determination of bavachin and isobavachalcone in fructus psoraleae by high-performance liquid...

Research Article

Determination of bavachin andisobavachalcone in Fructus Psoraleae byhigh-performance liquid chromatographywith electrochemical detection

A simple, sensitive and selective method of high-performance liquid chromatography with

electrochemical detection (HPLC-ECD) has been developed for simultaneous determi-

nation of bavachin and isobavachalcone in Fructus Psoraleae. At optimized conditions,

bavachin and isobavachalcone could be well separated within 15 min at a detection

potential of 10.80 V with 0.03 mol/L acetate buffer solution (pH 5.17)/acetonitrile (2:3,

v/v) as the mobile phase. The relationships between peak areas and concentrations were

linear from 8.26� 10�7 to 1.21� 10�4 mol/L for bavachin, and from 1.01� 10�8 to

1.61� 10�4 mol/L for isobavachalcone, respectively. The method offered excellent line-

arity with regression coefficient R240.995. The method presented detection limits

(S/N 5 3) of 8.81� 10�9 mol/L for bavachin and 1.17� 10�10 mol/L for isobavachalcone.

It indicates that the sensitivity of electrochemical detection is ten times higher than that of

diode array detection (DAD). The mean recoveries around 98% with a relative standard

deviation less than 3.1% for the two analytes have been obtained. The proposed separation

and detection procedures were successfully applied to the simultaneous determination of

bavachin and isobavachalcone in traditional Chinese medicine.

Keywords: Bavachin / Fructus Psoraleae / HPLC-ECD / IsobavachalconeDOI 10.1002/jssc.201000801

1 Introduction

Fructus Psoraleae is the dried ripe fruit of Psoralea corylifoliaL. (Fabaceae), which is traditionally used to alleviate asthma

and diarrhea and to treat vitiligo and alopecia areata [1] in

East Asian countries. Bavachin and isobavachalcone are two

typical flavonoid ingredients in Fructus Psoraleae. Their

structures are shown in Fig. 1. As we can see, both of them

have phenolic hydroxyl groups and an isopentenyl side

chain in their structures. They have many functions in

common such as inhibiting the release of b-hexosaminidase

in RBL-2H3 cells [2], platelet aggregation [3], a-glucosidase

activities [4], antioxidation [5, 6] and antibacterial [7, 8].

Besides, clinical studies have shown that bavachin can

stimulate bone formation [9] and isobavachalcone exhibits a

broad spectrum of biological activities, including inducing

apoptosis in neuroblastoma [10], inhibiting the accumula-

tion of nitrite (NO) as a marker for the production of NO

[11], enhancing cardiac contractility, preventing cardiac

fatigue due to lactic acid [12], inhibiting skin tumor

promotion [13] and inducing apoptosis in cancer cells [14].

As bavachin has structural similarity to isobavachalcone

and they have the same molecular weights, it is necessary to

include them in any quality assessment of traditional

Chinese medicine. The analysis of P. corylifolia L. has been

reported by LC [15–18] and HPLC-MS [19, 20]. For instance,

ultra performance liquid chromatography-diode array

detection (UPLC-DAD) [17] method has been used to

determine ten compounds in P. corylifolia L. with limits of

detection of 7.21� 10�8 mol/L for bavachin and

7.02� 10�8 mol/L for isobavachalcone.

Recently, more attention has been paid to use HPLC

coupled with electrochemical detection (ECD) to analyze

phenolic compounds. After a previous separation, a simple,

rapid and inexpensive method would be beneficial for

routine assay of flavonoids in natural products. ECD, which

often requires relatively simple preparation of samples

without extraction or preconcentration steps, can be used for

the analysis of phenolic compounds. The advantages of ECD

are shown in the recent papers for the determination of

flavonoids [21, 22].

On the basis of our literature search, no paper has been

reported to determine bavachin and isobavachalcone in

Yuan LiFang WangZilin Chen

Institute of PharmaceuticalAnalysis, College of Pharmacy,Wuhan University, Wuhan,P. R. China

Received November 15, 2010Revised December 9, 2010Accepted December 11, 2010

Abbreviations: ECD, electrochemical detection; GCE, glassycarbon electrode

Correspondence: Professor Zilin Chen, Institute of Pharmaceu-tical Analysis and Drug Screening, College of Pharmacy, WuhanUniversity, Wuhan 430072, P. R. ChinaE-mail: [email protected]: 186-27-68759850

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2011, 34, 514–519514

P. corylifolia L. by HPLC-ECD. In this work, a method is

proposed that takes advantage of LC and the selectivity and

sensibility of the ECD technique for the detection and

quantification of bavachin and isobavachalcone in P. coryli-folia L. Further, to evaluate the HPLC-ECD techniques

proposed, linearity, limits of detection and method precision

in terms of RSD were examined. The method has success-

fully been applied for the simultaneous determination of

bavachin and isobavachalcone in Fructus Psoraleae.

