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Molecularly imprinted polymer for solid-phase extraction of rutin

in complicated traditional Chinese medicines
Lin Peng, Yuzhi Wang,* Huan Zeng and Ya Yuan
Received 14th October 2010, Accepted 10th November 2010
DOI: 10.1039/c0an00798f

In the present work, an improved and direct approach for the preparation of molecularly imprinted
polymers (MIPs) was proposed. The MIPs were prepared based on bulk polymerization by water-bath
heating and ultrasonic elution of the template, using rutin as the template, acrylamide (AM) as the
Published on 13 December 2010 on http://pubs.rsc.org | doi:10.1039/C0AN00798F

functional monomer and 2,20 -azobisisobutyronitrile (AIBN) as the cross linker. Molecularly imprinted
polymers prepared by other elution methods, including microwave-assisted extraction and
conventional Soxhlet extraction, were used for comparison and the results showed that the ultrasonic
elution method is the best. The synthesized MIPs were characterized by Fourier transform infrared
(FT-IR) spectroscopy and scanning electron microscopy (SEM). High performance liquid
chromatography (HPLC) was used to evaluate the adsorption properties and recognition mechanism of
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the MIPs. Structurally similar compounds including quercetin and genistein were utilized for verifying
the molecular selectivity and characterizing the recognition capability of the MIPs. The MIPs were used
as a sorbent for the solid phase extraction of rutin, and the resultant cartridge showed a good extraction
performance. Thus, a molecularly imprinted solid-phase extraction (MISPE) procedure for selective
pre-concentration of rutin from complicated traditional Chinese medicine (TCM) samples was
proposed. Various elution parameters that affect the adsorption capacity of the polymer were evaluated
to optimize the selective pre-concentration of rutin. The characteristics of the MISPE method were
validated by HPLC. The recoveries ranged from 85% to 91% for TCMs, which demonstrated that this
MISPE-HPLC method could be applied to pre-concentrate and determinate rutin directly from
complicated TCM samples in the presence of other interfering substances.

1. Introduction samples.14–16 This technique is more rapid, economical,

Sample preparation processes are widely implemented in
analytical laboratories for trace analysis in complicated samples.
Attempts have been made to synthesize molecularly imprinted
polymers (MIPs) for enhancing the selectivity.
Molecularly imprinted polymers are man-made polymers with
a predetermined selectivity towards a given analyte or a group of
structurally related species.1,2 These materials have been
employed in fields where a certain degree of selectivity is required
such as separations,3 sensors,4 immunoassays5 and catalysis or
artificial enzymes.6 The most significant advantages of MIPs,
compared with biological receptors such as antibodies, enzymes,
nucleic acids, or cells, include their mechanical and chemical
stability, high selectivity, low cost of preparation, and wide range
of operating conditions.7,8 MIPs are gaining in importance as
robust molecular recognition elements in analytical and separa-
tion sciences.9,10 They are commonly synthesized by polymerizing
complexes of functional monomers and a template to be
imprinted with matrix-forming monomers to yield highly cross-
linked polymers.11–13
Solid-phase extraction (SPE) represents a suitable way to
clean-up and pre-concentrate biological, environmental or food
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of
Chemistry and Chemical Engineering, Hunan University, Changsha,
410082, P. R. China. E-mail: wyzss@hnu.edu.cn; Fax: + 86-731-
88821848; Tel: + 86-731-88821903
environmentally friendly and simpler than the traditional liquid–
liquid extraction. However, the main problem associated with
solid-phase extraction columns packed with ordinary stationary
phases is the low selectivity of the retention mechanism. In fact,
along with the desired analyte(s), many unwanted interfering
substances of similar hydrophobicity/hydrophilicity are also
retained and concentrated, due to the very limited selectivity of
the partition equilibria involved. Therefore, a separation
methodology with built-in selectivity for the target compounds is
urgently required.
Using MIPs as an SPE sorbent, it is possible to combine the
advantages of both molecular recognition and traditional sepa-
ration methods.17–19 Thus, molecularly imprinted solid-phase
extraction (MISPE) presents high specificity, selectivity and
sensitivity of the molecular recognition mechanism along with
the high resolving power of separation methods. It could be
expected to find use in the field of selective determination of
target analytes in complicated samples.
Traditional Chinese medicines (TCMs), as a class of compli-
cated samples, are playing an important role in the field of
medicine. Because of the complexity of the herb composition and
poor affinity and selectivity of conventional separation materials,
separation and extraction of the active components in TCMs are
tedious and inefficient. Much research has been directed towards
variation of the sorption materials in order to achieve selective
interaction with the target molecule.20 Recently, the application
of molecularly imprinted technology to TCMs has attracted

