Microchemical Journal 150 (2019) 104162

Contents lists available at ScienceDirect

Microchemical Journal
journal homepage: www.elsevier.com/locate/microc

Microextraction by packed sorbent using a new restricted molecularly T

imprinted polymer for the determination of estrogens from human urine
samples
Hanna Leijoto de Oliveira, Leila Suleimara Teixeira, Laíse Aparecida Fonseca Dinali,
Bruna Carneiro Pires, Nathália Soares Simões, Keyller Bastos Borges
⁎

Departamento de Ciências Naturais, Universidade Federal de São João del-Rei (UFSJ), Campus Dom Bosco, Praça Dom Helvécio 74, Fábricas, 36301-160 São João del-
Rei, Minas Gerais, Brazil

ARTICLE INFO ABSTRACT

Keywords: Microextraction by packed sorbent (MEPS) was employed with a new restricted access molecularly imprinted
Microextraction by packed sorbent polymer (MIP) for the determination of estrone (E1) and estriol (E3) from human urine samples. Several
Molecularly imprinted polymer parameters affecting the extraction efficiency during the procedure of sample preparation were systematically
Restricted access material optimized, including washing solvent, sample volume, amount of sorption material, elution solution, eluent
Estriol
volume, number of cycles of aspersion/dispersion and sample pH. Mean recoveries with their respective relative
Estrone
standard deviations (RSD%) for E1 and E3 were 79.5 ± 7.1% and 69.5 ± 1.5%, respectively. The method was
Human urine
linear in the concentration range of 100 to 1100 ng mL−1 for each hormone, with correlation coefficient of
0.9954 and 0.9952 for E1 and E3, respectively. The developed method was successfully applied in urine sample
from volunteer pregnant.

1. Introduction attracted the attention of researchers [3,4].

Hormones have two classifications as to their nature: natural or
artificial. The hormones produced from cholesterol have three main
groups: estrogens, androgens or progestogens [1]. Steroid hormones
have in common a structure composed of a pentagonal ring and three
hexagonal rings. Estrogens are characterized by their phenolic ring,
which is responsible for biological activity such as human growth and
reproduction. Estrone (E1) and estriol (E3) chemical structures are
shown in Fig. S1. Although they have similar composition, E1 has a
carbonyl at carbon 17 and E3 has two hydroxyls at carbons 16 and 17,
respectively [2]. Currently, these estrogens are a type of prescription
drug for the control of symptoms that involve menopause, physiological
disorders and in the treatment of prostate and breast cancer. Because of
the increasing use and potential risks to human health, estrogens have
Since the concentrations of these estrogens in human urine are very
low, it is important to isolate and pre-concentrate these analytes using
an efficient sample preparation method prior to analysis. In 2003,
Abdel-Rehim introduced a new sample preparation technique,
a miniaturization of conventional Solid Phase Extraction (SPE),
Microextraction by Packed Sorbent (MEPS). Both the sample and eluent
volume were reduced by about 10 to 100 times, i.e., from mL to μL
(10–1000 μL). MEPS is suitable for both small sample volumes (biolo-
gical samples) and also for larger sample volumes (environmental
samples). In this technique a micro-column is filled with 1–4 mg of
sorption material and connected to the needle of a micro-syringe. There
are selective phases with different extraction mechanisms such as ad-
sorption or partition, available commercially, among them being:
normal phase, reverse phase, mixed phase, strong cation exchanger,

Abbreviations: Bovine Serum Albumin, BSA; Estrone, E1; Estradiol, E2; Estriol, E3; Ethyleneglycol Dimethacrylate, EGDMA; Functional Monomers, FM; Fourier
Transform Infrared, FTIR; Hydrophilic Monomer, HM; Relative Selectivity, k'; Selectivity, k; Distribution Constants, kd; Limit of Detection, LOD; Limit of
Quantification, LOQ; Methacrylic Acid, MAA; Microextraction by Packed Sorbent, MEPS; Molecularly Imprinted Polymer, MIP; Mesoporous Molecularly Imprinted
Polymer, MMIP; Mesoporous Molecularly Imprinted Polymer encapsulated with Hydrophilic Monomer, MMIP-HM; Point of Zero Charge, PZC; Restricted Access
Material, RAM; Mesoporous Molecularly Imprinted Polymer double coated with Hydrophilic Monomer and Bovine Serum Albumin, RA-MMIP-HM-BSA; Mesoporous
Non-Imprinted Polymer double coated with Hydrophilic Monomer and Bovine Serum Albumin, RA-MNIP-HM-BSA; Relative Error, RE%; Relative Standard Deviation,
RSD%; Scanning Electron Microscopy, SEM; Solid-Phase Extraction, SPE; Thermogravimetric Analysis, TGA; Template Molecule, TM; UV–Vis, UV
⁎
Corresponding author.
E-mail address: keyller@ufsj.edu.br (K.B. Borges).

