanol, acetate buffer (pH 5 4.75) and

acetonitrile (21:58:21, v/v/v) and the flow rate 1.4 mL/min

were found to be suitable for an adequate chromatographic

separation of four compounds, DZ, GT, FNT, and BC, in the

presence of the internal standard (IS), spironolactone, in the

above-mentioned plant samples. It was observed that a good

agreement was reported for the chromatographic results

obtained by all of the proposed conventional and chemo-

metric HPLC approaches.

2 Theoretical aspect

This study contains the application of the PCR and PLS

algorithms to the multiple chromatographic data based on

the ratio of analyte/IS. For example, which wavelength point

or points are suitable for the multicomponent analysis of

four analytes having different maximum absorbance values

at the different wavelength points? As it is known, the

selection of the optimal wavelength is one of the chromato-

graphic analysis problems. However, chromatographic

multiple calibration (PLS and PCR) is appropriate for the

elimination of the above problem due to the use of

multiwavelength chromatographic measurements. Taking

into account maximum absorbances of analytes, single-

wavelength detection give main sensitivity problem for

the simultaneous quantification of four compounds in the

same quaternary mixture. The HPLC-chemometric

calibrations (HPLC-PCR and HPLC-PLS) based on the

multiple wavelength set instead of the single wavelength

detection provide desirable precise and accurate results for

the simultaneous chromatographic analysis. The brief

information of the HPLC-PCR and HPLC-PLS is explained

below.

2.1 HPLC-PCR method

The multiple chromatographic data (R 5 response)

consisting of the ratio of the analyte’s peak area and

analyte’s concentration set (C) were processed by mean-

centering as Ro and Co, respectively, and the covariance

dispersion matrix of the centered matrix Ro was computed.

The normalized eigenvalues and their eigenvectors were

obtained from the square covariance matrix of Ro. The

number of the optimal principal components (eigenvectors

(P)) corresponding to the highest values of the eigenvalues

was selected.

In the application of PCR algorithm, the coefficient

b defined as b 5 P� q is calculated, where P is the vectors of

eigenvectors and q is the C-loadings given by q 5

D�TT�Ro. TT indicates the transpose of the score matrix Tand D is a diagonal matrix corresponding to the inverse of

the selected eigenvalues. The analytes in samples were

predicted by using the formula, C 5 b�Rsamp. In this

equation, C is unknown concentration, b denotes the vectors

of the coefficients and Rsamp is chromatographic response of

multiple wavelength set.

HPLC-PCR calibration and data treatments were

performed by using PLS toolbox 3.0 in Matlab 7.0 software

and Microsoft Excel, respectively.

2.2 HPLC-PLS method

The PLS calibration based on the orthogonalized PLS

algorithm initiated by Wold [13] and extensively discussed

by Martens and Naes [14] consists of the simultaneous

decompositions or operations of both independent and

dependent variables. In the application of the PLS algorithm

to the multiple chromatograms, the HPLC-PLS calibration

is constructed by the decomposition of both concentration

data and ratio peak area data into latent variables, R 5

T�PT1E and C 5 U�QT1F. Here R is chromatographic

response (matrix), C is concentration set (vector). T and Uare scores, P and Q are loadings. The vector, b is calculated

by the expression b 5 W� (PT�W)�1�Q, where Wdenotes a weight matrix.

As in HPLC-PCR method, HPLC-PLS calibration and

data treatments were performed by using PLS toolbox 3.0 in

Matlab 7.0 software and Microsoft Excel, respectively.

3 Materials and methods

3.1 Chemicals and reagents

In our study, reagents (acetonitrile and methanol) used in

the HPLC analysis were of chromatographic grade (Merck,

Darmstadt, Germany). In the extraction procedure, metha-

nol and TFA were of analytical grade (Merck). Isoflavone

standards; DZ, GT, FNT, and BC were purchased from

Fluka (Buchs, Switzerland). DMSO (Merck) was used by

No Isoflavones Substituents

R1 R2

1 Daidzein H H 2 Formononetin H CH3

3 Genistein OH H 4 Biochanin A OH CH3

OHO

R1OR2

O

Figure 1. Molecular structures of isoflavones in the extract ofT. lucanicum.

J. Sep. Sci. 2010, 33, 2558–2567 Liquid Chromatography 2559


dissolving the isoflavone standards. Glacial acetic acid and

NaOH (Merck) were used to prepare the acetate buffer

solution (pH 5 4.75).

3.2 Plant material and sample preparation

The aerial parts of T. lucanicum Gasp. (Leguminosae) were

collected at the flowering stage from Van, C- atak, Dalbastı

village, surroundings of Seytanderesi in May 2003 in

Turkey. Plant material was provided from open oak forest

at the altitude of 1400 meters. Plant material was identified

by one of the authors (Barıs- Bani). Authenticated voucher

specimen (coded as B.B. 1603) was deposited in the

Herbarium of GAZI, Ankara, Turkey.