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considerable attention.21–25 It is expected that MIPs can be used
as separation materials for extracting certain active components
directly from herbs.
Rutin, 3,30 ,40 ,5,7-pentahydroxyflavone-3-(O-rhamnosylgluco-
side), has received much attention because of its pharmaceutical
use in phytotherapy. Its therapeutic action is mainly related to
the heart and the kidney. It has also been reported as an anti-
microbial and antihypertensive medicine.26 In view of the
importance of rutin and the high content in traditional Chinese
medicines,27 the separation of rutin from TCMs is very valuable.
In the present work, we proposed for the first time the appli-
cation of rutin-MIP to TCM sample purification and analyte
Published on 13 December 2010 on http://pubs.rsc.org | doi:10.1039/C0AN00798F
(rutin) enrichment. The MIPs were prepared based on bulk
polymerization, using rutin as the template, acrylamide as the
functional monomer, AIBN as the cross linker and THF as the
porogen solvent. The synthesized MIPs were characterized by
Fourier transform infrared (FT-IR) spectroscopy and scanning
electron microscopy (SEM). RP-HPLC was used to evaluate the
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adsorption properties and recognition mechanism of the MIPs.
Structurally similar compounds, including quercetin and genis-
tein, were utilized for verifying the molecular selectivity and
characterizing the recognition capability of the MIPs. In addi-
tion, the MIPs were used as the sorbent for the solid phase
extraction of rutin and the MISPE method was proven to be
applicable for pre-concentration and determination of rutin from
several traditional Chinese medicines.
2. Experimental
2.1. Chemicals and reagents
Rutin was purchased from Guoyao Group of Chemical Reagents
Ltd. Acrylamide (AM), supplied from Guanghua Chemical
Manufactory (Guangdong, China), was distilled under vacuum.
2,20 -azobisisobutyronitrile (AIBN) from Damao Chemical
Reagent Company (Tianjin, China), tetrahydrofuran (THF)
from Xilong Chemical Factory (Guangdong, China) and
ethylene glycol dimethacrylate (EGDMA) were of reagent grade.
Artemisia selengensis Turcz, Eucommia, Southernwood, and
Saururus chinensis, four complicated TCMs, were obtained from
a pharmacy (Hunan, China). All the other chemicals were of
analytical reagent grade. All solutions used for HPLC were
filtered through a 0.45mm filter before use.
2.2. Instrumentation
All chromatographic measurements were performed using an
Agilent 1100 liquid chromatograph (USA) with a diode-array
detector. A C18 column (250 mm  4.6 mm i.d., 5 mm) from
Agilent was used as the analytical column. The mobile phase was
methanol : water (45 : 55, v/v) at a flow rate of 1.0 mL min1.
Rutin was monitored at 356 nm.
The morphology of MIPs was studied using a Hitachi S-4800
scanning electron microscope (Tokyo, Japan). The infrared
absorption spectrum was obtained by the KBr method using
a Spectrum One NTS (Perkin Elmer, USA).
Adsorption experiments were carried out using a ZHWY-
100C Water Bath Isothermal Shaker (Shanghai, China).