https://doi.org/10.1016/j.microc.2019.104162
Received 12 April 2019; Received in revised form 31 July 2019; Accepted 2 August 2019
Available online 03 August 2019
0026-265X/ © 2019 Elsevier B.V. All rights reserved.
H.L. de Oliveira, et al.
and strong anions. However, the variety of extractive phases for com-
mercially available MEPS is less than the phase variation for conven-
tional SPE [5–7].
Molecular imprinted polymer (MIP) is provided with specific re-
cognition sites, stereochemically shaped from the template molecule
(TM) (e.g., a drug of interest) or the dummy template molecules (i.e.
analogues molecules). The synthesis step occurs after the formation of a
complex between the functional monomer (FM) and TM. With this, the
terminations of the ligands present in the FM are positioned at points
that correspond to the points from the TM. Routinely, the selection of
the best imprinting conditions are mainly performed in an empirical
way based on a trial-and-error method and chemical intuition [8].
Therefore, the computer-aided studies can be an effective and useful
tool to obtain the optimal pre-polymerization conditions, which can to
improve the properties of MIP and to save substantial labor and la-
boratory resources [8–11]. Subsequently there is formation of bonds,
and consequently the obtaining of a material with an important selec-
tive capacity for a TM or molecules with similar chemical structure
[12–15]. Currently there is a new adsorbent material that is the result of
the combination of MIP with Restricted Access Material (RAM) called
RAM-MIP [16]. In 2012, Figueiredo et al. introduced a restricted access
molecularly imprinted polymer coated with bovine serum albumin
(RAMIP-BSA). Initially the MIP was synthesized and then covered with
two hydrophilic monomers (hydroxy methyl methacrylate and glycerol
dimethacrylate). This material was subjected to a cross-linking reaction
with glutaraldehyde and BSA to obtain surface protein encapsulation of
MIP. The final material obtained was packaged in a column exchange
system, which showed an excellent and long-lasting ability to exclude
proteins from biological samples. However, currently no published
work can be found in the literature on RAMIP-BSA for use in MEPS,
particularly in urine samples [17,18].
Therefore, the aim of this work was to develop a new restricted
access MIP for the determination of E1 and E3 from human urine
samples by MEPS using the HPLC-UV system in the final stage of de-
termination. A novel material was synthetized using a surfactant to
obtain a restricted access mesoporous molecularly imprinted polymer
(MMIP) coated with hydrophilic monomers (HM) and bovine serum
albumin (BSA), termed RA-MMIP-HM-BSA. The main parameters that
affected the sample preparation were evaluated and discussed in detail.
After sample preparation and validation, the method was applied in the
analysis of a urine sample from a pregnant volunteer.
2. Experimental
2.1. Reagents and solvents
Methacrylic acid (MAA), ethyleneglycol dimethacrylate (EGDMA),
glycerol dimethacrylate, 2-hydroxyethyl methacrylate, benzalkonium
chloride, and hexane were purchased from Sigma-Aldrich® (St. Louis,
Missouri, USA). Prior to performing the adsorbent materials synthesis
procedure, MAA and EGDMA were purified according literature
[19–21]. 4,4′-azobis(4-cyanovaleric acid) from Santa Cruz Bio-
technology® (Dallas, Texas, USA), chloroform and dichloromethane
from Tedia® (Fairfield, Ohio, USA), sodium borohydride, gluter-
aldehyde, and tetraethyl orthosilicate (TEOS) from Merck® (Darmstadt,
Hessen, Germany), and BSA from Acros Organics® (Morris Plains, New
Jersey, USA). Acetonitrile and methanol were acquired from Dynamic®
(Diadema, São Paulo, Brazil). Ammonium hydroxide was purchased
from Quemis® (Indaiatuba, São Paulo, Brazil), sodium hydroxide
from Synth® (Diadema, São Paulo, Brazil) and acetic acid from Dy-
namic® (Diadema, São Paulo, Brazil). The water was distilled and
purified using a Millipore Milli-Q Plus® system (Bedford, Massachusetts,
USA). All reagents and solvents employed were of analytical and
HPLC grade.
2
Microchemical Journal 150 (2019) 104162
2.2. Preparation of stock and standard solutions
Estrogen standards (E1 and E3 - United States Pharmacopoeia - USP
Reference Standard) were obtained from Sigma Aldrich. A total of
0.0101 g of E1 was placed in a 10 mL volumetric flask and the volume
was quenched with methanol to give a standard solution of 1.0 mg mL−1.
The same procedure was also performed for E3 (0.0103 g). Standard
solutions of 1.0 mg mL−1 were diluted to yield: 100, 250, 400, 550, 700,
950 and 1100 ng mL−1 of each analyte. Standard solutions of both es-
trogens were stored in the absence of light (amber bottle) and in the
freezer at −20 °C.
2.3. Instrumentation
Chromatographic analyzes were performed using Agilent HPLC (Palo
Alto, CA, USA), consisting of a quaternary pump model 1260 (G1311B),
a thermostat model 1290 (G1330B), an automatic injector model 1260
Hip ALS (G1367E), a model 1290 TCC column oven (G1316C) and a
VWD model 1260 ultraviolet (UV) detector (G1314F). Data was acquired
and the instrument was software controlled by an Agilent Open LAB
Chromatography Data System. Chromatographic analysis of estrogens
was obtained by a C18 column (250 mm × 4.6 mm, 5 μm) (Gemini
Phenomenex®) with mobile phase composed of methanol: acetonitrile:
ultrapure water (60: 20: 20, v/v/v), flow rate of 1.0 mL min−1, wave-
length at 290 nm, room temperature, and injection volume of 20 μL.
Fourier Transform Infrared (FTIR) analyzes were performed using a
Fourier transform spectrometer (Shimadzu, IRAffinity-1, Kyoto, Japan),
operating between 4000 and 400 cm−1, by the conventional method
(KBr pellet), using about 5% by weight. Scanning Electron Microscopy
(SEM) images were obtained using a TM3000 Hitachi Analytical Table
Top microscope (Tarrytown, NY, USA) with acceleration of tension at
20 kV; the sample was applied on a carbon tape. Thermogravimetric
Analyzes (TGA) were performed in a thermocouple 2950 Thermal
Analysis Instrument, (TA Instruments, New Castle, DE, USA) with a
heating rate of 10 °C min−1 under nitrogen flow (50 mL min−1) from 25
to 600 °C. Adsorption-desorption isotherms of N2 were obtained
by a surface area and porosity analyzer, using Autosorb-iQ2. The
Barrett–Joyner–Halenda (BJH) estimated pore volumes and sizes. The
specific areas were determined using the Brunauer–Emmett–Teller (BET)
equation.
Protein exclusion analysis was determined by molecular absorption
spectroscopy in the visible region employing a Varian-Agilent Cary
5000 Probe UV–vis spectrophotometer (Agilent Technologies, Palo
Alto, California, USA) and a quartz cuvette with an optical path of
10 mm. Data were acquired between the range of 200 to 400 nm.
2.4. Synthesis of adsorbent materials
For the preparation of the MMIP, 1 mmol of E1, 1 mmol of ben-
zalkonium chloride (surfactant), and 4 mmol of MAA were dissolved in
10 mL of chloroform and sonicated for 5 min to form the pre-poly-
merization complex. This solution was transferred to an amber flask
containing 84 mg of 4,4′-azobis(4-cyanovaleric acid) and 20 mmol of
EGDMA, and placed in an ultrasonic bath for 10 min. It was then left in
an oven at 80 °C for 24 h. The obtained polymer was triturated, sieved
in a 100-mesh sieve and washed for E1 removal with methanol: acetic
acid solution (9: 1, v/v). After washing, the MIP was dried in oven at
60 °C for 48 h.
To obtain a restricted access mesoporous molecularly imprinted
polymer coated with hydrophilic monomers (RA-MMIP-HM), the MMIP
was coated only with HM as follows: 1.0 g MMIP, 7.5 mmol of 2-hy-
droxyethyl methacrylate, 0.5 mmol of glycerol dimethacrylate and
35 mL of chloroform were placed in a 50 mL Falcon tube. The tube was