Two-hundred milligrams powdered materials were

extracted by using 20 mL of 80% MeOH (acidified to pH 3

with TFA) for 15 min under reflux at 851C. This procedure

was repeated two times as a sequence treatment. After

filtering the collected extract samples, 1 mL of filtrate was

diluted with 9 mL water and loaded on to a Sep-Pak C18

cartridge (Waters). Isoflavones were retained on the Sep-Pak

C18 cartridge which was then washed with 10 mL of water

twice and eluted with 80% methanol. This procedure was

repeated five times. Four isoflavones, DZ, GT, FNT, and BC

in the final extract samples were determined by using the

proposed HPLC-chemometric and conventional HPLC

methods.

3.3 Chromatographic instrumentation and software

Chromatographic analysis was performed by using an

Agilent 1100 series HPLC system (Agilent Technologies,

California, USA), a thermostatted autosampler, a thermo-

statted column compartment, and a multi wavelength diode

array detector (DAD). HP Chem Station for LC (Rev. A0.01

[403]) software (Hewlett–Packard) was used for the HPLC

data processing. Chromatographic separation was carried

out by using the HPLC column, a SupelcosilTM LC-18

(4.6� 150 mm, 5 mm).

Multiple HPLC data consisting of the ratio of analyte/IS

were processed by PLS toolbox in Madlab 7.0 software to

obtain HPLC-PCR and HPLC-PLS calibration. For the

conventional single HPLC, linear regression analysis and

statistical calculations were achieved by Microsoft Excel

software.

3.4 Chromatographic conditions and settings

Column temperature was 301C. Flow rate was maintained at

1.7 mL/min and the injection volume was 10 mL. The mobile

phase consisting of methanol, acetate buffer (pH 5 4.75),

and acetonitrile (21:58:21, v/v/v) was prepared daily, filtered

through a 0.45 mm membrane filter and degassed before

use. Tab

le1.

Cali

bra

tio

nse

tan

dco

rresp

on

din

gra

tio

peak

are

as

for

the

HP

LC

-ch

em

om

etr

icca

lib

rati

on

mo

dels

mg/m

LR

atio

ofpe

akar

eas

(DZ/

IS)

Rat

ioof

peak

area

s(G

T/IS

)R

atio

ofpe

akar

eas

(FN

T/IS

)R

atio

ofpe

akar

eas

(BC

/IS

)

Set

no.

DZ

GT

FNT

BC

IS24

024

825

626

424

024

825

626

424

024

825

626

424

024

825

626

4

12.

52.

54.

04.

025

.00.

2093

0.25

040.

3529

0.72

500.

1074

0.18

130.

3933

0.94

670.

3153

0.38

030.

5432

1.11

660.

1384

0.24

420.

5506

1.35

91

25.

05.

08.

08.

025

.00.

3773

0.46

510.

6593

1.37

050.

2105

0.35

080.

7671

1.87

370.

6220

0.76

731.

1018

2.29

370.

3041

0.50

981.

1277

2.80

62

37.

57.

512

.012

.025

.00.

5800

0.71

601.

0234

2.14

340.

3254

0.53

891.

1861

2.91

210.

8998

1.11

571.

6177

3.38

970.

4652

0.76

671.

6966

4.24

59

410

.010

.016

.016

.025

.00.

7616

0.97

911.

4005

2.95

930.

4160

0.72

871.

6030

3.97

491.

1595

1.51

582.

2024

4.65

670.

6715

1.06

022.

3442

5.91

37

512

.512

.520

.020

.025

.01.

0299

1.23

701.

7689

3.76

310.

5662

0.92

182.

0302

5.06

251.

6791

2.03

392.

9503

6.27

880.

8407

1.36

693.

0193

7.66

10

615

.015

.024

.024

.025

.01.

2187

1.47

712.

1158

4.54

140.

6552

1.07

132.

3598

5.93

701.

9193

2.34

173.

4020

7.30

400.

9902

1.59

383.

5317

9.04

46

J. Sep. Sci. 2010, 33, 2558–25672560 N. Kuc- ukboyacı et al.


3.5 Standard solutions

Stock solutions of 5 mg/20 mL of DZ, GT, FNT, BC,

and IS (spironolactone) were prepared in a mixture of

DMSO and methanol (1:19, v/v). A standard concentration

set of the mixture solutions containing four compounds in

the range between 2.5–15 mg/mL of DZ and GT,

and 4.0–24 mg/mL of FNT and BC using 25 mg/mL

of IS were prepared from the above stock solutions. As a

validation set, nine solution samples consisting of the

quaternary synthetic herbal mixtures were prepared by

using the same stock solutions. Methanol was used, as a

solvent, for the preparation of both concentration set and

validation set.