Template elution was performed using an AS2060B ultrasonic
instrument (Tianjin, China). A MDS-2002AT pressure
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self-control microwave decomposition system (Shanghai, China)
was used for the crude extraction of TCMs.
2.3. MIPs and NIPs preparation with bulk polymerization
The template molecule, 0.01 g rutin, was dissolved in 5 mL THF,
then 0.2 g acrylamide was added to the mixture. The mixture was
stirred in the water bath isothermal shaker for half an hour to
ensure sufficient reaction of acrylamide with rutin. Then
EGDMA (3.5 mL) and the cross linker AIBN (0.01 g) were
successively added to the solution. The solution was purged with
oxygen-free nitrogen for 15 min. The glass tube was sealed under
nitrogen and then placed in a water bath at 60  C. The poly-
merization was allowed to proceed for 24 h. The formed gel was
sieved to obtain particles with a size range of 70 to 80 mm. The
resulting particles were sonicated for 1 h in 100 mL
methanol : acetic acid (9 : 1, v/v) to remove the template and
then washed with methanol to remove acetic acid. Finally, the
resultant polymer was dried at 60  C and placed in a vacuum
drying oven before use in the following studies.
In order to verify that retention of the template was due to
molecular recognition rather than non-specific binding, a non-
imprinted polymer (NIP) was prepared following the same
procedure, including washing but in the absence of the target
molecule.
2.4. Preparation of the MISPE column
A PVDF film (0.045 mm pore size) was placed on the bottom of
an empty column (5 mL total volume, 1 cm diameter) which was
employed as the MISPE column. Then the column was packed
with 500 mg of rutin-MIPs in 5 mL methanol solution using a wet
packing method. Prior to loading the sample, the cartridge was
washed with a mixture of methanol and acetic acid until rutin
could not be detected by HPLC analysis.
2.5. Determination of the MISPE selectivity
In order to investigate the selectivity of the extraction protocol,
a mixture containing rutin and its analogue genistein was
prepared in methanol. 2 mL of the mixture (0.05 mg mL1 for
each) was loaded onto the SPE cartridge. Subsequently, the
cartridge was consecutively rinsed with 5 mL methanol and 5 mL
of the mixed solution of methanol : acetic acid (v/v, 9 : 1). The
schematic diagram of the MIPSE procedure is presented in
Fig. 1. The collected column packing solution, rinsing solution
and eluates were evaporated to 1 mL before analysis by HPLC.
2.6. Application of the MISPE method
The extraction of rutin from the four complicated TCM samples
(mentioned in Chemicals and reagents) was performed by
microwave decomposition. In brief, each TCM sample (1.0 g)
mixed with 30 mL methanol was sealed in a vessel and placed
into the pressure self-control microwave decomposition system
for 10 min. The extracts were centrifuged in a TGL-16C centri-
fuge (Shanghai, China) at 10 000 rpm for 10 min and filtered
using 0.45 mm polypropylene membranes. Then 5 mL of filtrate
was loaded onto the MISPE cartridge. Similarly, the collected
Analyst, 2011, 136, 756–763 | 757

