Chinese Chemical Letters 25 (2014) 1492–1494

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Chinese Chemical Letters

journal homepage: www.elsevier.com/locate/cclet

Original article

Rapid, simple and selective determination of 2,4-dinitrophenol by

molecularly imprinted spin column extraction coupled with
fluorescence detection
Min Fang a,b, Fang Lei a, Jia Zhou a, Yong-Ning Wu c, Zhi-Yong Gong a,b,*
a
Institute of Food Science and Engineering, Wuhan Polytechnic University, Wuhan 430023, China
b
Hubei Collaborative Innovation Center for Processing of Agricultural Products, Wuhan Polytechnic University, Wuhan 430023, China
c
China National Center for Food Safety Risk Assessment, Beijing 100050, China

A R T I C L E I N F O A B S T R A C T

Article history: A rapid, simple and selective method based on molecularly imprinted, spin column extraction coupled
Received 11 April 2014 with fluorescence detection was successfully established for the determination of 2,4-dinitrophenol in
Received in revised form 16 May 2014 serum samples. The 2,4-dinitrophenol imprinted polymers exhibited highly selective recognition for the
Accepted 11 June 2014
template molecule and the maximum adsorption capacity was 138.9 mg/g. The results indicated that
Available online 20 June 2014
when water is used as the loading solution, only 2,4-dinitrophenol could be adsorbed on the spin column
without the remaining structural analogs (2-nitrophenol, 4-nitrophenol and phenol). After eluting with
Keywords:
acetonitrile/acetic acid (9/1, v/v), 2,4-dinitrophenol in serum samples could be determined by using
Molecularly imprinted solid phase
extraction
the fluorescence spectrometer, based on the fluorescence enhancement of fluorescein by the template
Indirect fluorescence detection molecule. Under the optimal conditions, the spiked recovery ranged from 95.8% to 103.4% and the
Spin column detection limit was 1 nmol/L. The results confirmed the reliability and practicality of the protocol and
2,4-Dinitrophenol revealed a good perspective of this method for biological sample analysis.
ß 2014 Zhi-Yong Gong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights
reserved.

1. Introduction Currently, several strategies have been reported for the

The desire to look attractive is universal. Many young people
are trying every possible way to lose weight and keep fit. In the
1930s, 2,4-dinitrophenol (2,4-DNP) had been used as an oral
weight-control drug, since studies showed it can obviously
increase the basal metabolic rate, but soon its use for this purpose
was banned by the US FDA because of serious adverse effects,
including hyperthermia, cataracts and death [1,2]. Last year, an 18
year-old Indian student died in the UK after apparently taking the
banned slimming drug (2,4-DNP) that was popular among
bodybuilders to get rid of extra fat (http://www.dailymail.co.uk/
femail/article-2315433/Sarah-Houston-cause-death-Boiled-alive-
internet-slimming-pills-DNP.html). To date, there have been 62
published deaths in the medical literature attributed to 2,4-DNP
[3]. Thus, developing a rapid, simple and selective analytical
method is very important for monitoring of trace 2,4-DNP in
biological samples to avoid adverse events.
* Corresponding author at: Institute of Food Science and Engineering, Wuhan
Polytechnic University, Wuhan 430023, China.
E-mail address: gongzhiyong2013@gmail.com (Z.-Y. Gong).
determination of 2,4-DNP, such as high performance liquid
chromatography (HPLC) coupled with different detectors [4], gas
chromatography (GC) [5] and electrochemical sensor [6]. Although
chromatographic analysis has good sensitivity and accuracy,
they are not accessible in non-professional laboratories for routine
analysis due to the high cost of equipment and the requirement of
a skillful operator. For the electrochemical sensor, interference
of structural analogs is unavoidable because of the absent of
effective separation. To the best of our knowledge, few studies to
date have developed a rapid and low cost method for fluorescence
detection of 2,4-DNP. However, due to the lack of selectivity, a
more highly selective and effective sample pretreatment method
is required for the determination of 2,4-DNP.
Solid-phase extraction (SPE) has been proposed as a well-
established method for sample cleanup and concentration at trace
level [7,8]. Regarding spin column extraction, procedures such as
sample loading, washing, and elution of target analytes can be
accomplished by simple centrifugation and multiple samples can
be processed simultaneously in the same centrifuge. Thus, sample
pretreatment by using the spin column has many advantages: it is
simple to perform, requires a low-volume of extraction solvent,
and does not involve evaporation [9,10]. However, the main