sonicated for 10 min and then left in an oven at 60 °C for 24 h. After
drying, the RA-MMIP-HM was washed again with ultrapure water:
methanol solution (1: 1, v/v) and finally dried at 60 °C for 24 h.
H.L. de Oliveira, et al.
To obtain RA-MMIP-HM-BSA, the material was first coated with HM
as previously described. This material was then encapsulated with BSA
as follows: 1.0 g RA-MMIP-HM and 20 mL of BSA solution (1%, m/v).
The solution was stirred by vortex for 1 min and then left for 30 min on
a beaker. Finally, excess BSA was withdrawn and 5.0 mL of 25% glu-
taraldehyde solution were added. The tube was vortexed for 1 min and
then left on a bench for 5 h. Finally, excess glutaraldehyde was with-
drawn. Thereafter, 10.0 mL of sodium borohydride (1%) was added to
the material and the solution was vortexed for 1 min and allowed to
stand for 15 min. The excess sodium borohydride was removed. The
Falcon tube was placed in an oven at 60 °C for 24 h. After drying, the
RA-MMIP-HM-BSA was washed again with ultrapure water: methanol
solution (1:1, v/v) and finally dried at 60 °C for 24 h. All procedures
were performed in the same manner for the mesoporous non-imprinted
polymer (MNIP), but in the absence of the TM (E1).
2.5. Point of zero charge
Initially the solutions were adjusted to the following pH values: 2, 4,
6, 8 and 10 using solutions of HCl (0.1 mol L−1) or NaOH (0.1 mol L−1).
The exact initial pH of the samples was measured with the aid of a pH
meter. Subsequently, 25 mg of each synthesized material (MMIP, RA-
MMIP-HM and RA-MMIP-HM-BSA) were placed separately in a Falcon
tube along with 10 mL of the solutions with different initial pH. The
tubes were left on the bench for 24 h. The final pH was obtained after
this time through a new measurement of the pH of the solutions. The
PZC was determined by the intersection of the curve, in which pH
initial = pH final. All determinations were performed in triplicate.
2.6. Protein exclusion test
The protein exclusion test is intended to exclude macromolecules,
which may be present in biological matrices, for example in human
urine samples. For this test, 40 mg of each material (MMIP, RA-MMIP-
HM, and RA-MMIP-HM-BSA) were placed in tubes containing a protein
solution (3 mL of BSA solution at 0.1%, m/v). The tubes were shaken by
vortex at 2000 rpm for 1 min. After stirring, the solutions were analyzed
by UV–Vis in a range of 200 to 400 nm to verify if the proteins are
adsorbed or excluded by the materials. All tests were performed in
triplicate.
2.7. Angle of contact and wettability
Measurement of the contact angle to evaluate the wettability of the
materials was performed by placing a drop of water on the surface of
the material, which was photographed using a Nikon D90 camera and
50 mm lens. This test was performed to reveal the hydrophilicity of
MMIP, RA-MMIP-HM, and RA-MMIP-HM-BSA by the interception re-
sulting from the interaction of their surfaces with water. Thus, hydro-
philicity was determined based on the contact angle (θ) between the
surface of each material exposed in a Petri dish with a drop of water.
The water was used to reflect its wettability and the hydrophilitic
property of each of the material was then determined.
2.8. Imprinting effect studies
The selectivity was evaluated for the imprinted (RA-MMIP-HM-BSA)
and non-imprinted (RA-MNIP-HM-BSA) polymers. Some interferents
were studied: E3, phenacetin, caffeine, lidocaine, procaine, sulfa-
methoxazole and trimethoprim. The tests were performed by adding
4 mg of adsorbent material to the test tubes containing 250 μL of spiked
urine samples (40 μg mL−1). The tubes were shaken at 2000 rpm using
vortex for 1 min. After stirring, the solutions were analyzed by HPLC-
UV. Based on these tests the parameters that are related to the se-
lectivity of the adsorbent material as distribution coefficient (kd), se-
lectivity coefficient (k) and relative selectivity coefficient (k') were
3
Microchemical Journal 150 (2019) 104162
determined. The kd of E1 and other drugs (interferents) were calculated
by Eq. 1.
(Ci Cf ) V (mL)
kd = ×
Cf Adsorbent mass (g ) (1)
in which Ci, Cf and V correspond to the initial, final and volume con-
centration of the solution, respectively.
The K and K′ was calculated from Eqs. 2 and 3, respectively.
k d E1
k=
kd Interferents (2)
kimprinted
k =
knon imprinted (3)
2.9. Microextraction in packed sorbent
Initially, RA-MMIP-HM-BSA was packaged in the MEPS syringe, but
to evaluate the applicability of the material on MEPS, it is essential that
the factors capable of influencing the extraction efficiency should be
evaluated. Some parameters such as the effect of pH, amount of ma-
terial, aspersion and dispersion cycles, elution solvent, volume of
sample and eluent, and reuse of the material were evaluated. Therefore,
some conditions to initiate sample preparation were chosen: one as-
persion and dispersion cycle, 3 mg of material (RA-MMIP-HM-BSA),
100 μL of water for conditioning, 200 μL of human urine sample
(40 μg mL−1 of each hormone), 200 μL of water (wash solvent), and
200 μL of methanol: acetic acid (9:1, v/v) solution (elution solvent).
After the extraction step, the eluent solvent was collected and evapo-
rated in an oven at 60 °C. Finally, the analytes were re-suspended in
methanol and an aliquot of 20 μL was analyzed by HPLC-UV.
2.10. Method validation
The validation parameters for the analysis of the hormones were:
selectivity, linearity (calibration curve), precision, accuracy, limit of
detection (LOD), limit of quantification (LOQ), robustness, and stability
[22–25]. The selectivity of the method was evaluated by analyzing a
pool of hydrolyzed human urine (blank) and hydrolyzed urine spiked
with the hormones (extraction), in order to observe the presence of
possible interferents from the matrix in the same retention times of the
analytes. The linearity of the method was determined by the analytical
curve with seven different concentrations (linear range of 100 to
1100 ng mL−1). The correlation coefficient, r, was also calculated
which is a parameter that allows to evaluate the quality of the obtained
curve, because the closer to 1.0, the less the dispersion of the experi-
mental points in the set and the uncertainty of the calculated regression
coefficients. The limits of detection (LOD) and quantification (LOQ)
were investigated experimentally for the targets in the standard solu-
tions under the optimized conditions.
The precision of the developed method represents the dispersion of
results between independent assays. The assays were repeated from the
same sample, considering the defined conditions. Repetition was de-
termined by analyzing human urine samples (n = 6) at three different
concentrations of each hormone (250, 550 and 950 ng mL−1) on the
same day and under the same experimental conditions. Precision should
be expressed by means of the relative standard deviation (RSD%), not
exceeding 15%. Accuracy represents the agreement between the result
of an analysis and a reference value. It was measured as the percentage
of the difference between the nominal concentration and the mea-
surement. The accuracy was expressed by the relative error (RE%), and
values outside the range of ± 15% of the nominal value are not al-
lowed.
The robustness of the chromatographic method was evaluated by
varying the parameters such as flow rate (0.95, 1.00 and
H.L. de Oliveira, et al.
1.05 mL min−1), proportion of mobile phase methanol: acetonitrile:
water (65: 10: 5, 60: 20: 20, 60: 25: 15, v/v/v) and injection volume
(37, 40, and 43 μL). The ANOVA test was applied with a significance
level set at p-value < 0.05. A stability test was performed using human