mAU

0

10

40

-10

20

30

-20

-30

-40

Calibration set no.1

DAD1 A, Sig=240 nmDAD1 B, Sig=248 nm DAD1 C, Sig=256 nmDAD1 D, Sig=264 nm

DZ

Time (min)0-50

AB

CD

mAU

0

10

40

-10

20

30

50

-20

-30

-40


DAD1 A, Sig=240 nmDAD1 B, Sig=248 nm DAD1 C, Sig=256 nmDAD1 D, Sig=264 nmDZ

Time (min)0

AB

CD

Time (min)0

mAU

0

10

40

60

-10

20

30

50

70

-20

-30-40


DAD1 A, Sig=240 nmDAD1 B, Sig=248 nm DAD1 C, Sig=256 nmDAD1 D, Sig=264 nmGTDZ

AB

CD



mAU

0

20

40

60

80

-40

-20

Time (min)5 10 15 20 2505 10 15 20 25

5 10 15 20 250 5 10 15 20 25

DZ

AB

CD

mAU

0

20

40

60

80

100

-40

-20


Time (min)5 10 15 20 25


DZ GT

A

B

CD

0 Time (min)5 10 15 20 25

mAU

0

20

40

60

80

100

-20 DAD1 A, Sig=240 nmDAD1 B, Sig=248 nm DAD1 C, Sig=256 nmDAD1 D, Sig=264 nmA

B

CD


SICBTNFTGZD

120

0

Time (min)5 10 15 20 25

mAU

0

20

40

60

80

100

-20 DAD1 A, Sig=240 nmDAD1 B, Sig=248 nm DAD1 C, Sig=256 nmDAD1 D, Sig=264 nm

A

B

C

D


2.89

6

4.60

5

7.69

3

15.6

20

17.1

74

SICBTNFTGZD

120

0

BC IS

BC IS

BC IS

FNT

FNT

GT FNT

BC ISGT FNT

BC ISGT FNT

Figure 2. Multiple chromato-grams of DZ (2.5, 5.0, 7.5,10.0, 12.5, and 15 mg/mL) andGT (2.5, 5.0, 7.5, 10.0, 12.5, and15 mg/mL), FNT (4.0, 8.0, 12.0,16.0, 20.0, and 24 mg/mL), BC(4.0, 8.0, 12.0, 16.0, 20.0, and24 mg/mL) in the presence ofthe constant amount of IS(25 mg/mL) in the calibrationset 1–6. The calibration set 6shows the multiple chromato-grams of DZ (15 mg/mL), GT(15 mg/mL), FNT (24 mg/mL), BC(24 mg/mL), and IS (25 mg/mL) atthe multiple wavelength set,240 nm (A), 248 nm (B),256 nm (C), and 264 nm (D).



4 Results and discussion

4.1 HPLC-chemometric method development

The main problem of the chromatographic analysis of

complex mixtures is the optimization of the chromato-

graphic settings and conditions to obtain separation or to

reach the best recovery results. For this aim, in our case,

several mobile-phase systems were tested to find appropriate

chromatographic separation of DZ, GT, FNT, and BC

together with IS. The mobile phase consisting of methanol,

acetate buffer (pH 5 4.75) and acetonitrile (21:58:21, v/v/v)

at the flow rate 1.4 mL/min on the reversed phase column,

SupelcosilTM LC-18 (15 cm� 4.6 mm id) were found to be

suitable for the chromatographic separation of the target

isoflavones, DZ, GT, FNT, and BC with IS in the extract of

the aerial parts of T. lucanicum. In the chromatographic

studies, another chromatographic problem is the finding of

an optimal detection wavelength because multiple

compounds in a given sample have the different maximum

absorbances at different wavelength points. In shortly, the

use of the single wavelength detection may not give better

determination results due to bad selection of the working

wavelength in some cases. This drawback can be eliminated

by HPLC-chemometric calibrations using the multiple

wavelengths set corresponding to the maximum absorbance

values of the analytes in a large region of wavelength.

In our study, HPLC-PCR and HPLC-PLS based on the

multiple chromatograms at the wavelength set, 240, 248,

256, and 264 nm were applied to the simultaneous quanti-

fication of DZ, GT, FNT, and BC in our plant samples. For a

comparison of the results obtained by HPLC-PCR and

HPLC-PLS methods, conventional HPLC method based on

single wavelength detection was used for the same problem.

4.2 HPLC-PCR application

The brief information related to PCR algorithm was given in

Section 2.1. In our application, PCR algorithm was applied

to the multiple chromatograms of the calibration set

containing isoflavones between 2.5–15 mg/mL for DZ and

GT and 4.0–24 mg/mL for FNT and BC in the presence of

the constant amount of IS (25 mg/mL) prepared by

dissolving 5 mg of each compound in 20 mL in the

solvent system consisting of DMSO and methanol (1:19,

v/v). A calibration set consisting of six mixture samples

including the related compounds and IS is summarized in

Table 1.