http://dx.doi.org/10.1016/j.cclet.2014.06.015
1001-8417/ß 2014 Zhi-Yong Gong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
 M. Fang et al. / Chinese Chemical Letters 25 (2014) 1492–1494
problem associated with spin column extraction packed with
common stationary phases is the low selectivity for the analytes.
Molecularly imprinted polymers (MIPs) with selective recognition
cavities, compared with other recognition elements, possess
many promising characteristics, such as low cost and easy
synthesis, high stability to harsh chemical and physical conditions,
and excellent reusability [11,12]. Thus, MIPs have been
recognized as the promising and innovative adsorbents of spin
column in the determination of target molecules.
In this study, molecularly imprinted spin column extraction
coupled with fluorescence detection has been developed for the
determination of 2,4-dinitrophenol in serum samples. Compared
with the reported method, this method exhibited a lower detection
limit and excellent extraction efficiency.
2. Experimental
Hydrophilic MIPs were synthesized according to the literature
[13]. Briefly, MIPs were synthesized using 2,4-DNP as a template,
acrylamide and glycidyl methacrylate as functional monomers,
trimethylolpropane trimethylacrylate as a cross-linker and 2,2-
azobisisobutyronitrile as an initiator. Subsequently, polymeric
particles were dispersed in perchloric acid solution (10%, w/w) to
obtain a hydrophilic layer by opening the epoxide ring. Finally, the
particles were filtered, washed with water at pH 7.0 and dried
under vacuum overnight. Hydrophilic non-imprinted polymers
(NIPs) were prepared in the same way without the addition of the
template molecule. Human serum samples were kindly provided
by a team of volunteers, which were stored at 20 8C until analysis.
Serum samples were spiked with different concentration of the
analytes and then interfering proteins were removed by precipi-
tation and centrifugation (10,000 rpm, 5 min). The hydrophilic
MIPs were packed into empty spin column (GL Sciences, Tokyo,
Japan) and then this column was installed into a microtube (2 mL)
for sample loading and washing. Prior to extraction, conditioning
using acetonitrile and an aqueous solution was carried out and the
column was centrifuged at 5000 rpm for 1 min, respectively. The
samples (1 mL) were then applied to the conditioned spin column
and centrifuged at 5000 rpm for 2 min. The loading procedure
could be repeated by 5 times to obtain a high enrichment factor.
Subsequently, the spin column was rinsed with 1.0 mL of
acetonitrile to remove the sample matrix and structural analogs
by centrifugation. Finally, the spin column was installed into a new
microtube, and 2,4-DNP were eluted with 0.5 mL of acetonitrile/
acetic acid (9/1, v/v). After evaporating the solvent under a
nitrogen stream to dryness at 40 8C, 0.2 mL of sampling buffer (pH
9.0), composed of 10 mmol/L borate and 80 mmol/L sodium
dodecylsulfate, were added to redissolve the residue.
Because nitrophenol compounds could enhance the fluores-
cence intensities of fluorescein, sampling buffer and 0.21 mmol/L
fluorescein solution (5 mL) was transferred into 96-well plate and
determined by TECAN Infinite 200 (Tecan, San Jose, CA). Enhanced
fluorescence intensity of 2,4-DNP was represented as F = F1 F0.
Here, F1 and F0 were the fluorescence intensities of the systems
with and without 2,4-DNP, respectively. The standard curve
method was employed in quantification of trace amounts of 2,4-
DNP in spiked serum samples. Furthermore, serum samples were
analyzed using the proposed method and traditional LC–MS/MS
method [4] to verify the performance of the developed method for
2,4-DNP detection.
3. Results and discussion
The MIPs are tailor-made, stable polymers with molecular
recognition abilities, so that they are excellent materials for
providing selectivity in sample preparation. The synthesized
1493
Fig. 1. The effect of solvent on the extraction efficiency of hydrophilic MIPs-packed
spin column.
hydrophilic MIPs exhibited highly selective recognition for the
template molecule (imprinting factor was 3.34) and the maximum
adsorption capacity was 138.9 mg/g. For the phenol compounds,
the acidity decreased in the order of 2,4-DNP (pKa = 3.96), 4-
nitrophenol (4-NP, pKa = 7.16), 2-nitrophenol (2-NP, pKa = 7.17)
and phenol (pKa = 9.89). Therefore, ion-pair interaction, in addition
to hydrogen bonding, could be formed to enhance the recognition
ability of hydrophilic MIPs for 2,4-DNP. It was shown that 4-NP, 2-
NP and phenol could be removed in the washing procedure, which
provided an effective way for the sole determination of 2,4-DNP in
serum samples.
In this study, the extraction conditions were optimized by
analyzing 0.1 mmol/L sample buffer. Firstly, the effect of solvent on
the adsorption efficiency and recognition ability of hydrophilic
MIPs-packed spin column was investigated (Fig. 1). The results
indicated that nitrophenol compounds could be completely
retained on the spin column using water as a solvent. Acetonitrile
or methanol was unfavorable for the adsorption of 2-NP and 4-NP
on the hydrophilic MIPs-packed spin column, because of the low