Published on 13 December 2010 on http://pubs.rsc.org | doi:10.1039/C0AN00798F
Fig. 1 Schematic diagram of the MIPSE procedure for selective adsorption of rutin.
column packing solution, rinsing solution and eluates were
evaporated to 1 mL before analysis by HPLC.
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2.7. Reversed-phase liquid chromatography
RP-HPLC analysis was used for quantification of the analytes
after solid-phase extraction. Because of the complexity and
difficulty in the separation of rutin from Artemisia selengensis
Turcz, RP-HPLC analysis for this TCM was carried out on an
Agilent Series 1100 liquid chromatograph with a diode array
detection (DAD) system. A reversed-phase C18 column (150 mm
 4.6 mm i.d., 5 mm) from Agilent was used with the mobile
phase consisting of methanol (A) and water (B). The optimized
gradient elution was performed using the following linear
gradient: 0 min: 30% A; 5 min: 45% A; 35 min: 70% A; 65 min:
70% A.
HPLC analysis for Eucommia, Southernwood, and Saururus
chinensis was carried out on an Agilent Series 1100 liquid chro-
matograph with UV detection system, connected to a reversed-
phase column (Diamonsil C18, 150 mm  4.6 mm i.d., 5 mm,
Agilent, USA). The absorbance was measured at a wavelength of
356 nm for the detection of rutin.
The concentrations of rutin (0.0125, 0.025, 0.05, 0.1, and
0.2 mg L1) were analyzed and peak areas were plotted against
concentration. The regression equation was A ¼ 63637.706 +
1.333  107 C and the correlation coefficient of the standard
rutin was equal to 0.9996 (n ¼ 5).
3. Results and discussion
3.1. Selection of the best elution method
This work focused on the optimization of the elution process,
following the polymerization, to improve the capability of the
sorbent for selective pre-concentration of rutin from samples.
Initially, the effect of ultrasonic elution conditions was opti-
mized, since this controlled the degree of elution. Thus, the
polymers were eluted by different elution liquid-solid ratios (5,
10, 20, 40 and 80 mL g1) and for various elution times (0.25, 0.5,
1, 2 and 4 h). The elution capacity continuously increases with
the elution liquid-solid ratio, and reaches equilibrium at a ratio
of 40 mL g1. Next, by increasing the ultrasonic time at the
optimal elution liquid-solid ratio (40 mL g1), the elution
capacity was found to continuously increase with time, until 1 h
when the elution capacity reaches almost 825 mg g1. Hence, the
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ultrasonic time of 1 h and the elution liquid-solid ratio of 40 mL
g1 were set as the optimum conditions for the elution.
Next, the microwave-assisted extraction and conventional
Soxhlet extraction elution conditions, including liquid-solid ratio
and elution time, were optimized. Following this the three elution
approaches were compared to optimize the elution process
(elution parameters listed in Table 1). This was performed with
MIP particles after sieving (described in Section 2.3), followed by
elution by the three methods in their optimized conditions. The
eluent was analyzed by RP-HPLC. The elution capacity Q, which
was calculated from the concentration of rutin in the eluent, was
used as the analytical signal. From Table 1, it is clearly seen that
MIPs after ultrasonic elution obtain the highest elution capacity.
Thus, ultrasonic elution was chosen as the optimal method for
the process.
3.2. Characteristics of the rutin-MIPs and NIPs
The surface morphologies of MIPs and NIPs were studied by
SEM (shown in Fig. 2). Although they both have rough surfaces,
the pores of the MIPs are greater in number and larger in size
(Fig. 2a and 2b) than those of the NIPs (Fig. 2c and 2d). It can be
concluded that the surface of the MIPs exhibits larger pores and
therefore a larger volume of rough structure than that of the
NIPs. The uniform and more open structure is obviously
favorable for the embedding of the large template molecules of
rutin.
To further ascertain that the rutin-MIPs have been made, we
performed additional FT-IR analysis. A strong absorption peak
of AM (monomer) situated around 1633 cm1 is assigned to
Table 1 Selection of the elution method for MIPs
Elution
liquid-
solid Elution
ratio/mL Elution capacity
Elution methods g1 Time/h Qa/mg g1
Soxhlet extraction 100 24 547
Ultrasonic extraction 40 1.0 815
Microwave-assisted 40 0.5 326
extraction
a
Q, elution capacity; Q ¼ Ce  eluent volume [mL]/adsorbent mass [g],
where Ce represents the concentration of analyte in the eluent.
This journal is ª The Royal Society of Chemistry 2011