urine samples spiked with hormones at two different concentrations
(250 and 950 ng mL−1), which were subjected to 12 h at room tem-
perature, freeze/thaw cycles, 48 and 96 h of freezing before finally
being analyzed. The t-test (student) was applied to compare with fresh
samples, with significance level p-value < 0.05.
2.11. Human urine samples
Human urine samples (51 mL) were obtained from one pregnant
volunteer (eighth month of gestation) and stored in a freezer until use
(24 h after collection). For the studies that involved urine testing, in-
formed consent was obtained from the recruited volunteers and the
study protocol was implemented and approved in accordance with the
Commission on Ethics with Research Involving Human Beings of the
Federal University of São João del-Rei (Universidade Federal de São João
del-Rei) (CEPSJ) (CAAE: 96382618.4.0000.5151). Initially in a 10 mL
flask, 150 μL of hydrochloric acid (1.0 mol L−1) was added and the
volume was completed with urine samples. Subsequently, the solution
was left in a water bath for 1 h at 65 °C, then the pH was adjusted to
10.0 using a solution of sodium hydroxide (1.0 mol L−1). Finally, the
solution was centrifuged for 3 min at 2000 rpm.
3. Results and discussion
3.1. Development of RA-MMIP-HM-BSA material
Firstly, the MMIP was synthesized by free radical polymerization,
which is the most used method to obtain the MIPs, with the synthesis
type being in bulk, which among the types of existing syntheses is
certainly the most used. In this type of synthesis, it is important to note
that the shape of the particles obtained is not uniform. Secondly, de-
pending on the application of the material, it is interesting that other
types of synthesis procedures such as precipitation or suspension are
adopted. However, since the application is on sample preparation, the
bulk synthesis provides the desired adsorbent material. In this type of
synthesis, the reactants (MAA, E1, EGDMA, benzalkonium chloride and
4,4′-azobis(4-cyanovaleric acid) were solubilized in porogenic solvent
(chloroform) and the mixture placed in an amber bottle. Double im-
printed polymers (template and surfactant molecules) have better mass
transfer than MIP and NIP synthetized without any surfactant [26,27].
Furthermore, benzalkonium chloride exhibited higher adsorptive ca-
pacity that other surfactants for adsorption of pharmaceuticals [26,27].
After removal of O2, this vial was sealed, and the heat induced a re-
action for polymerization to occur. After 24 h a monolith was formed,
which was crushed and sieved to standardize the particle size. The
MMIP obtained was washed to remove E1. Subsequently, the HM and
BSA were used to cover the MMIP [28–32]. HM were responsible for
increasing the hydrophilicity on the surface of the MMIP, decreasing
the rate of protein retention during extractions of urine samples
[17,18]. The material was enveloped by the BSA layer, because binding
between the BSA amine groups and glutaraldehyde crosslinking elim-
inates proteins by electrostatic repulsion of the proteins present in the
urine samples and the BSA layer, due to the positive or negative charges
of both the proteins at pH values different from their isoelectric point
[18,33]. Fig. 1 shows a scheme of the synthesis of RA-MMIP-HM-BSA.
3.2. Characterization
3.2.1. FTIR analysis
Fig. 2A shows the FTIR spectra of adsorbent materials (MMIP, RA-
MMIP-HM, and RA-MMIP-HM-BSA), which are very similar. In addition
to the presence of the characteristic absorption bands of the MAA and
4
Microchemical Journal 150 (2019) 104162
EGDMA functional groups used in the synthesis of MMIP, there is also
the presence of bands referring to HM and BSA used in the MMIP
coating stage. For example, the bands at approximately 3500 cm−1
occur because of stretching of the hydroxyl groups from the MAA. This
can be confirmed because of the intense band at about 1750 cm−1 re-
ferring to the stretching of the C]O bond of the carboxyl group of MAA
and also of HM for RA-MMIP-HM and RA-MMIP-HM-BSA. There is also
in this region (3500 cm−1) an overlap with the vibration of elongation
of the hydroxyl group from the adsorbed water. In addition, also at
approximately 3500 cm−1, there are overlapping bands corresponding
to vibrations (NH2 and NH) from BSA to RA-MMIP-HM-BSA. Bands at
approximately 3000 cm−1 refer to the CeH stretch of the CH3 and CH2
groups. The bands at 1260 cm−1 and 1160 cm−1 were attributed to the
symmetrical and asymmetric stretching of the CeO bond of the ester
functional group from EGDMA and also from HM to RA-MMIP-HM and
RA-MMIP-HM-BSA [33].
With these results, it was possible to indicate that the polymeriza-
tion of MMIP employing MAA and EGDMA was obtained as desired. In
addition, characteristic bands of HM and BSA are present in the coated
materials (RA-MMIP-HM and RA-MMIP-HM-BSA), proving that the
coating of such materials has been satisfactorily achieved.
3.2.2. TGA results
Fig. 2B shows the TGA results of the adsorbent materials (MMIP,
RA-MMIP-HM, and RA-MMIP-HM-BSA). For all materials it was pos-
sible to observe a thermal event below 100 °C because of the loss of
mass (about 10%) relative to moisture and compounds that were not
consumed during the synthesis of the materials. For the MMIP and RA-
MMIP-HM-BSA, it was possible to observe the second thermal event
around 350 °C, referring to the rapid mass loss (about 70%) related to
the decomposition of these materials. Soon after the second event, it
was possible to observe a mass loss of approximately 20% that is related
to the degradation of possible solids (products) that were obtained after
the decomposition of the materials. As for the RA-MMIP-HM, it was
possible to observe the second thermal event with a mass loss (around
20%) between 100 and 150 °C referring to a small degradation of the
material, probably due to the presence of HM on the polymer surface. It
was also possible to observe the third thermal event at approximately
350 °C, related to the greater loss of mass of this material (around 60%)
related to its decomposition. After the third event, it was possible to
observe a mass loss of approximately 10%, related to the degradation of
possible solids that were obtained after the decomposition of the ma-
terials.
The different thermal behavior for the RA-MMIP-HM may be be-
cause of the HM present on the MIP surface, and for the RA-MMIP-HM-
BSA may be because of the large amount of interactions (electrostatic
force, dipole-dipole interaction, London, hydrogen bonding, and non-
polar bond), between the BSA, HM and the functional groups present in
the polymer chain [34,35], favoring the interconnection of the polymer
chains.
3.2.3. Determination of PZC
The determination of the PZC of the three appropriate prepared
materials was carried out before sample preparation, because the PZC
provides an analysis of the behavior of the electric charges present on
the surfaces of the adsorbent materials. Therefore it is possible to in-
dicate the pH value where the balance of positive and negative charges
on the surfaces of the materials is zero. As an example, if the pH value
of the solution is above pHPZC, the surface will be negatively charged,
i.e., deprotonated, and theoretically favoring the adsorption of cationic
species. Conversely, if the pH value is below pHPZC, the surface of the
adsorbent material will be protonated, i.e., positively charged, and fa-
voring the adsorption of anion species. From Fig. 2C, it is possible to
observe the PZC of the three adsorbent materials synthesized, which is
given by the point of intersection of the curve (pHinitial = pHfinal) [36].
Table S1 shows the exact values for each of these materials.
H.L. de Oliveira, et al. Microchemical Journal 150 (2019) 104162

