980 J. Sep. Sci.

2013, 36, 980–985

Ziru Lian Research Article

Jiangtao Wang

Key Laboratory of Marine Determination of crystal violet in seawater

Chemistry Theory and
Technology, Ministry of
EducationOcean University of
China, Qingdao, China
Received October 8, 2012
Revised November 14, 2012
Accepted November 21, 2012
and seafood samples through off-line
molecularly imprinted SPE followed by HPLC
with diode-array detection
A highly selective sample cleanup procedure combined with molecularly imprinted SPE
was developed for the isolation of crystal violet from seawater and seafood samples. The
molecularly imprinted polymer was prepared using crystal violet as the template molecule,
methacrylic acid as the functional monomer, and ethylene glycol dimethacrylate as the
cross-linker. The crystal violet-imprinted polymer was used as the selective sorbent for the
SPE of crystal violet. An off-line molecularly imprinted SPE method followed by HPLC with
diode-array detection for the analysis of crystal violet was also established. Good linearity on
the molecularly imprinted SPE columns was obtained from 0 to 200 ␮g/L (R2 > 0.99). The
result demonstrated that the proposed method can be used for the direct determination of
crystal violet in seawater and seafood samples. Finally, five samples were analyzed and the
following crystal violet concentrations were obtained: 0.92 and 0.52 ␮g/L in two seawater
samples, as well as 0.36 and 0.27 ␮g/kg in two seafood samples. There is no crystal violet
detected in the third seawater sample.

Keywords: Crystal violet / Molecularly imprinted polymer / Seafood / Seawater /

SPE
DOI 10.1002/jssc.201200939

1 Introduction MS [10–14]. However, these methods are troublesome and

Crystal violet (CV; also known as Methyl Violet 10B and
hexamethyl pararosaniline chloride) is a triphenylmethane
dye [1, 2] extensively used in animal feeds because of its ef-
fective resistance to important protozoan and fungal infec-
tions [3,4]. CV is readily absorbed by fish tissue through water
exposure and is metabolically reduced by fish into leucocrystal
violet [5]. Studies have shown that CV and leucocrystal vio-
let are potential carcinogens, teratogens, and mutagens [6–9].
Thus, CV is highly restricted as an aquaculture veterinary
drug in many countries, but is still used illegally because of
its low cost and high efficacy. Considering the adverse effect
of CV on aquatic animals and human health, sensitive and
selective analytical methods are needed for CV determination
in real samples, such as water samples and fish feed samples.
Several methods of determining CV have been re-
ported, including immunoassay method, LC-MS, and GC–
Correspondence: Dr. Jiangtao Wang, Key Laboratory of Marine
Chemistry Theory and Technology, Ministry of Education, Ocean
University of China, Qingdao 266100, China
E-mail: jtwang@ouc.edu.cn
Fax: +8653266782506
Abbreviations: AIBN, 2,2-azoisobutyronitrile; CV, crystal
violet; EGDMA, ethylene glycol dimethacrylate; MAA,
methacrylic acid; MIP, molecularly imprinted polymer;
MISPE, molecularly imprinted SPE; NIP, nonimprinted poly-
mer
involve time-consuming sample preparation. SPE is an ex-
traction method that uses a solid phase and a liquid phase
to isolate one or one type of analyte from a solution. SPE is
usually used to clean up a sample before performing a chro-
matographic or analytical method to quantify the amount
of analyte(s) in the sample [15, 16]. One of the main disad-
vantages of the classical SPE sorbents (C8, C18, etc.) is the
low selectivity. An increasing number of new sorbents such
as molecularly imprinted polymers (MIPs) and immunosor-
bents are being developed to meet the requirement of selec-
tivity. Coupling MIP with SPE can combine the advantages of
both molecular recognition and traditional separation meth-
ods. Thus, molecularly imprinted SPE (MISPE) has the high
specificity, selectivity, and sensitivity of the molecular recog-
nition mechanism, as well as the high resolving power of
separation methods [17–19]. At present, most methods for
CV determination are established for the analysis of seafood
and environmental samples such as water supply and river
water samples [20,21]; however, few methods are for seawater
samples.
In this study, CV-imprinted polymer was prepared by
bulk polymerization using CV as the template molecule,
methacrylic acid (MAA) as the functional monomer, and
ethylene glycol dimethacrylate (EGDMA) as the cross-linker.
The characteristics of the obtained polymers were analyzed
under a scanning electron microscope. The imprinted poly-
mer showed high affinity to CV, and was successfully ap-
plied as a special SPE sorbent for the selective extraction