polarity of nitrophenol compounds. Thus, water was used as the
loading solution and acetonitrile was used as washing solution.
Furthermore, addition of acetic acid solution could decrease the
ion-pair interaction of the template-functional monomer, and then
further decrease the adsorption efficiency of spin column for the
2,4-DNP. So, acetonitrile/acetic acid (9/1, v/v) was fixed as eluting
solution in this study. Subsequently, volumes of washing and
eluting solutions were also optimized (Fig. S1 in Supporting
information). Under the optimized condition, the recovery of 2,4-
DNP (0.01, 0.1, 1 mmol/L, respectively) on MIPs-packed spin
column ranged from 89.5% to 92.1%. In addition, the spin column
can be employed for 5 consecutive cycles without more treatment
being required between cycles. The column is only needed to be
preconditioned with acetonitrile and aqueous solution between
extractions. The target molecules (2,4-DNP) were not detected in
blank serum extracts from the reused spin column, indicating that
there was no carryover effect.
The results of chromatographic analysis indicated the sample
matrix was removed and the nitrophenol compounds were
retained on the spin column in the loading procedure (Fig. 2). In
the washing step, it was interesting to note that phenol, 4-NP and
2-NP were completely removed from the spin column using
acetonitrile as the washing solution, which was beneficial for
the sole detection of 2,4-DNP in serum samples and then
the elimination of fluorescence interference. Due to the fluores-
cence quenching caused by the acetonitrile/acetic acid solution,
1494 M. Fang et al. / Chinese Chemical Letters 25 (2014) 1492–1494
Fig. 2. The chromatograms obtained by direct injection of the spiked serum samples
(1 mmol/L) (A), washing solution (B), eluting solution (C) and standard solution
(D). Peaks: (1) phenol, (2) 4-NP, (3) 2,4-DNP, (4) 2-NP. HPLC-UV condition:
SunFireTM C18 column (150 mm  4.6 mm i.d., particle size 5 mm, Waters, Milford,
USA), acetonitrile/0.01 mol/L phosphate solution as the mobile phase and 279 nm
as measurement wavelength.
the eluting solution was evaporated to dryness and redissolved
using sampling buffer. The results demonstrated that an efficient
cleaning and high recovery of 2,4-DNP were achieved by the
hydrophilic MIPs extraction procedure, because of the highly
specific recognition ability of MIPs.
The mechanism of indirect fluorescence detection can be
described by the displacement of fluorescein by the nitrophenol
compounds. Subsequently, the experimental condition of fluores-
cence detection was optimized (Fig. S2 in Supporting information).
The pH of the sample buffer played an important role and the
maximum signal intensities for 2,4-DNP were obtained at pH 9.0.
Then the effect of the fluorescein concentration on the change of
fluorescence intensities was studied. The optimized concentration
was fixed at 0.21 mmol/L, which provided a relatively better signal-
to-noise ratio and high sensitivity.
Under the optimal conditions, analytical parameters such as the
linear range, the correlation coefficient and the detection limit
(LOD) were studied by analyzing spiked serum samples. It was
shown that the change of fluorescence intensities against 2,4-DNP
concentration was linear in the range of 2.5 nmol/L to 5000 nmol/L,
and the correlation coefficient was 0.992. The relative standard
deviation (RSD) for the measurement of each data point was less
than 8.7%. The detection limit (LOD) was 1 nmol/L, which was
lower than those of the reported method for 2,4-DNP detection
using MIPs as the sorbents (40, 50 and 400 nmol/L, respectively
[6,14,15]). Assay reproducibility was investigated by analyzing a
spiked serum sample (containing 10 nmol/L) five times. The
results showed that RSD value was 8.6%, indicating an acceptable
level of precision.
Spiked serum samples with different concentrations of 2,4-DNP
were assayed to evaluate the analytical applicability of the
proposed method. The results indicated that spiked recoveries
were changed from 95.8% to 103.4%. To further investigate the
practical perspective of the proposed method, eight serum samples
were analyzed by this method and the traditional LC–MS/MS
method. The results obtained by the proposed method were
linearly correlated to those by traditional method (r = 0.9964) (Fig.
S3 in Supporting information). Thus, this method provided a
promising method for monitoring of 2,4-DNP in serum samples to
control the banned slimming drug.
4. Conclusion
A rapid, simple and selective method has been developed for
the determination of 2,4-dinitrophenol in serum samples based on
molecularly imprinted, spin column extraction coupled with
fluorescence detection. It was shown that only 2,4-dinitrophenol
remained on the spin column without the interference of structural
analogs. In a word, this method can be used for monitoring of 2,4-
DNP in serum samples to control the banned slimming drug.
Acknowledgment
This work was supported by National Key Technology R&D
Program in the 11th Five-Year Plan of China (No. 2009BADB9B02)
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cclet.2014.06.015.
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