Published on 13 December 2010 on http://pubs.rsc.org | doi:10.1039/C0AN00798F
Fig. 2 The morphologies of the MIPs and NIPs. (a) Scanning electron
microscope images of MIPs. (b) An enlarged view of a representative
sample section of image a. (c) Scanning electron microscope images of
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NIPs. (d) An enlarged view of a section of image c.
C]C stretching. For MIPs, the weak absorbance peak of C]C
(1638 cm1) suggests that most of the AM was cross-linked and
only a few monomers remained. For NIPs, the characteristic
signals of FT-IR are almost the same, except for the intensity of
the stretching vibration peak of C]O (1726 cm1), distinctly
lower than that of MIPs. A possible reason is that the template
molecules, which are assembled with the monomer via the
hydrogen-bonded interaction in preparing MIPs, are helpful for
the co-polymerization of monomer, and thus stable imprinting
cavities are formed by ordered distribution of functional groups
containing a carbonyl group. The FT-IR results confirmed that
there were functional groups in the MIPs which would interact
with the template molecule, namely the acrylamide group in the
rutin-MIPs.
3.3. Adsorption capability of rutin-MIPs and NIPs
Fig. 3 shows the dynamic curves for the adsorption of rutin onto
the MIPs and NIPs. The adsorption capabilities of rutin-MIPs
and rutin-NIPs were investigated with 0.05 mg mL1 of rutin
standard solution. The mass of MIP was 100 mg and the volume
of the standard solution was 5 mL. The mixture was incubated
with continuous stirring on the magnetic stirring apparatus at
Fig. 3 Dynamic curves for the adsorption of rutin onto MIPs and NIPs.
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room temperature for different time intervals (0.5, 1.0, 1.5, 2.0,
2.5, 4.0, and 8.0 h).
The adsorption capacity, Q, was calculated on the basis of the
difference of rutin concentration before and after adsorption, the
volume of methanol solution and the weight of the beads
according to
ðC0  Ce Þ
Adsorption capacity Q ¼ V (1)
W
where C0 is the initial rutin concentration (0.05 mg mL1), Ce is
the rutin concentration (mg mL1) after adsorption, V is the
solution volume (mL), and W is the weight of the MIPs (g).
The experiment reveals that the adsorption capacity continu-
ously increases with increasing time, and almost reaches satu-
ration within 1 h. Obviously, the adsorption capacity of the
imprinted polymer is higher than that of the non-imprinted
polymer. For the adsorption equilibrium, the adsorption time of
1 h was chosen as optimum for the process. It is assumed that
when these imprinted sites in the surface are occupied, it becomes
difficult for rutin to implant into the MIPs. This would cause the
adsorption to slow down. The various contributions of polymers
towards the template molecular recognition would be attributed
to the hydrogen bonds between template and monomer and
complementary cavity to the template molecules. Although NIPs
show a similar trend to MIPs towards rutin adsorption, their
adsorption capacities are much lower. This is likely due to the
non-specific adsorption of NIPs caused by van der Waals forces
and the absence of suitable recognition sites and imprinting
cavities in the NIPs. However, there are numerous and precise
imprinting sites in the MIPs, resulting in specific adsorption, and
thus they produce higher adsorption capacities. So, the presence
of complementary cavities to the template molecules in the MIPs
is vital for the specific adsorption.
The adsorption capabilities of rutin-MIPs for rutin and
structurally similar reference compounds were investigated by
using standard solutions of 0.05 mg mL1 (as shown in Fig. 4). It
is obvious that the adsorption capacity of the rutin-MIPs for the
three flavonoids is within the range of 948–250 mg g1, which is
much higher than the range of 300–100 mg g1 for the NIPs. The
interaction between rutin-MIPs and the template can enable
selective adsorption of rutin and its analogues, which contain
a benzene ring and several hydroxyl groups. The degree of
molecular analogy to the template is relative to the adsorption
capabilities of the rutin-MIPs. The adsorption capability of
MIPs for rutin, which has a large substituent in the g-OH posi-
tion, is 948 mg g1. This result indicates that the interaction is not
only based on hydrogen bonding between the polymeric matrix
and the template, but also on the complementarity to the
template in size and shape.
From Fig. 4 we can see that the interaction between quercetin-
MIPs and the template is possibly weaker than that of rutin-
MIPs because quercetin can be easily removed during the
washing step. There is not an obvious difference among the
adsorption capabilities of the NIPs for rutin and the reference
compounds, which likely depends on the same mechanism of
non-specific adsorption.
In order to further investigate the competitive recognition
coefficients of the particles, quercetin and genistein as reference
compounds were chosen for measurement in a mixed solution.
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3.4. Binding isotherms