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J. Sep. Sci. 2013, 36, 980–985
of CV from seawater and seafood samples. To our knowl-
edge, this work is the first attempt in using MIP as selec-
tive SPE sorbents for the determination of CV in seawater
samples.
2 Experimental
2.1 Standards and reagents
CV was purchased from Sinopharm Chemical Reagent Com-
pany (Chengdu, China). MAA and 2,2-azoisobutyronitrile
(AIBN) were obtained from Kermel Chemical Co. (Tianjin,
China). EGDMA was from Alfa. MAA and EGDMA were pu-
rified before use to remove the polymerization inhibitor, and
AIBN was recrystallized prior to use. ACN and methanol were
HPLC grade from Merck. Unless specified, all reagents were
analytical grade. All water used was obtained from a Milli-Q
purification system (Millipore, Bedford, MA, USA). A 1.0 g/L
CV (Aladdin Co., Shanghai) stock solution was prepared in
methanol and placed in the dark at 4⬚C.
2.2 HPLC conditions
HPLC analysis was performed on a Hitachi L-2000 series
HPLC system containing an L-2130 binary pump, an L-2200
autosampler, an L-2300 column compartment, and an L-
2455 diode-array detector monitoring the effluent at 588 nm.
The analytical column was a LaChrom C18 column (250 ×
4.6 mm, 5 ␮m; Hitachi, Japan). The column thermostat was
set at 25⬚C. The mobile phase was HAc/NH4 Ac (0.1 mmol/L,
pH 4.5)–ACN (3:7 v/v), and the flow rate was 1.0 mL/min.
2.3 Preparation of the MIP and nonimprinted
polymer (NIP)
MIP was prepared using the bulk polymerization method
by dissolving 0.5 mmol of CV, 2 mmol of the functional
monomer MAA, and 20 mmol of the cross-linker EGDMA
in 5 mL of methanol in a 50 mL borosilicate glass bottle
with a rubber cap. The mixture was rotated at 150 rpm for
6 h at 50⬚C to form a complex of the template molecule
and monomers. After adding 20 mg of AIBN, the solu-
tion was saturated with dry nitrogen for 15 min and the
bottle was placed in a thermostated water bath at 60⬚C
for 24 h. After polymerization, the polymer was ground
with a mortar and pestle, sieved between 140- and 200-
mesh screens to obtain particles with sizes between 75 and
106 ␮m, and then repeatedly suspended in acetone to remove
the small particles. The product was extracted with methanol
containing 5% acetic acid using a Soxhlet apparatus until
the template was removed. Subsequently, the product was
washed with methanol thrice and dried in a vacuum at room
temperature.

C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Other Techniques 981
The NIP particles were prepared and washed using the
same formulation but without the addition of the template
CV.
2.4 Morphology observation
The surface morphology of the particles was observed using a
Hitachi S-4800 cold field emission scanning electron micro-
scope (Tokyo, Japan). All samples were prepared by wetting
the slide glass with a small drop of the diluted particle dis-
persion. Before SEM experiments, the dried specimen was
coated under vacuum with a thin layer of gold.
2.5 Steady-state binding studies
About 20 mg of the bulk polymer particles was weighed and
placed in a 4 mL vial. Then, 2 mL of a CV methanol–water
(40:60 v/v) solution with known concentration (0–150 mg/L)
was mixed with the polymer. The mixture was slightly shaken
in a horizontal shaker for 24 h at 25⬚C, and then centrifuged
for 5 min at 4000 rpm. The final CV concentrations were
determined by HPLC with a diode-array detector at 588 nm.
The amount of CV bound to the polymer was calculated by
subtracting the amount of free CV from the initial amount
added to the mixture. CV standard solutions in methanol–
water (40:60 v/v) were prepared, and a standard calibration
curve for CV analysis was constructed. The curve was linear
(R2 = 0.998) within the range 0–150 mg/L.
2.6 Preparation of the MISPE column
The MISPE column was prepared by packing the dry MIP
(50 mg) in a 1.0 mL glass syringe (2 cm × 0.9 cm id). The
syringe tube was thoroughly cleaned and dried, and two sieve
plates were attached to the bottom and top ends, respectively.
2.7 Sample preparation
Seawater samples were collected from three sites: #1, Shi-
laoren fish farms (120.49 E, 36.08 N); #2, Qingdao coastal sea
(120.29 E, 36.01 N); and #3, northwest Pacific (117.40 E, 20.06
N); 10 mL of seawater samples was filtered through a 0.22 ␮m
filter before use, and spiked with CV at concentrations of 5,
10, 15, and 20 ␮g/L for loading.
Two kinds of seafood were obtained from a supermarket
in Qingdao: #4, fish (Psetta maxima); and #5, shrimp (Trachy-
penaeus curvirostris). The edible parts were stripped away and
a meat chopper was used to grind these parts; 1 g of seafood
samples was mixed with 5 mL of ACN and spiked with CV at
concentrations of 1, 3, 5, and 10 ␮g/L. The mixture was stirred
to homogeneity with a glass rod, and then ultrasonicated for
10 min at room temperature. Afterwards, it was centrifuged
at 4000 rpm for 5 min. The extract was vaporized to dryness
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982 Z. Lian and J. Wang J. Sep. Sci. 2013, 36, 980–985