Fig. 1. Scheme of the RA-MMIP-HM-BSA synthesis.

3.2.4. Protein exclusion test 3.2.5. SEM analysis

By means of the protein exclusion test it is possible to verify if the
obtained material was coated with BSA correctly and if the exclusion of
the macromolecules was efficient. The absorption curves obtained by
UV–Vis analyze of the standard BSA solution and the three materials
synthesized after contact with the BSA (protein) solution are shown in
Fig. 2D. The percentage of protein exclusion for each material was 85%
MMIP, 91% RA-MMIP-HM, and 99% RA-MMIP-HM-BSA. The results
indicate that the doubly coated material excludes almost completely the
proteins, while the uncoated material adsorbs a small amount of pro-
teins, and RA-MMIP-HM presented an intermediate exclusion.
Fig. 3 (A-F) shows the SEM images obtained from the adsorbent
materials synthesized (MMIP, RA-MMIP-HM, and RA-MMIP-HM-BSA),
making possible an evaluation of the structural morphology of these
materials with a magnification at 50 and 1000 times. Fig. 3 (A-F) shows
that both materials have heterogeneous, irregular, and indefinite
morphologies. This result was already expected due to the synthesis of
the MMIP being of the in bulk type induced by heating [8]. However,
the irregularities are not limiting for the application of these materials
as efficient adsorbents in sample preparation.