The polymer particles (100 mg) were mixed with 5 mL of a rutin

standard solution of a known concentration (0.5, 1, 2, 4 and 8 mg
L1) and the mixtures were incubated for 1 h at room tempera-
ture. After incubation, the adsorption capacity Q, described in
section 3.3, was calculated on the basis of the difference of rutin
concentration before and after adsorption.
Information about the adsorption site distribution (mono-
layer or double-layer) can be obtained with more reliability when
the adsorption isotherms are built and compared with models
(e.g. Langmuir and Freundlich models). The Langmuir (L)
adsorption isotherm is able to model homogeneous systems (eqn
Published on 13 December 2010 on http://pubs.rsc.org | doi:10.1039/C0AN00798F

(2)) while the Freundlich (F) adsorption isotherm successfully

models non-homogeneous systems (eqn (3)).

1/Qe ¼ 1/Qmax + 1/(Qmax  b  Ce) (2)

logQ ¼ nlogCe + logk (3)

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where Qe is the poised adsorption capacity (mg g1), Qmax is the

Fig. 4 Chemical structures of the evaluated flavonoids and their
adsorption capacity Q on MIPs and NIPs.
The distribution coefficient (Kd), selectivity coefficient (k), and
relative selectivity coefficient (k0 ) of the particles were evaluated
herein. Table 2 shows the measured values of the three para-
meters for the tested compounds. The distribution coefficient
(Kd) is defined as the ratio of the concentrations of the solute in
the two phases in the equilibrium state, suggesting the adsorption
capability of a substance, and we obtained 30.65 mL g1 of rutin
for MIPs, compared to 4.35 mL g1 for NIPs. The selectivity
coefficient (k) is defined as the ratio of the Kd values of the two
competitive solutes and indicates the difference of two substances
adsorbed by the polymers. The relative selectivity coefficient (k0 )
is defined as the ratio of the k values of the two competitive
solutes. The k value of the MIPs (2.80 or 5.46) is 1.61-fold (2.62-
fold) greater than that for the NIPs (0.64 or 2.09) for quercetin
and genistein, respectively. It is evident that the MIPs have a high
selectivity for rutin over the reference compounds of quercetin or
genistein, depending upon the imprinted effect in the matrix.
saturant adsorption capacity (mg g1), b is the equilibrium
constant (L mg1), and Ce is the poised rutin concentration (mg
L1), k and n are empirical constants which are determined by the
temperature, solvent and the nature of adsorbent.
The fitting coefficients are mean values calculated from two
independent experimental binding isotherms. From eqn (2), we
obtained the Langmuir model: 1/Qe ¼ 0.0158  0.0035/Ce, where
Qmax was 63.29 mg g1 and b was 4.5 L mg1. From eqn (3), we
obtained the Freundlich model: logQ ¼ 0.32logCe + 1.6800,
where k and n were 47.86 mg g1 and 0.32 L mg1, respectively.
From the results, we can see that the F isotherm gave a better fit
which means that the adsorption site distribution on rutin-MIPs
is non-homogeneous.
3.5. SPE specificity of the MIPs for rutin
The prepared rutin-polymer was employed as the sorbent of the
SPE column for the pretreatment of rutin and structurally similar
genistein. Fig. 5 shows the HPLC-UV chromatograms of the
standard mixtures. Good baseline separation is obtained without
the SPE procedure and rutin is eluted first (as shown in Fig. 5a).
No apparent peak is observed in Fig. 5b in the determination of
the solutions after MISPE, which indicates that genistein and
rutin were almost completely adsorbed onto the MIPs. Fig. 5c
shows that almost all the genistein, but only minor amounts of
rutin, was eluted by rinsing the MISPE column with methanol;
rutin was not easily eluted with methanol. When using the more
polar eluting solutions, namely methanol : acetic acid (9 : 1, v/v),

Table 2 Binding constants of the MIPs and NIPsa

C0/mg mL1 Ce/mg mL1 Kd/mL g1 k k0

Sample Rutin Quercetin Genistein Rutin Quercetin Genistein Kd1 Rutin Kd2 Quercetin Kd3 Genistein k1 k2 k10 k20

MIP 0.05 0.05 0.05 0.031 0.041 0.045 30.65 10.97 5.56 2.80 5.46 1.61 2.62
NIP 0.05 0.05 0.05 0.046 0.044 0.048 4.35 6.82 2.08 0.64 2.09
a
Kd, distribution coefficient; Kd ¼ (C0Ce)/(C0)(solution volume [mL]/adsorbent mass [g]),where C0 and Ce represent the initial and final
concentrations, respectively; k, selectivity coefficient, k1 ¼ Kd1/Kd2, k2 ¼ Kd1/Kd3; k0 , relative selectivity coefficient, k10 ¼ k1MIP/k1NIP, k20 ¼ k2MIP/k2NIP.