under vacuum at 40⬚C. The residue was then reconstituted in

5 mL of methanol–water (40:60 v/v) for loading.

2.8 SPE for CV standard solutions

Before loading the analyte, the MISPE column was previously

conditioned with 2 mL of deionized water and methanol suc-
cessively. After the CV standard solutions at different con-
centrations were passed through the columns at a flow rate
of 0.25 mL/min, the columns were washed with 1 mL of
methanol–water (70:30 v/v) at the same flow rate. The analyte
retained on the sorbent was eluted with 1 mL of methanol
containing 5% acetic acid for further HPLC analysis.

2.9 SPE for spiked seawater and seafood samples

To validate the performance of MISPE, the MIP and cor-

responding NIP were individually packed into cartridges to
compare their efficiency in extracting CV from seawater and
seafood samples. Three replicate spiked seafood and seawa-
ter samples in Section 2.7 were passed through the MISPE
(or non-imprinted solid-phase extraction; NISPE) column,
and then the columns were washed and eluted as described.
Finally, the elution fractions were collected for subsequent
HPLC analysis.
Figure 1. SEM photograph of MIP (A) and NIP (B).
3 Results and discussion

3.1 Characterization of the MIP and NIP
For successful imprinting, adequate template: functional
monomer molar ratios must be used [22, 23]. In most cases,
the molar ratio between template and functional monomers
can be approximately set from 1:3 to 1:5 (1:4 in this exper-
iment). Considering the restrictions of the MIP mechanical
strength, the molar ratio of monomer to cross-linker was
set at 1:10. MAA was selected as the functional monomer
because it is suitable for hydrogen bond or ionic bond in-
teraction in the porogen prior to polymerization [24]. Thus,
a stable donor–receptor complex between the template and
functional monomer was formed during the imprinting pro-
cess. The existence of the complex led to the formation of
well-defined specific binding sites in the MIP, followed by
thermal polymerization with AIBN as a free radical initiator.
The half-life of AIBN at 60⬚C was about 10 h, which was appro-
priate for polymerization. The cross-linker EGDMA formed
a “frozen” spatial structure. In this study, the MIP for CV
was prepared by bulk polymerization. The ionic bond inter-
action between the functional monomer (carboxy group) and
the target (amine) was probably responsible for the selective
binding of CV [25].
The surface morphologies of polymers were studied by
SEM (Fig. 1). The SEM images revealed that the MIP seemed
more dense and homogenous with more 3D pores, whereas
the surface of the NIP was irregular and globular without
dense 3D cavities. The bound specificity of the MIP depended
on the selectivity of the binding cavities toward CV. Thus,
the uniform and more open structure obviously favored the
embedding of the template molecule.
3.2 Evaluation of the MIP recognition efficiency
After synthesis, equilibrium binding experiments were per-
formed at room temperature with MIP and NIP. Figure 2
shows the experimental binding isotherms of the MIP (or
NIP) amount versus the CV concentrations. Within 0–150
mg/L CV, the MIP exhibited a higher capacity for CV than
the NIP. The weak adsorption of template onto the NIP was
due to the nonspecific interaction with the polymer matrix.
The data of the static absorption experiment were further pro-
cessed with the Scatchard equation to estimate the binding
parameters of the MIP. A Scatchard plot was constructed by
plotting the ratios of the bound CV to free CV concentration
against the bound concentration.
As shown in Fig. 3, one straight line fitting the Scatchard
equation B/F = (Bmax − B)/Kd can be drawn, and yielded
two typical binding parameters. The dissociation constant Kd
(90.91 mg/L) and maximum number of binding sites Bmax
(8.78 mg/g) for the MIP were calculated from the Scatchard
equation. Likewise, the dissociation constant (117.78 mg/L)
and maximum number of binding sites (6.99 mg/g) for NIP