Fig. 2. FTIR, TGA, PZC and protein exclusion of materials synthesized (MMIP, RA-MMIP-HM and RA-MMIP-HM-BSA).

5
H.L. de Oliveira, et al.
Fig. 3. SEM at magnification of 50 and 1000× for MMIP (A and D), RA-MMMIP-HM (B and E) and RA-MMIP-HM-BSA (C and F) and wettability property of MMIP
(G), RA-MMMIP-HM (H) and RA-MMIP-HM-BSA (I).
3.2.6. Angle of contact and wettability
From Fig. 3 (G-I), it was possible to observe a drop of water in
contact with the surface of each of the three synthesized materials. It is
important to note that when the value of θ is > 90° the material is
considered hydrophobic; on the other hand, if θ is less than or equal to
90° the material is hydrophilic; and if θ is equal to 0°, the material is
considered superhydrophilic [37]. In Fig. 3G and H, the drops of water
in contact with the surfaces of the materials form contact angles equal
to 0°, indicating that they are superhydrophilic, that is, the attractive
force between the surfaces of the materials and the molecules of water
is stronger than the repulsive force. In Fig. 3I, the angle formed between
the contact of the water droplets with the surface of the material is
higher than 90°, presenting hydrophobic properties, in which case the
attractive force between the surfaces of the materials and the water
molecules is weaker than the repulsive force. An important observation
is that the hydrophobic material is the doubly coated material, in-
dicating that the BSA coating is present on the surface of the material
and that, because of this coating, the materials become more hydro-
phobic.
3.2.7. Specific surface area, porosity and pore size distribution
The specific surface areas, volume and pore size of MMIP, RA-
MMIP-HM and RA-MMIP-HM-BSA were determined by the BET ad-
sorption equation and also by the BJH method using N2 adsorption/
desorption isotherms. The results obtained for the three materials are
shown in Table S2 and the adsorption-desorption isotherms of N2 are
shown by Fig. S2. In agreement with SEM images, these results show
that RA-MMIP-HM-BSA presents a large surface area (155.8 m2 g−1)
and mesoporous (112.7 Å). These results make the material with good
characteristics that explain the great capacity of extraction and re-
covery of the analytes in the sample preparation.
6
Microchemical Journal 150 (2019) 104162
3.3. Optimization of MEPS preparation process
3.3.1. Washing solvent
The solvents evaluated for the removal of interferents were: ultra-
pure water, dichloromethane, and hexane (200 μL). After the analysis of
the results, the ultrapure water presented better results in relation to
the others tested, since it removed more interferents from the matrix
(data not shown). In addition, the analytes were recovered from the
matrix with values lower than 4% E3 and 8% E1, since using hexane the
recoveries were 15% E3 and 19% E1; and dichloromethane 27% E3 and
42% E1 (Fig. 4A). The ultrapure water was then chosen to wash the
material, reducing the interference present in the sample and re-
covering less analytes from the matrix.
3.3.2. Sample volume
As shown in Fig. 4B, three sample volumes were evaluated: 150, 200
and 250 μL. The results showed that with increasing sample volume the
recovery of the analytes also increased. This indicates that larger
sample volumes favored recovery because of the larger (bulk) amount
of the analytes. In addition, the results indicate that there is no sa-
turation of the binding sites of the material due to a greater amount of
the analytes, because it has a large surface area and it is mesoporous.
Thus, the volume of 250 μL of sample was selected for the subsequent
parameters, since it presented greater recovery for E3 (53%) and E1
(50%).
3.3.3. Amount of material
Adsorption occurs by the interaction of the analyte with the selec-
tive sites of recognition of the RA-MMIP-HM-BSA, and the amount of
sorption material used can influence. Thus, 2, 3 and 4 mg of adsorbent
material were analyzed. As shown in Fig. 4C, the 4 mg of RA-MMIP-
HM-BSA corresponded to the higher recovery of the analytes (56% E3
and 52% E1), due to the greater amount of material presenting more
H.L. de Oliveira, et al. Microchemical Journal 150 (2019) 104162

Fig. 4. (A) Effect of washing solvent; (B) Effect of sample volume; (C) Effect of amount of adsorbent material (MIP); (D) Effect of elution solvent; (E) Effect of eluent
volume; (F) Effect of number of cycles of aspersion/dispersion (G) Effect of pH sample; (H) Comparison of with other adsorbent material (RA-MNIP-HM-BSA and
C18).

selective recognition sites that would consequently increase the inter-
action with the analyte.
3.3.4. Elution solution
Another very important parameter of the MEPS technique is the
choice of the appropriate elution solvent, which must elute the analyte
without affecting the adsorbent material. Thus, the following elution
solvents were evaluated: methanol: acetic acid (9: 1, v/v), ethanol:
acetic acid (9: 1, v/v), and acetonitrile: acetic acid (9: 1, v/v). As shown
in Fig. 4D, the results indicate that the methanol: acetic acid solution
(9: 1, v/v) presented the best recovery (56% E3 and 52% E1) among the
solvents tested, probably because methanol exhibits the highest polarity
between solvents tested, in addition to more efficiently breaking the
interaction between the analytes and the adsorbent material.