2 Materials and methods

2.1 Materials and reagents

The ripe seed of P. corylifolia L. was purchased from

Liuyouyu Drugstore in Wuhan (Hubei, China). Bavachin

(purity: Z99%) and isobavachalcone (purity: Z99%) were

obtained from Shanghai Shunbo Bio-engineering Technol-

ogy (Shanghai, China). Methanol and acetonitrile were

HPLC grade and were purchased from Tedia (USA) and

VBS (USA), respectively. Sodium acetate, acetic acid,

disodium hydrogen phosphate and concentrated hydrochlo-

ric acid were obtained from Sinopharm Chemical Reagent

(Shanghai, China), potassium dihydrogen phosphate was

purchased from Shantou Xilong Chemical Factory (Guang-

dong, China), sodium hydroxide was from Tianjin Feng-

chuan Chemical Reagent (Tianjin, China), citric acid was

purchased from Tianjin Guangfu Jingxi Chemical Research

Institute (Tianjin, China). The supporting electrolyte solu-

tions were prepared by deionized water (Chongqing

Qianyan Water Disposal Equipment Chongqing, China).

For all experiments analytical grade chemicals and solvents

were used.

2.2 HPLC conditions

The HPLC system was equipped with LC-20AT (Shimadzu,

Japan), a sample injector equipped with a 20-mL loop, a Sepax

amethyst C18-P column (5 mm, 4.6� 250 mm; Sepax Tech-

nologies, USA) and a CHI 842B electrochemical analyzer

(Shanghai Chenhua Instrument, Shanghai, China) with a

thin layer radial flow cell (Shanghai Chenhua Instrument). In

the HPLC system, a diameter of 1-mm glassy carbon

electrode (GCE) was used as a working electrode, the

stainless steel served as counter electrode and an Ag/AgCl

(saturated KCl) electrode was used as a reference electrode.

The data acquisition and treatment is controlled from a CHI

842B electrochemical analyzer equipped with CHI software

package (Shanghai Chenhua Instrument). The pH buffers

were prepared using a PHSJ-3F acidity meter purchased from

Shanghai Jingmi Scientific Instrument (Shanghai, China).

The mobile phase was 0.03 mol/L acetate buffer solution

(pH 5.17)/acetonitrile (2:3, v/v). They were filtered through

0.22-mm nylon filter membranes (Shanghai Xingya Jinghua

Materials Factory, Shanghai, China) and degassed in ultra-

sonic bath before being used. The flow rate was adjusted to

0.80 mL/min. The working electrode was set at a potential of

10.80 V versus the Ag/AgCl reference electrode.

2.3 Standard solutions

Stock solutions of 1.54� 10�3 mol/L bavachin and isoba-

vachalcone were prepared by dissolving appropriate amount

of standard samples in acetonitrile. These solutions were

stored in dark bottles at 41C. Working solutions were

prepared by mixing and diluting appropriately of each of the

stock standard solutions with mobile phase.

2.4 Preparation of samples

Extraction of the flavonoids from Fructus Psoraleae was

performed by ultrasonication, using hydrochloric acid in

methanol as an extraction solvent. The fruit of P. corylifoliaL. was pulverized into appropriate powder. The powder

sample (1.00 g) was weighed and mixed with 10 mL of

methanol/concentrated hydrochloric acid 5:1 v/v for extrac-

tion solvent. The sample was subjected to ultrasound

treatment at 201C during 45 min. Then, the sample was

taken out of the ultrasonic bath and left at room

temperature for 30 min. The extract was centrifuged at

14 000 rpm for 20 min, the supernatant was collected and

diluted to 10 mL with extraction solvent and stored in a

refrigerator at 41C.

2.5 Analytical procedure

The GCE was polished with alumina slurry to mirror finish,

and then it was rinsed with water. All samples were filtered

O

H3C

CH3

OHHO

O

bavachin

H3C CH3

HO OHOH

Oisobavachalcone

Figure 1. Molecular structures of bavachin and isobavachal-cone.

J. Sep. Sci. 2011, 34, 514–519 Liquid Chromatography 515


through 0.22-mm nylon filter membranes and degasified in

ultrasonic bath before their injection (20 mL) in the

chromatographic system. The system was equilibrated for

at least 30 min prior to injection of the prepared samples.