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J. Sep. Sci. 2013, 36, 980–985
Figure 2. Binding isotherm for MIP and NIP particles. Experiment
was conducted by the addition of 20 mg of particles in 2 mL of
CV methanol–water (40:60 v/v) solution at 25⬚C.
Figure 3. Scatchard plot over 60–150 mg/L concentration range.
were calculated from the NIP binding data. A large dissoci-
ation constant meant low affinity with the target. Compared
with NIP, MIP had a higher binding capacity and higher affin-
ity to CV. The difference between the CV binding affinities of
the MIP and NIP clearly indicated the role of the imprinting
process in the formation of specific binding sites. The linear-
ity on the MIP indicated that a class of binding sites existed
in the imprinted polymers and that the recognition sites for
the template molecules were selective.
3.3 SPE for CV standard solutions
To minimize the nonspecific component of the interaction
between CV and polymeric matrices, a washing step was per-
formed and methanol with different percentages of water (0,
5, 10, 30, 50, 80, and 100%) was tested. The results showed
that 11% CV was recovered in the washing fraction of the MIP
columns using methanol–water (70:30 v/v) as the washing so-
lution. In the NIP columns, this percentage increased to 85%

C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Other Techniques 983
Figure 4. HPLC chromatograms of standard solution at the con-
centration of 25 ␮g/L (A), no. 5 shrimp sample spiked with
1 ␮g/L CV after extracted by MISPE column (B), and NISPE (C)
with an enrichment factor of 5.
because of the absence of specific interactions between the
template and polymeric materials. Thus, methanol contain-
ing 30% water was chosen to optimize the washing condition.
The optimized elution was increased using methanol–acetic
acid (95:5 v/v), and the highest eluting effect was achieved
under this condition.
The recoveries of the standard solutions containing CV
(0–200 ␮g/L) were calculated. The results showed that the
MIP columns had good recoveries (>89.4%, data not shown)
for CV at different concentrations. A standard calibration
curve for CV analysis was constructed and found to be linear
(R2 > 0.99) within 0–200 ␮g/L. Thus, this method can be
used to detect some polluted samples.
3.4 SPE for spiked seawater and seafood samples
Figure 4A shows the HPLC chromatogram of the CV stan-
dard solution. The extraction from the #5 shrimp sample
successfully cleaned up the seafood matrix and enriched it
after MISPE, thereby allowing the extraction of CV with high
selectivity (Fig. 4B). On the other hand, the NIP columns
showed no such selectivity (Fig. 4C) because most of the CV
was washed away during the washing process. The result
indicated that the recovery of the spiked samples at differ-
ent concentrations was 74.27–92.96%, and the RSDs ranged
from 1.72 to 4.69% (n = 3). The high percentage of recovery
indicated no interference in the seafood samples at differ-
ent formulations, which confirmed the high efficiency of the
extraction procedure.
As shown in Fig. 5A, the complexity of the seawater back-
ground was evident and no signal response from CV was
detected. The extraction from the MIP column successfully
cleaned up the seawater matrix and showed a high selec-
tivity to CV (Fig. 5B). By contrast, the NIP column had no
clear signal response (Fig. 5C). Hence, MIP was a simple and
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984 Z. Lian and J. Wang J. Sep. Sci. 2013, 36, 980–985

Table 2. Regeneration property experiment of the MISPE column;

the CV concentration used for binding was 100 ␮g/L

Added Detected Recovery RSD

conc. (␮g/L) conc.a) (␮g/L) (%) (%)

100 472.6 94.5 1.83

478.4 95.7
481.2 96.2
461.5 92.3

a) The extractions were conducted with an enrichment factor of

5.