The multiple chromatograms of the calibration set

corresponding to Table 1 were recorded at four-wavelength

set (240, 248, 256, and 264 nm) by using DAD system under

the above-optimized chromatographic conditions. The

multiple chromatograms of the calibration set containing

four compounds with IS are shown in Fig. 2. As shown in

Fig. 2, the retention times of DZ, GT, FNT, BC, and IS were

observed as 2.896, 4.605, 7.693, 15.620, and 17.174 min,

respectively. We concluded that this chromatographic

Table 2. Linear regression analysis and its statistical results for the conventional HPLC analysis

l ma) Nb) Rc) SE(m)d) SE(n)e) SE(r)f) LODg) LOQh)

DZ 240 0.0809 �0.0198 0.9990 1.35� 10�3 1.27� 10�3 1.43� 10�2 0.14 0.47

248 0.0966 �0.0032 0.9992 1.46� 10�3 1.37� 10�3 1.54� 10�2 0.13 0.43

256 0.1359 0.0089 0.9982 3.06� 10�3 2.88� 10�3 3.24� 10�2 0.19 0.64

264 0.2789 0.0870 0.9946 1.10� 10�2 1.03� 10�2 5.56� 10�2 0.33 1.11

GT 240 0.0447 �0.0084 0.9992 9.20� 10�4 8.96� 10�4 9.63� 10�3 0.18 0.60

248 0.0726 �0.0031 0.9994 1.21� 10�3 1.18� 10�3 1.27� 10�2 0.15 0.49

256 0.1604 �0.0140 0.9994 2.72� 10�3 2.64� 10�3 2.84� 10�2 0.15 0.49

264 0.4066 �0.1070 0.9996 6.03� 10�3 6.99� 10�3 6.30� 10�2 0.15 0.52

FNT 240 0.0818 �0.0460 0.994VN4 4.35� 10�3 6.78� 10�3 7.29� 10�2 0.75 2.49

248 0.1000 �0.0416 0.9982 3.01� 10�3 4.70� 10�3 5.04� 10�2 0.42 1.41

256 0.1459 �0.0729 0.9983 4.23� 10�3 6.58� 10�3 7.07� 10�2 0.41 1.35

264 0.3154 �0.2427 0.9982 9.48� 10�3 1.48� 10�2 1.59� 10�1 0.42 1.40

BC 240 0.0434 �0.0392 0.9992 8.74� 10�4 1.36� 10�3 1.46� 10�2 0.28 0.94

248 0.0687 �0.0377 0.9994 1.23� 10�3 1.91� 10�3 2.06� 10�2 0.25 0.84

256 0.1516 �0.0777 0.9994 2.61� 10�3 4.06� 10�3 4.37� 10�2 0.24 0.80

264 0.3904 �0.2942 0.9994 6.94� 10�3 1.08� 10�2 1.16� 10�1 0.25 0.83

a) m, slope of regression equation.

b) n, intercept of regression equation.

c) r, correlation coefficient.

d) SE(m), standard error of slope.

e) SE(n), standard error of intercept.

f) SE(r), standard error of correlation coefficient.

g) LOD (mg/mL)

h) LOQ (mg/mL).



Tab

le3.

Reco

very

resu

lts

of

the

syn

theti

cp

lan

tsa

mp

les

by

the

pro

po

sed

con

ven

tio

nal

HP

LC

meth

od

Qua

tern

ary

mix

ture

(mg/

mL)

Foun

d(m

g/m

L)

DZ

GT

FNT

BC

No.

DZ

GT

FNT

BC

240

248

256

264

240

248

256

264

240

248

256

264

240

248

256

264

12.

5012

.50

4.00

20.0

02.

502.

442.

402.

2812

.74

12.7

412

.70

12.9

34.

314.

184.

254.

5321

.12

21.5

421

.58

21.9

4

27.

507.

5012

.00

12.0

07.

457.

307.

287.

287.

427.

347.

897.

2811

.87

11.6

711

.60

11.5

611

.35

11.6

211

.57

11.5

3

312

.50

2.50

20.0

04.

0012

.20

12.2

312

.10

11.9

72.

592.

312.

482.

3519

.72

19.3

336

.85

14.7

04.

023.

783.

773.

89

410

.00

2.50

8.00

12.0

09.

819.

9610

.00

10.0

42.

332.

442.

652.

608.

007.

867.

897.

9811

.29

11.5

011

.56

11.6

0

510

.00

12.5

08.

0024

.00

10.1

910

.29

10.5

110

.39

12.8

412

.88

13.3

013

.17

8.23

7.94

8.03

8.33

23.6

324

.37

24.6

125

.08

68.

008.

004.

0016

.00

8.02

7.83

8.21

8.08

7.81

7.76

9.19

7.84

4.13

4.03

4.10

4.32

15.6

215

.67

15.6

415

.74

78.

0010

.00

24.0

016

.00

8.29

8.30

8.18

8.30

10.1

010

.26

10.5

010

.42

23.9

423

.80

23.8

423

.92

15.5

915

.71

15.7

915

.93

815

.00

6.00

16.0

08.

0015

.64

15.8

815

.05

14.9

46.

276.

026.

125.