Chromatograms of sample were collected and the mean of

peak area (n 5 3) was used as an analytical signal. The

determination of bavachin and isobavachalcone were

performed on a GCE at a detection potential of 10.8 V

with 0.03 mol/L acetate buffer solution adjusted to pH 5.17/

acetonitrile (2:3, v/v) as the mobile phase. The content of

bavachin and isobavachalcone in Fructus Psoraleae were

calculated by the peak areas of each target analyte from the

calibration curves under the same conditions. All experi-

ments were carried out at room temperature.

For each standard sample, three replicates were made.

Peak area was selected as analytical signal to determine the

concentration of two analytes. In this work, the retention

times and resolution were used to evaluate the influence of

different chromatographic parameters on the separation of

bavachin and isobavachalcone.

3 Results and discussion

3.1 Selection of the mobile phase

First, the optimization of the acetonitrile contents in the

mobile phase was investigated. The acetonitrile content in the

mobile phase varied between 80 and 40%, the retention times

decreased with increase in the amount of acetonitrile and

small differences in the acetonitrile content had significant

effect on the separation behavior. Mobile phases with higher

percent of acetonitrile cannot elute less polar flavonoid

substances, and the retention time was too short to separate

bavachin and isobavachalcone in Fructus Psoraleae completely.

Many overlap peaks appeared. When the acetonitrile content

was lower than 55%, the two compounds were eluted very

slowly. Considering the retention time and resolution, 60% of

acetonitrile was used for the analysis.

Then, the effect of the electrolyte and its concentration on

S/N was studied. Several buffer solutions including sodium

acetate–acetic acid, disodium hydrogen phosphate–potassium

dihydrogen phosphate and citric acid–sodium hydroxide were

tested. In general, the background noise was higher in the

case of using disodium hydrogen phosphate–potassium

dihydrogen phosphate and citric acid–sodium hydroxide

buffer solutions. To check the influence of the concentration

of acetate buffer, the concentration of acetate buffer in mobile

phase was varied from 0.00 to 0.05 mol/L. In the range

examined, the amount of acetate buffer in the mobile phase

has no significant effect on the retention times. The sensi-

tivity for all the analytes improved as the concentration of

acetate buffer was increased, but an increase of the back-

ground noise was observed at the highest concentration of

acetate buffer. It is because the noise level decreases with the

baseline increases. About 0.03 mol/L acetate buffer was

chosen as the optimal electrolyte.

Finally, the influences of buffer pH on migration

behaviors were examined using buffer solution between pH

3.40 and 6.00. Little change was observed between pH 4.50

and 5.50. With further decrease or with further increase in

the pH, the retention times decrease. Thus, we chose pH

5.17 as the running buffer pH.

Based on the consideration of the retention times,

resolution and sensitivity of detection, the mobile phase

consisted of an aqueous solution containing 0.03 mol/L

acetate buffer solution adjusted to pH 5.17 and acetonitrile

(2:3, v/v).

3.2 Selection of flow rate

Bavachin (6.20� 10�5 mol/L) and isobavachalcone

(2.30� 10�5 mol/L) were injected in the chromatographic

system in order to study the influence of flow rate on the

efficiency of the separation of the two compounds. The flow

rate was evaluated from 0.60 to 1.20 mL/min. The peak

width increased when the flow rate was adjusted to 0.80 mL/

min. With increase of flow rate, effective number of

theoretical plates (N) also varies. With further decrease in

the flow rate, a decrease in the peak width was observed.

According to these results, a value of 0.8 mL/min was

chosen due to the advantage of short times of analysis and

acceptable resolution for the separation of bavachin and

isobavachalcone effectively.

3.3 Selection of electrode potentials

The influence of detection potential over the range of

0.50–1.00 V on peak areas of the analytes was investigated.

The variation in peak areas of bavachin and isobavachalcone

with detection potential is shown in Fig. 2. Bavachin and

isobavachalcone showed an increased response close to

500 600 700 800 900 10000

3

6

9

12

15

18

21

Pea

k ar

ea

Applied potential /mV

bavachinisobavachalcone

Figure 2. Hydrodynamic voltammograms of (6.20� 10�5 mol/L)bavachin (m) and (2.30� 10�5 mol/L) isobavachalcone (�).Mobile phase: 0.03 mol/L acetate buffer solution (pH 5.17)/acetonitrile (2:3, v/v). Flow rate: 0.80 mL/min.