the sample loading solution, Y is the signal intensity of CV in

the elution, and the ratio of intercept to slope represents the
Figure 5. HPLC chromatograms of no. 1 seawater sample spiked
with 5 ␮g/L CV before (A) and after extracted by MISPE column
actual concentration of CV in the samples. The CV concentra-
(B), and NISPE (C) with an enrichment factor of 10. tions in seawater from the Shilaoren fish farms and Qingdao
coastal sea were 0.92 and 0.52 ␮g/L, and the RSDs (n = 3)
Table 1. Calibration curve and detected concentrations in real were 3.80 and 4.62%, respectively. There is no CV detected
samples after MISPE enrichment from northwest Pacific seawater sample. The CV concentra-
tions in fish and shrimp tissues were 0.36 and 0.27 ␮g/kg,
Sample Calibration R2 Detected RSD (%) and the RSDs (n = 3) were 4.31 and 2.74%, respectively. The
no. curve concentrations concentrations were below the maximum residue limits es-
tablished by the US Food Drug Administration (FDA) and
1 y = 267.16 x + 247 0.995 0.92 ␮g/L 3.80
the European Union. [26, 27].
2 y = 349.58 x + 181 0.997 0.52 ␮g/L 4.62
The LOD was obtained from the signal-to-noise ra-
3 y = 472.82 x – 12 0.996 N.D -
4 y = 284.18 x + 103.1 0.991 0.36 ␮g/kg 4.31
tio (S/N). In this work, the baseline noise was measured
5 y = 312.86 x + 84.6 0.992 0.27 ␮g/kg 2.74 from a chromatogram of seawater and seafood samples
without CV. S/N = 3 was used to calculate LOD of the
calibration curve. The LOD of the seawater sample was
0.1 ␮g/L and the LOQ (S/N = 10) was 0.35 ␮g/L. The LOD
straightforward technique for the direct analysis of CV in sea- of the seafood sample was 0.05 ␮g/kg and the LOQ was
water without requiring a time-consuming sample cleanup 0.17 ␮g/kg.
process. The result showed that the recoveries of the spiked
samples at different concentrations ranged from 32.6 to
62.3%, and RSDs ranged from 2.11 to 4.71% (n = 3). The 3.6 Reproducibility of the MIP
mean values of the recoveries in samples #1 and #2 were
34.2 and 45.1%, respectively. By contrast, the mean value Regeneration is one of the important advantages of MIP. The
in sample #3 at different concentrations was 60.6%, which MIP can be used again to adsorb the analyte of interest after
was obviously higher than the others. The salinity, pH, and regeneration. To reuse the polymers after each extraction, a
dissolved organic matter in seawater possibly influenced the short regeneration step was performed by washing the poly-
recovery. Both seawater samples #1 and #2 collected from mers with 4 mL of methanol–acetic acid (95:5 v/v). The results
the coastal sea in Qingdao whose matrices were more com- (Table 2) showed that the MIP can be used up to four times
plicated than that of sample #3 from northwest Pacific, were and maintain their adsorption capacity at an almost constant
seriously affected by anthropogenic activities. This may be the value. This result confirmed the reliability and efficacy of the
crucial factor of different recoveries in different area. This ap- proposed method for the analysis of CV residues in polluted
plication is the first attempt on seawater samples, and further samples.
investigations are warranted.
4 Conclusion

3.5 Determination of CV in seawater and seafood
samples
The proposed method was used to analyze CV in seawater
and seafood samples. CV was detected in five samples. The
calibration curves at different concentrations of spiked sam-
ples are listed in Table 1, where X is the CV concentration of
CV-imprinted polymer was prepared by bulk polymerization
using MAA as the functional monomer and EGDMA as the
cross-linker. The obtained MIP showed good selectivity and
affinity for CV. Thus, a method was successfully developed for
the analysis of CV in seawater and seafood samples using the
MIP as SPE sorbents coupled with HPLC. The recovery values
and precision proved that the proposed method was valid for


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J. Sep. Sci. 2013, 36, 980–985 Other Techniques 985

the analysis of CV in different matrices. The detected CV
concentrations in seawater and seafood samples were below
the maximum residue limits established by the US FDA and
European Union.
This work was supported by the Natural Science Foundation
of China (41076065), the Major State Basic Research Develop-
ment Program of China (973 Program; 2010CB428701) and
the Fundamental Research Funds for the Central Universities
(201261004). The authors gratefully acknowledge Associate Prof.
Junhui Chen of the Qingdao Key Laboratory of Analytical Tech-
nology Development and Standardization of Chinese Medicines,
The First Institute of Oceanography, for his valuable suggestions
and assistance in revising this paper.
The authors have declared no conflict of interest.
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