8915

.85

15.5

915

.44

15.0

97.

627.

547.

557.

57

95.

0015

.00

20.0

012

.00

4.71

5.11

5.09

4.91

14.2

315

.17

15.4

414

.87

18.4

919

.67

19.5

119

.36

11.7

211

.52

11.6

011

.68

Rec

over

y(%

)

DZ

GT

FNT

BC

No.

240

248

256

264

240

248

256

264

240

248

256

264

240

248

256

264

110

0.18

97.5

495

.83

91.3

910

1.92

101.

9010

1.63

103.

4310

7.78

104.

5210

6.35

113.

3110

5.58

107.

7010

7.88

109.

71

299

.29

97.3

897

.05

97.0

098

.97

97.9

210

5.20

97.0

698

.92

97.2

496

.67

96.3

394

.60

96.8

196

.44

96.1

2

397

.61

97.8

696

.82

95.7

710

3.48

92.2

099

.28

93.9

498

.58

96.6

718

4.26

73.5

010

0.44

94.5

994

.34

97.3

4

498

.09

99.5

510

0.03

100.

4493

.12

97.4

910

5.94

103.

9999

.94

98.3

098

.62

99.8

194

.05

95.8

696

.31

96.6

3

510

1.95

102.

9410

5.13

103.

8610

2.73

103.

0810

6.38

105.

3410

2.86

99.2

910

0.38

104.

1798

.48

101.

5310

2.54

104.

50

610

0.28

97.8

310

2.63

101.

0497

.59

96.9

811

4.83

97.9

810

3.24

100.

6910

2.39

107.

9697

.60

97.9

497

.72

98.3

7

710

3.57

103.

7010

2.20

103.

7410

0.95

102.

6510

4.96

104.

2199

.75

99.1

599

.34

99.6

697

.44

98.1

998

.67

99.5

8

810

4.26

105.

8810

0.32

99.6

310

4.54

100.

3410

2.04

98.2

399

.05

97.4

796

.50

94.3

295

.23

94.2

694

.38

94.6

0

994

.11

102.

1910

1.84

98.2

594

.87

101.

1210

2.93

99.1

592

.47

98.3

397

.57

96.7

997

.70

96.0

096

.67

97.3

5

Mea

n99

.93

100.

5410

0.20

99.0

199

.80

99.3

010

4.80

100.

3710

0.29

99.0

710

9.12

98.4

397

.90

98.1

098

.33

99.3

6

SD

3.15

3.19

3.11

3.96

3.95

3.49

4.42

3.97

4.17

2.38

28.3

511

.15

3.52

4.21

4.35

4.79

RS

D3.

153.

173.

104.

003.

963.

524.

223.

954.

162.

4025

.98

11.3

23.

594.

304.

424.

82



separation was sufficient to perform to quantify four

compounds in their samples.

PCR algorithm explained in the theoretical aspect was

applied to the multivariate HPLC data which correspond to

the ratio of DZ/IS, GT/IS, FNT/IS, and BC/IS areas at the

four-wavelength set (Table 1). The chemometric calibration

obtained by applying PCR was denoted as HPLC-PCR

method.

The multiple chromatographic areas for the extracts of

T. lucanicum containing IS were obtained at the above-

mentioned multiple wavelength set for DZ, GT, FNT, and

BC with IS. The ratio of the peak areas, DZ/IS, GT/IS, FNT/

Table 4. Recovery results of the synthetic plant samples by the proposed HPLC-chemometric methods

Quaternary mixture (mg/mL) Found (mg/mL)

DZ GT FNT BC

No. DZ GT FNT BC PLS PCR PLS PCR PLS PCR PLS PCR

1 2.50 12.50 4.00 20.00 2.55 2.57 12.79 12.79 4.06 4.07 20.53 20.63

2 7.50 7.50 12.00 12.00 7.30 7.29 7.36 7.40 11.69 11.79 11.52 11.55

3 12.50 2.50 20.00 4.00 11.80 11.79 2.40 2.41 20.62 20.66 3.88 3.88

4 10.00 2.50 8.00 12.00 9.77 9.75 2.48 2.50 7.96 7.95 11.48 11.49

5 10.00 12.50 8.00 24.00 10.18 10.18 12.97 12.99 8.16 8.20 24.30 24.40

6 8.00 8.00 4.00 16.00 7.92 7.90 7.81 7.81 4.11 4.10 15.66 15.76

7 8.00 10.00 24.00 16.00 8.08 8.09 10.20 10.28 23.81 23.87 15.75 15.74

8 15.00 6.00 16.00 8.00 14.60 14.55 6.06 6.09 15.49 15.59 7.88 7.86

9 5.00 15.00 20.00 12.00 5.01 5.02 14.84 14.84 19.23 19.29 11.63 11.63

Recovery (%)