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Fig. 5 Chromatograms of the standard mixtures. (a) Initial solutions
before MISPE. (b) Solutions after MISPE. (c) Solutions after rinsing with
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methanol. (d) Eluates after rinsing with methanol : acetic acid (9 : 1, v/v).
rutin was almost completely eluted (shown in Fig. 5d). This result
can be easily explained by considering that imprinted polymers
usually show specific retention behavior based on non-covalent
interactions between the bulk of the polymer and the hydroxyl
groups of the analyte molecules. However, the rinsing step
caused the elution of most of the genistein, whereas this was not
the case for rutin. After the elution step, the elution ratio of rutin
following MISPE was greater than 90% (shown in Table 3), while
genistein was only partially recovered, due to the limited reten-
tion during the rinsing step.
These results show that the MISPE exhibited highly selective
binding affinity for rutin and demonstrated that the adsorption
of rutin was due to imprinted binding sites, rather than non-
specific binding.
3.6. Application of rutin-MIPs to TCM samples
The aim of this work was to provide a simple process, using
MISPE, which can be applied in the separation and determina-
tion of analytes from complicated samples. Accordingly, four
TCMs, Artemisia selengensis Turcz, Eucommia, Southernwood,
and Saururus chinensis, were extracted by the microwave
decomposition system described in Section 2.6. Satisfactory
sample clean-up was achieved by the MISPE protocol. The
results were shown in Fig. 6. When comparing the chromato-
grams it can be seen that, although rutin is extracted in high
yields (25.67 mg mL1, 57.20 mg mL1, 37.21 mg mL1 and 43.14
mg mL1 in Artemisia selengensis Turcz, Eucommia,
Table 3 Elution ratio of rutin after MISPEa
Compd. C0/mg mL1 C1/mg mL1 C2/mg mL1 C3/mg mL1
Rutin 0.0500 0.0035 0.0032 0.0435
Genistein 0.0500 0.0047 0.0414 0.0028
a
Q1, adsorption capacity, Q1 ¼ (C0C1)  (loading solution volume [mL]/adsorbent mass [g]); Q2, rinsing capacity, Q2 ¼ C2  rinsed solution volume
[mL]/adsorbent mass [g]; Q3, elution capacity, Q3 ¼ C3  elution solution volume [mL]/adsorbent mass [g], where C0, C1, C2 and C3 represent the
loading, column, rinsed and elution solution concentrations, respectively.
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Southernwood and Saururus chinensis, respectively) from TCM
samples, it cannot be individually separated in each TCM extract
without the MISPE procedure (as shown in Fig. 6a). No
apparent peak is observed in Fig. 6b in the analysis of the solu-
tions after MISPE, which indicates that the components in the
TCM extracts were almost completely adsorbed onto the MIPs.
Fig. 6c shows that, although most of the unknown components
were eluted by rinsing the MISPE column with methanol, rutin is
not easily eluted. When using more polar eluting solutions,
namely methanol : acetic acid (9 : 1, v/v), rutin was almost
completely eluted (as shown in Fig. 6d). It was obvious that
a high yield of rutin was obtained from the TCM samples by the
proposed MISPE, which cannot be individually separated in
TCM extracts without the MISPE procedure. This efficient
method allowed cleaner extracts to be obtained and interfering
peaks arising from the complex biological matrix to be sup-
pressed. The results from the HPLC analyses showed that
because rutin is a polar constituent, only strong polar solvents
could elute it from the complicated samples. This conclusion
achieved the selective extraction and separation of rutin from the
crude extracts of complicated TCM samples.
From the study, conclusions can be drawn as follows: Tradi-
tional Chinese medicines (TCMs) are examples of complex herbs.
Because of the poor affinity and selectivity of conventional
separation materials, separation and extraction of active
components in TCMs are tedious and inefficient. The MISPE
technology we promoted in this study presents the high speci-
ficity, selectivity and sensitivity of molecular recognition mech-
anisms and the high resolving power of separation methods. It
could expect to be used in the field of selective determination of
target analytes in complicated samples.
3.7. The validation of the MISPE method
Considering that slow release of the template molecule from the
MISPE column could affect the accuracy of the determination of
rutin in the TCM sample extraction step, possible bleeding of the
MISPE column was examined. After adding 0.5 mL of rutin (0.05
mg mL1) onto the MISPE column, 5 mL of methanol : acetic acid
(9 : 1, v/v) was used for elution. The collected eluates were
evaporated to 1 mL before analysis by HPLC. Apparatus preci-
sion was evaluated by the analysis of five successive injections of
the same sample solution. The RSD was 0.31% which showed that
the precision of the apparatus was satisfactory.
The limits of detection and quantification were 8.0 ug g1 and
25.0 ug g1 respectively. The lifetime of the MIP cartridge was
one week, which indicated that after repetitive use for seven days,
it would lose activity.
Q1/mg g1 Q2/mg g1 Q3/mg g1 Elution ratio (%)
465 32 435 93.5
453 414 28 8.2
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Fig. 6 Chromatograms of complicated TCM samples. (a) Initial solutions before MISPE. (b) Solutions after MISPE. (c) Solutions after rinsing with
methanol. (d) Eluates after rinsing with methanol : acetic acid (9 : 1, v/v).