J. Sep. Sci. 2011, 34, 514–519516 Y. Li et al.


0.75 V and there was a marked increase in the response up

to 0.80 V; however, further increase resulted in increased

baseline noise. Therefore, 0.80 V was chosen as an optimum

detection potential.

Under the optimum conditions, a typical chromatogram

for the separation of bavachin and isobavachalcone with

ECD are shown in Fig. 3.

3.4 Linearity, detection limits and reproducibility

Under the optimum chromatographic conditions, a linear

relationship between peak area and concentration of

standard samples was found. The chromatograms of

bavachin and isobavachalcone with different concentrations

on GCE are shown in Fig. 4. A linear calibration curve was

obtained for standard analyte at different concentration

levels. The analytical characteristics of the calibration plots

are summarized in Table 1. The linear range of bavachin

was from 8.26� 10�7 to 1.21� 10�4 mol/L and the linear

regression equation was Q/nA � s 5 0.329 1 0.148c/mmol/L,

R2 5 0.9978 (n 5 9). The calibration curve of isobavachal-

cone was linear in the range of 1.01� 10�8 mol/L to

1.61� 10�4 mol/L. The linear regression equation of

isobavachalcone was Q/nA � s 5 0.36710.837c/mmol/L,

R2 5 0.9954 (n 5 9). The calibration curves exhibit excellent

linear behaviors over the concentration range of about three

orders of magnitude. The limit of detection (S/N 5 3) was

0 200 400 600 800 1000 1200-120

-100

-80

-60

-40

-20

0

Cu

rren

t/n

A

Time/s

1 2

Figure 3. Chromatogram of standard solution of (1) bavachin(6.20�10�5 mol/L) and (2) isobavachalcone (2.30� 10�5 mol/L)obtained by ECD. Mobile phase: 0.03 mol/L acetate buffersolution (pH 5.17)/acetonitrile (2:3, v/v). Flow rate: 0.80 mL/min.Potential: 10.80 V.

0 200 400 600 800 1000-450

-400

-350

-300

-250

-200

-150

-100

-50

0

0 20 40 60 80 100 120 140 160 1800

20

40

60

80

100

120

140

isobavachalconebavachin

Pea

k ar

ea

Concentration/µmol L

Cu

rren

t/n

A

Time/s

1

2

Figure 4. Chromatograms of (1) bavachin and(2) isobavachalcone with different concentra-tions on GCE. Insert: calibration curve ofbavachin (m) and isobavachalcone (�) on GCE.Mobile phase: 0.03 mol/L acetic acetate buffersolution (pH 5.17)/acetonitrile (2:3, v/v). Flowrate: 0.80 mL/min. Potential: 10.80 V.

Table 1. Validation of the method concerning linearity, the limits of detection and intra- and inter-day precision

Analyte Calibration parameters Limit of detection

(nM)

Precision (%RSD)

Slope Intercept R2 Intra-day precisiona) Inter-day precisionb)

Bavachin 0.148 0.329 0.9978 8.81 4.21 4.71

Isobavachalcone 0.837 0.367 0.9954 0.117 4.05 4.52

a) Represents the intra-day precision that was obtained by analyzing mixed standards at 0, 0.5, 1, 2, 4, 6 and 8 h in the same day.

b) Represents the inter-day precision that was obtained by analyzing mixed standards during sequential 7 days.



8.81� 10�9 mol/L for bavachin and 1.17� 10�10 mol/L for

isobavachalcone, respectively.

The relative standard deviation (RSD) peak areas of the

two analytes was 4.34 and 3.82% for seven successive

determinations of 6.20� 10�5 mol/L bavachin and

2.30� 10�5 mol/L isobavachalcone, respectively. These

results illustrated that this method showed good reprodu-

cibility for the determination of bavachin and isoba-

vachalcone.

Besides, intra- and inter-day precisions were performed.

To estimate intra-day analytical variance, a standard mixture

was determined seven times over an 8-h working period and

the analytical variance in the peak areas of each analyte was

assessed. Intra-day precision was 4.21 and 4.05% for bava-

chin and isobavachalcone, respectively. Inter-day analytical

variance was evaluated by determining a standard mixture

each day for a period of seven days. Inter-day precision was

4.71% for bavachin and 4.52% for isobavachalcone, respec-

tively. As shown in Table 1, the data illustrate that the

HPLC-ECD method has advantage of high precision.