DZ GT FNT BC

No. PLS PCR PLS PCR PLS PCR PLS PCR

1 102.18 102.90 102.34 102.34 101.42 101.66 102.67 103.17

2 97.33 97.19 98.07 98.60 97.38 98.22 96.02 96.27

3 94.41 94.33 95.88 96.28 103.09 103.30 96.97 96.97

4 97.72 97.52 99.06 99.86 99.53 99.41 95.65 95.74

5 101.84 101.84 103.77 103.93 102.03 102.53 101.25 101.67

6 99.03 98.78 97.59 97.59 102.73 102.62 97.90 98.52

7 101.03 101.15 101.96 102.80 99.19 99.44 98.46 98.40

8 97.36 97.00 100.94 101.44 96.78 97.41 98.46 98.22

9 100.16 100.37 98.96 98.96 96.17 96.47 96.95 96.95

Mean 99.00 99.01 99.84 100.20 99.81 100.12 98.26 98.43

SD 2.54 2.76 2.57 2.58 2.63 2.50 2.34 2.48

RSD 2.57 2.79 2.57 2.57 2.64 2.49 2.38 2.52

Table 5. Statistical parameters in the application of the HPLC-chemometrics method

Steps Parameter DZ GT FNT BC

PLS PCR PLS PCR PLS PCR PLS PCR

Calibration m 1.0486 1.0431 1.0000 0.9778 1.0390 1.0316 0.9338 0.9324

n �0.2802 �0.2592 0.0000 0.1223 �0.3325 �0.3023 0.9675 0.9579

r 0.9983 0.9986 0.9994 0.9990 0.9995 0.9995 0.9965 0.9966

SECa) 0.2952 0.2583 0.2126 0.2002 0.3638 0.2995 0.3591 0.3502

Prediction m 1.0486 1.0505 0.9778 0.9781 1.0123 1.0092 0.9665 0.9936

n �0.2802 �0.2997 0.1460 0.1207 �0.0598 �0.0651 0.6075 0.0514

r 0.9983 0.9983 0.9989 0.9988 0.9986 0.9987 0.9989 1.0000

SEPb) 0.2952 0.3095 0.1993 0.2130 0.3956 0.3689 0.3212 0.3186

a) SEC, standard error of calibration.

b) SEP, standard error of prediction.



IS, and BC/IS, were calculated. In the following step, the

amounts of four compounds in the extract and synthetic

samples were computed by inserting multiple peak area

ratio data to the PCR calibration equation for each

compound.

4.3 HPLC-PLS application

As in HPLC-PCR calibration, the PLS algorithm as

described above was subjected to the construction of the

HPLC-PLS calibration. A calibration set of the mixture

sample solutions of compounds between 2.5–15 mg/mL for

DZ and GT and 4.0–24 mg/mL for FNT and BC was used to

obtain the concentration data set. This concentration data

matrix was named as calibration set numbered from 1 to 6.

Table 1 summarizes the outline of the calibration set. In this

chromatographic study, the volume corresponding to 25 mg/

mL of IS was added to each sample.

For the calibration set summarized in Table 1, the

multiple HPLC chromatograms were recorded by using

DAD system. The multiple HPLC chromatograms for the

sequence of the calibration set are shown in Fig. 2. As can

be seen, a good chromatographic separation was observed

between DZ, GT, FNT, BC with IS.

HPLC-PLS calibration in the application of the PLS

algorithm to the peak area ratio data set was provided to

quantify the content of DZ, GT, FNT, and BC compounds in

samples.

4.4 Conventional single HPLC method

In the study, conventional single HPLC method is based on

the relationships between concentration and the ratio of

area (compound)/area (IS) at single-wavelength detection

for each compound in samples. In the our application,

standard series of the analyzed compounds were prepared

between 2.5–15 mg/mL for DZ and GT and 4.0–24 mg/mL

for FNT and BC using methanol. A 25 mg/mL IS was used

during the analysis of samples. The registration of DZ, GT,

FNT, BC, and IS chromatograms was performed by an

Agilent 1100 series HPLC system and DAD detections at

four different wavelengths. As in HPLC-chemometric

approaches, single-wavelength HPLC separation was accom-

plished by an isocratic mobile phase system as described in

Section 3.3. Different flow rates were tried to reach desirable

separation of the related compounds with IS. It was

observed that the flow rate at 1.4 mL/min provides a good

chromatographic separation. In case of this chromato-

graphic analysis, retention times of 2.896 min for DZ,

4.605 min for GT, 7.693 min for FNT, 15.620 min for BC,

and 17.174 min for IS are shown in Fig. 2.

Calibration equations for each compound in the

subjected samples were calculated by using the linear

regression analysis based on the relationship between

concentration and peak area ratio values at four different

wavelengths as explained above. Linear regression analysis

and its statistical results are summarized in Table 2. The

calculated linear regression functions for each compound

were used for the quantitative evaluation of DZ, GT, FNT,

and BC in synthetic and plant samples.