7
H.L. de Oliveira, et al.
3.3.5. Eluent volume
Three different volumes of eluent were evaluated: 150, 200 and
250 μL. According to the results shown in Fig. 4E, the volume of 150 μL
of elution solvent extracted a smaller amount of analytes than the other
volumes studied (39% E3 and 31% E1). The volumes of 200 and 250 μL
presented similar recovery values (56% E3 and 52% E1; and 55% E3
and 52% E1, respectively). Thus for a lower solvent expenditure, 200 μL
was chosen for the next experiments.
3.3.6. Number of cycles of aspersion/dispersion
The number of aspersion and dispersion cycles was analyzed to
verify if the increase in the number of cycles would increase the re-
covery of the analytes. According to Fig. 4F, it was possible to observe
that there had been a gradual increase of recovery of the analytes with
the increase of the cycles. Therefore four aspersion and dispersion cy-
cles were used in the subsequent experiments (60% E3 and 61% E1).
3.3.7. pH sample
Fig. 4G shows that the results of the pH effect of the sample were
very important for the retention of the analytes in the adsorbent ma-
terial. This can be explained because pH plays an important role in the
extraction of organic compounds in biological samples. Thus, it is
possible to determine whether the organic compound (analyte) is in its
molecular or ionic form. The pH values 1, 4, 7, 10 and 13 were chosen
to evaluate the effect of the pH of the sample. E1 and E3 have the same
pKs, 10.33 [38]. At pH values below 10 (1, 4 and 7) both drugs are
present in their neutral forms (100%), at pH 10 both drugs are present
in their neutral (approximately 68%) and ionic (about 32%) forms, and
at pH 13, the drugs are in greater quantity in their anionic forms (81%
E3 and 100% E1) [38]. Because of the neutral forms of the analytes
which favor recovery of the analytes (69% E3 and 79% E1), pH 4 was
chosen.
3.3.8. Other materials
After obtaining all parameters in MEPS using RA-MMIP-HM-BSA, a
study was performed for extractions with RA-MNIP-HM-BSA and C18 in
order to determine and compare the efficiency of these materials' ad-
sorbents. As expected, RA-MMIP-HM-BSA was much more efficient
(69% E3 and 79% E1), showing higher recovery for hormones than RA-
MNIP-HM-BSA (55% E3 and 58% E1) and C18 (15% E3 and 39% E1), as
can be seen in Fig. 4H.
3.4. Reuse
The reuse of RA-MMIP-HM-BSA was performed to evaluate the
ability of the adsorbent material to maintain the recoveries of the
analytes near the recoveries obtained when the material was used only
once. However, as is shown in Fig. S3A, reuse makes recovery even
lower, principally for E1 which varied by about 15% using the same
material for the second time (from 79% to 65%). This is probably be-
cause the elution solution uses acid and this destroys the selective sites
of recognition of the adsorbent material, in addition to the amount of
material used being very small. The recovery of the material is also
affected by the use of organic solvents, such as methanol, which are
chemical substances that do not react with the polymer, but because
they have chemical affinity (present similar structures), solubilizing in a
certain degree and it typically occurs in amorphous materials, as is the
case of RA-MMIP-HM-BSA, which is basically composed of C and N.
Methanol may have interfered in cohesive forces, reducing the inter-
action between the molecules of the material and increasing local
molecular mobility. Thus, the increase in the number of extractions
using the same material can promote modifications in the polymer
macromolecular chain irreversibly, which can alter the performance of
the material. Therefore, RA-MMIP-HM-BSA should be used only once in
the MEPS.
8
Microchemical Journal 150 (2019) 104162
3.5. Imprinting effect studies
It is possible to establish the distribution constants (kd), selectivity
(k) and relative selectivity (k') of the material by the adsorbent print
test (RA-MMIP-HM-BSA and RA-MNIP-HM-BSA) [39]. The test was
performed with seven different drugs, among them E3, which presented
the highest recovery, around 70% for RA-MMIP-HM-BSA and 55% for
RA-MNIP-HM-BSA, while caffeine obtained lower, 11% recovery for
both materials, as can be observed in Figs. S3B and S3C.
Thus, the parameters of kd, k and k' were determined for each drug
analyzed. The calculated values are set forth in Table S3. The kd re-
presents the affinity and migration of the analyte to the adsorbent
material used. According to the table, the value of kd is higher for the
TM (E1) and this leads to a strong indication that the polymer synthesis
occurred correctly. The k values obtained for RA-MMIP-HM-BSA con-
firm the ability of the polymer to recognize the analyte used as the TM.
Also, according to the results, it is possible to observe that the values of
k varied in increasing form of the drugs that presented greater recovery
than those of less recovery, that is, E3 obtained lower value of k (1.70)
for RA-MMIP-HM-BSA and (1.12) for RA-MNIP-HM-BSA; and greater
caffeine (30.33) for RA-MMIP-HM-BSA and (10.36) for RA-MNIP-HM-
BSA. Furthermore, it is observed that Kd values for E1 are higher than
for other drugs, indicating that success in molecular imprinting has
been achieved, but Kd values for E3 are also high (142.18) for RA-
MMIP-HM-BSA and (77.40) for RA-MNIP-HM-BSA, but can be ex-
plained because of the similarity between chemical structures. The k' is
the calculated value to evaluate the effectiveness of the chemical im-
print of the polymer, as all values obtained were k' > 1, thus the results
confirm the positive effect.
3.6. Method validation
A suitable chromatographic separation for estrogens was obtained
after an intense bibliographical review and a systematic optimization of
the analytical method. Fig. S4B shows the analysis of standard solution at
40 μg mL−1 and human urine hydrolyzed spiked with E3 and E1, being
possible to observe well separated peaks and with good retention times,
in addition to acceptable values for the chromatographic system com-
pliance parameters (Table S4). From Fig. S4A it is possible to observe
that no interferent present in the samples was in the same retention time
of the analytes, indicating that the method was selective, besides
showing the chromatograms and acceptable recoveries of analytes using
MEPS with RA-MMIP-HM-BSA. The data obtained by the validation of
the analytical method are shown in Table 1. The results obtained in the
linearity parameter are in accordance with the requirements of ‘Guidance
for Industry, Validation of the Bioanalytical Method [25], because the
obtained values of r of each of the analytes by the calibration curves are
above 0.99 and the RSD% values of the slopes of the calibration curves
are below 15%. In addition, RSD% of the LOQ of each hormone is below
20% considering that both the LOD and the LOQ were obtained experi-
mentally. Accuracy and precision values for intra- and inter-day analyzes
are presented in Table S5. It is possible to observe that the RSD% values
are between 1.2 and 3.5% (intra-day) and between 0.2 and 1.7% (inter-
day). The RE% (accuracy test) values are between −2.6 and 14% (intra-
day) and between −2.1 and 14% (inter-day). These values are satisfac-
tory since all values are below 15% [25]. Table S6 presents the data
obtained for the robustness test (RE%, RSD% and p-value), as well as the
chromatographic conditions and the investigation intervals. The results
showed values of RSD% below 15% (ranging from 3.0 to 4.7% for E3 and
3.3–12% for E1). In addition, the p-values are above 0.05 (ranging from
0.12 to 0.78 for E3 and 0.33–0.68 for E1); summarizing, the values ob-
tained represent statistically the same results for all groups evaluated.
The results of the stability study are shown in Table S7. All RSD% values
are < 15% (ranging from 3.6 to 14% for E3 and 3.6–15% for E1) and the
p-values are higher than 0.05 (ranging from 0.06 to 0.70 for E3 and
0.20–0.81 for E1).
H.L. de Oliveira, et al. Microchemical Journal 150 (2019) 104162