3.5 Recovery and sample analysis

In order to evaluate the accuracy of proposed method,

recovery was tested. Accurate amounts of mixed standards

were added to powder sample, and then extracted as

described in Section 2.4 and analyzed. The recovery values

were obtained using their peak areas from the calibration

curves under the same conditions. As shown in Table 2, the

recovery of bavachin changes from 95.38 to 101.32% and the

recovery of isobavachalcone changes from 96.20 to 101.40%,

and the results indicated that this method was suitable for

the sample analysis.

The proposed method was applied to the determination

of bavachin and isobavachalcone in Fructus Psoraleae.

A chromatogram for separating bavachin and isoba-

vachalcone in Fructus Psoraleae is shown in Fig. 5. The

content of bavachin and isobavachalcone were analyzed.

The content of bavachin and isobavachalcone in FructusPsoraleae was calculated as 0.092 and 0.21%, respectively, by

using the peak areas of each target analyte from the cali-

bration curves under the same conditions. The developed

HPLC-ECD method is suitable for the detection of all these

electro-active compounds in Fructus Psoraleae.

Moreover, a comparison between UV detection method

(optimized to a wavelength of 234 nm) and ECD on GCE at

0.80 V was made. Chromatograms obtained by UV detection

are given in Fig. 6. As compared, the results obtained by

ECD (in Fig. 5) with that obtained by UV detection (in

Fig. 6) indicate that the analytes are detected well in FructusPsoraleae by ECD, and we obtained two single-target peaks.

On the contrary, UV detection gave some overlap peaks

owing to the presence of many UV-absorbing components

in Fructus Psoraleae that co-eluted with analytes. Thus, ECD

was selected for the determination of bavachin and isoba-

vachalcone.

4 Concluding remarks

In this work, a simple, sensitive and accurate method for

simultaneous separation and determination of bavachin and

isobavachalcone in P. corylifolia L. has been proposed by

HPLC-ECD. The procedure of ultrasonication can decrease

the loss of the analytes, which compares with distillation

and solid-phase extraction. Excellent detection limits of

8.81� 10�9 mol/L for bavachin and 1.17� 10�10 mol/L for

isobavachalcone have been achieved. Average recoveries are

higher than 98%. The sensitivity for the two compounds

Table 2. Recovery of bavachin and isobavachalcone in samples

Constituents Contained (mg) Added (mg) Found (mg) Recovery (%) Average recovery (%) RSD (%)

Bavachin 0.054 0.011 0.062 95.38 98.29 3.02

0.053 0.056 0.107 98.17

0.051 0.101 0.154 101.32

Isobavachalcone 0.155 0.108 0.253 96.20 98.31 2.78

0.151 0.148 0.291 97.32

0.153 0.204 0.362 101.40

0 200 400 600 800 1000 1200-600

-500

-400

-300

-200

-100

0

Cu

rren

t/n

A

Time/s

2

1

Figure 5. Chromatograms of (1) bavachin and (2) isobavachal-cone in Fructus Psoraleae obtained by ECD. Mobile phase:0.03 mol/L acetate buffer solution (pH 5.17)/acetonitrile (2:3, v/v).Flow rate: 0.80 mL/min. Potential: 10.80 V.

J. Sep. Sci. 2011, 34, 514–519518 Y. Li et al.


detected by ECD is ten times higher than those obtained by

diode array detection. Moreover, in this work, a comparison

between ECD and UV detection was made. ECD can provide

a high selectivity for the analysis of bavachin and

isobavachalcone in P. corylifolia L. The developed HPLC-

ECD method is suitable for the detection of all these electro-

active compounds in P. corylifolia L. without complicated

sample preparation procedure, ECD can be applied to

analysis of trace quantities of electro-active antioxidants in

natural products.

The authors gratefully acknowledge the financial supportfrom the National Natural Science Foundation of China (Nos.20775055, 30973672 and 90817103) and start-up funding forZ.C.’s Luojia chair professorship of Wuhan University, NationalMega Project on Major Drug Development (2009ZX09301-014-1) and the Fundamental Research Funds for the CentralUniversities.

The authors have declared no conflict of interest.

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A B Figure 6. Chromatograms of(1) bavachin and (2) isobava-chalcone by UV detection. (A)Chromatograms of standardsolution of bavachin (2.80�10�5 mol/L) and isobavachal-cone (4.10� 10�5 mol/L) by UVdetection; (B) chromatogramsof bavachin and isobavachal-cone in Fructus Psoraleae byUV detection. Mobile phase:0.03 mol/L acetate buffer solu-tion (pH 5.17)/acetonitrile (2:3,v/v). Flow rate: 0.80 mL/min.Wavelength: 234nm.



determination of bavachin and isobavachalcone in fructus psoraleae by high-performance liquid...

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