4.5 Analytical method validation

The method optimization and method validation are the

main problems of the chromatographic analyses and

applications. In this context, after the chromatographic

method optimization as explained above, analytical method

2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0

mAU

0

25

50

75

100

125

150

175

200

225

250

22.5 25.0 27.5 Time (min)

Extracted sample of Trifolium lucanicum


GT SICBTNFDZ

-25

A

BC

D

0.0

Figure 3. Multiple chromato-grams of DZ, GT, FNT, BC,and IS in the extracted sampleof T. lucanicum at the multiplewavelength set, 240 nm (A),248 nm (B), 256 nm (C), and264 nm (D).



validation was carried out by the observation of the linearity,

precision, accuracy, LOD, and LOQ parameters, etc.

As summarized in Table 2, good linearities for calibra-

tions with their high correlation coefficients were reported

in our concentration ranges by applying the conventional

HPLC method.

Recovery studies on the synthetic plant samples of DZ,

GT, FNT, and BC containing IS were done to observe the

precision and accuracy of the results provided by the

proposed HPLC-chemometric and conventional HPLC

methods. Mean percent recoveries, SDs, and RSDs were

calculated and listed in Tables 3 and 4. From Tables 3 and 4,

the results indicated that adequate accuracy and precision,

defined by percent mean recovery and RSD, respectively,

were sufficient for applicability and validity of HPLC-PCR,

HPLC-PLS, and conventional HPLC methods for the

analysis of the plant samples.

In addition, the LOD (S/N is 3:1) and the LOQ (S/N is

10:1) values were calculated by using the slope (m) of linear

regression equation and the SD of linear regression equa-

tion’s intercept. The LOD and LOQ values are listed in

Table 2.

The ability of the proposed HPLC-multivariate calibra-

tions was performed on the parameters defined by the

standard error of calibration and standard error of predic-

tion based on the relationship between the actual and the

predicted concentrations in the calibration and prediction

steps, respectively. The statistical results obtained by

applying least squares linear regression analysis to the

actual and predicted concentrations are summarized in

Table 5. As summarized in Table 5, in the application of

HPLC-PCR and HPLC-PLS calibrations, good correlation

coefficients between the actual and the predicted concen-

trations were observed.

Cross-validation procedure in the application of PCR

and PLS algorithms to the multiple chromatographic areas

was used to identify the optimal factor number for the

construction of HPLC-PCR and HPLC-PLS. From cross-

validation procedure, first one factor was found to be

suitable for both HPLC-PCR and HPLC-PLS [14, 15]. The

root mean square error of cross-validation for the HPLC-

PCR and HPLC-PLS calibrations were obtained as 0.5447

and 0.5446 for DZ, 0.2218 and 0.2222 for GT, 1.3201 and

1.3187 for FNT, and 0.7825 and 0.7827 for BC indicating

good accuracy and precision. In our calculation, units of the

results are mg/mL.

4.6 Analysis of plant samples

As described in the context of this study, HPLC-PCR and

HPLC-PLS approaches were applied to the multicomponent

determination of the amount of DZ, GT, FNT, and BC in

the extracts of T. lucanicum. For a comparison of the above

Table 6. Assay results obtained by applying the HPLC-chemometric and conventional HPLC to the extracts of Trifolium lucanicum

Conventional-HPLC Chemometric method Conventional-HPLC Chemometric method

No. 240 248 256 264 HPLC-PCR HPLC-PLS No. 240 248 256 264 HPLC-PCR HPLC-PLS

DZ (mg/mg) FNT (mg/mg)

1 2.524 2.314 2.391 2.396 2.3817 2.4137 1 0.0908 0.0803 0.0801 0.0903 0.0898 0.0900

2 2.471 2.389 2.378 2.510 2.4125 2.3819 2 0.0883 0.0797 0.0818 0.0916 0.0882 0.0891

3 2.387 2.393 2.391 2.450 2.4269 2.4271 3 0.0832 0.0820 0.0842 0.0932 0.0892 0.0882

4 2.404 2.388 2.568 2.396 2.3599 2.3601 4 0.0870 0.0787 0.0826 0.0922 0.0881 0.0892

5 2.367 2.258 2.373 2.485 2.4692 2.4499 5 0.0857 0.0795 0.0784 0.0882 0.0886 0.0889

Average 2.4307 2.3485 2.4203 2.4476 2.4100 2.4066 Average 0.0870 0.0800 0.0814 0.0911 0.0888 0.0891

SD 0.0653 0.0606 0.0831 0.0515 0.0421 0.0358 SD 0.0028 0.0012 0.0022 0.0019 0.0007 0.0007

RSD 2.69 2.58 3.43 2.11 1.75 1.49 RSD 3.27 1.54 2.73 2.11 0.81 0.76

SE 0.0292 0.0271 0.0371 0.0230 0.0188 0.0160 SE 0.0013 0.0006 0.0010 0.0009 0.0003 0.0003

CL 0.0572 0.0531 0.0728 0.0452 0.0369 0.0314 CL 0.0025 0.0011 0.0019 0.0017 0.0006 0.0006

GT (mg/mg) BC (mg/mg)