The standard addition method was used to evaluate the
recoveries of the MISPE-HPLC process. TCM extracts were
spiked with rutin and processed by the MISPE-HPLC procedure.
The test samples were prepared by mixing 1 mL of TCM extract
with 1 mL of standard rutin solution (0.05 mg mL1). The
recoveries of rutin in spiked TCM extracts are shown in Table 4.
The recoveries of the spiked Artemisia selengensis Turcz,
Eucommia, Southernwood, and Saururus chinensis samples are
91.3%, 89.0%, 85.8%, and 89.1%, respectively, which demon-
strates that the MISPE process is a suitable method for the
sample pretreatment and the determination of rutin in TCMs.
Georgios Theodoridis28 evaluated for the first time several
conditions for MIP synthesis (rutin imprinted polymer) in terms
of the nature and proportion of reagents, with successful appli-
cation to food samples. Based on this information, in our work
we proposed an analogous method to apply polymeric rutin-MIP
to TCM sample purification and analyte (rutin) enrichment. The
comparison between the results obtained in this study and the
results related in ref. 28 is as follows: Firstly, we both prepared
the rutin-MIPs based on bulk polymerization and water-bath
heating. Secondly, the template elution process after the poly-
merization was ultrasonic extraction in our work, which was

Table 4 Recovery of rutin using methanol loading, methanol rinsing and acidic elution (n ¼ 3)a

Mean recovery (%)

Fractions Artemisia selengensis Turcz Eucommia Southernwood Saururus chinensis

Loading (methanol, 5 mL) 0.4 1.6 1.0 2.1

Rinsing (methanol, 5 mL) 5.6 7.2 5.2 6.8
Elution (methanol : acetic acid 85.3 80.2 79.2 80.2
(9 : 1, v/v), 5 mL)
Total 91.3 89.0 85.8 89.1
a
Spiked amount is the amount in the 5 mL sample solutions. The amount of rutin in 4 mL of four TCM extracts before spiking was 0.0168, 0.0208,
0.0196 and 0.0190 mg, respectively. The MISPE process was performed as in Section 2.5.

762 | Analyst, 2011, 136, 756–763 This journal is ª The Royal Society of Chemistry 2011

proven to be a more effective and time saving method than
Soxhlet extraction which was applied in ref. 28. Thirdly, the
proposed methods were both applied for the determination of
flavonoids in real samples. The recoveries analyzed in our work
were in the range of 85.8–91.3% in the determination of the TCM
samples, but this was not illustrated in the determination of the
real food samples in ref. 28.
4. Conclusion
The prepared rutin-MIPs were confirmed by SEM and FTIR,
characterized by HPLC-UV, and applied for the separation and
the enrichment of the active ingredient from traditional Chinese
Published on 13 December 2010 on http://pubs.rsc.org | doi:10.1039/C0AN00798F
medicines. Optimal elution parameters for enhanced recognition
properties of MIPs toward rutin were attained, which were very
important for saving time in the preparation of the MIPs. The
obtained polymer showed good selectivity and high adsorption
capacity. Thus, a method was successfully developed by using
MISPE coupled with HPLC for enrichment and analysis of rutin
Downloaded by University of Sussex on 18 June 2012
in TCMs. The high recoveries (85–91%) from the TCM samples
proved that the method was valid for the analysis of rutin in
different TCMs. From this point of view, the proposed MISPE-
HPLC method for selective separation and enrichment of rutin in
TCMs might develop into an analytical tool for various potential
applications.
Acknowledgements
The authors gratefully acknowledge the financial support of the
present work by the National Natural Science Foundation of
China (No. 20875025) and PCSIRT.
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