1 0.1316 0.1333 0.1324 0.1387 0.1314 0.1324 1 0.2697 0.2406 0.2401 0.2564 0.2509 0.2507

2 0.1388 0.1300 0.1282 0.1354 0.1316 0.1318 2 0.2619 0.2407 0.2439 0.2522 0.2507 0.2525

3 0.1361 0.1232 0.1267 0.1411 0.1326 0.1326 3 0.2549 0.2457 0.2467 0.2643 0.2535 0.2532

4 0.1380 0.1322 0.1369 0.1392 0.1332 0.1332 4 0.2557 0.2489 0.2482 0.2628 0.2532 0.2519

5 0.1360 0.1351 0.1324 0.1421 0.1360 0.1342 5 0.2552 0.2533 0.2519 0.2643 0.2519 0.2515

Average 0.1361 0.1307 0.1313 0.1393 0.1330 0.1328 Average 0.2595 0.2458 0.2462 0.2600 0.2521 0.2520

SD 0.0028 0.0046 0.0040 0.0026 0.0019 0.0009 SD 0.0064 0.0055 0.0045 0.0055 0.0013 0.0010

RSD 2.05 3.52 3.06 1.86 1.40 0.69 RSD 2.46 2.22 1.81 2.10 0.51 0.38

SEa) 0.0012 0.0021 0.0018 0.0012 0.0008 0.0004 SE 0.00285 0.00244 0.00199 0.00244 0.00058 0.00043

CLb) 0.0024 0.0040 0.0035 0.0023 0.0016 0.0008 CL 0.0056 0.0048 0.0039 0.0048 0.0011 0.0008

a) SE, standard error of slope.

b) CL, confidence limits.



HPLC-multivariate calibrations, the conventional HPLC

methods based on the single detection for each wavelength

point, 240, 248, 256, and 264 nm, respectively, were

subjected to the quantitative analysis of DZ, GT, FNT, and

BC amount in the same plant samples (Fig. 3). The assay

results provided by HPLC-chemometrics and conventional

HPLC are summarized in Table 6. In this table, the

experimental results provided by all methods are the average

of five replicates. As summarized in Table 6, the RSD values

obtained by HPLC-chemometric approaches were a little

lower than those obtained by conventional HPLC methods.

These small RSD values indicate that the precision of

HPLC-chemometric approaches is better than that of the

conventional HPLC method.

5 Concluding remarks

In this study, new HPLC multivariate calibration models

named HPLC-PLS and HPLC-PCR were improved for the

chromatographic multicomponent determination of DZ,

GT, FNT, and BC in the extract of T. lucanicum. In addition

to this, a conventional HPLC based on the single detection

was developed to compare the results obtained from the

HPLC-chemometric approaches. In comparison of the

proposed methods in our study, the HPLC-chemometric

methods give better recovery results with small RSD values

than those of conventional HPLC (Tables 3 and 4). This

investigation shows that the application of the HPLC

multivariate calibrations and conventional HPLC

approaches can be used safely for the quantitative analysis

of T. lucanicum containing phytoestrogen isoflavones.

This study was supported by Gazi University ResearchFoundation (No:02/2006-02).

The authors have declared no conflict of interest.

6 References

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[2] Cos, P., Bruyne, T. D., Apers, S., Berghe, D. V., Pieters,L., Vlietinck, A. J., Planta Med. 2003, 69, 589–599.

[3] Davis, P. H., Mill, R. R., Tan, K., in: Davis, P. H. (Ed.),Flora of Turkey and the East Aegean Islands, Vol. 10,Edinburgh University Press, Edinburgh 1988, p. 126.

[4] Byfield, A. J., in: Guner, A., Ozhatay, N., Ekim, T.,Bas-er, K. H. C. (Eds.), Flora of Turkey and the EastAegean Islands, Vol. 11, Edinburgh University Press,Edinburgh 2000, p. 95.

[5] Ozhatay, N., Kultur, S., Turk. J. Bot. 2006, 30, 281–316.

[6] Ozhatay, N., Kultur, S., Aslan, S., Turk. J. Bot. 2009, 33,191–226.

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[9] Ramos, G. P., Dias, P. M. B., Morais, C. B., Froehlich, P.E., Dall’Agnol, M., Zuanazzi, J. A. S., Chromatographia2008, 67, 125–129.

[10] Dinc- , E., Ustundag, O., Ozdemir, A., Baleanu, D., J. Liq.Chrom. Rel. Technol. 2005, 28, 2179–2194.

[11] Tosun, A., Bahadır, O., Dinc- , E., Chromatographia 2007,66, 677–683.

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[13] Wold, H., in: Johnson, N. L., Kotz, S. (Eds.), Encyclo-paedia of Statistical Sciences, Wiley and Sons, NewYork 1984.

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New HPLC-chemometric approaches to the analysis of isoflavones in Trifolium lucanicum Gasp

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