Table 1
Linearity and LOQ for hormones in human urine by the developed method.
Linearity LOQ

a −1 b
Analytes Linear equation Correlation coefficient (r) Range (ng mL ) RSD (%) Nominal (ng mL−1) Analyzed (ng mL−1)c RSD c
(%) RE d
(%)

E3 y = 611× + 86,072 0.9952 100–1100 4.21 100 105.81 1.21 5.81

E1 y = 579.39× + 99,788 0.9954 100–1100 1.76 100 85.79 2.04 −14.22

Calibration curves determined in triplicate (n = 3) for concentrations of 100, 250, 400, 550, 700, 950 and 1100 ng mL−1; y = ax + b, where y is the analyte peak
a

area of analytes, a is the slope, b is the intercept and x is the concentration of the measured solution ng mL−1.
b
RSD = relative standard deviation of the slope of the calibration curve.
c
RSD = relative standard deviation of the limit of quantification.
d
RE = relative error with mean of six replicates.

Table 2
Determination of the analytes in real human urine samples.
Analyte E3 E1

Human urine from volunteer pregnant

Concentration found (ng mL−1) 719.90 ± 0.10 507.01 ± 1.92

Spiked human urine (pool)

Concentration added (ng mL−1) 250 550 950 250 550 950
b
Concentration determined ± SD (ng mL−1) (n = 6) a
283.86 ± 0.40 540.98 ± 0.46 962.27 ± 0.82 277.28 ± 1.72 538.74 ± 0.44 973.81 ± 0.16
Recovery ± SD b (n = 3) a 70.02 ± 2.68 67.02 ± 2.19 70.20 ± 2.14 74.49 ± 3.45 83.87 ± 1.46 76.98 ± 2.94

a
n, number of repetitions.
b
RSD (%), relative standard deviation.

3.7. Methods previously reported in literature
In general, some authors have developed methods to separate E1,
E3, and other estrogenic hormones in biological fluids, as in the case of
urine. The preparation of the most commonly used sample was SPE and
also its modifications, such as SPME and MSPE. In Table S8, some of
these papers published between 2015 and 2018 are shown, as well as
the recoveries of analytes, LOQ, LOD, linear range, analytical techni-
ques, and adsorbent materials used. It is important to emphasize that no
studies that used RA-MMIP-HM-BSA as adsorbent in MEPS for extrac-
tion of E1 and E3 in any type of sample were found. In addition, even
using less amount of adsorbent material, this work presented recoveries
in the same range as for other extraction methods, including LOD and
LOQ higher than the described in literature. The theoretical was ob-
tained using the signal-to-noise equation ((LOD = 3 × S/N and LOQ
=10 × S/N) and the experimental by analyzing the sample with
smaller concentration analytes, until it is no longer possible to quantify
them with RE% below 20% (ICH). This difference was because the other
methods from literature (see Table S8) described limits determined by
the signal-to-noise ratio and in this work were determined experimen-
tally, which are considered more real.
3.8. Real samples analyses
E2 is a natural hormone and is related to the development of female
sexual characteristics and reproduction. This estrogen may be naturally
produced or be taken in contraceptive use and also in cases of hormone
replacement [48]. In addition to excretion in its unaltered form, E2 is
also excreted in urine as metabolites, such as: glucuronides (glucur-
onides of estrone, 2-hydroxy-estrone, E2, estriol and 16α-hydroxy-es-
trone), and sulfate (estrone sulfate) [49]. E1 is eliminated daily by the
human body of between 2 and 20 μg day−1, in addition to being present
in a higher concentration in women during menopause and gestation.
E3, a hormone that is present in the bloodstream of women, is also
eliminated by the human body of between 3 and 65 μg day−1 and its
concentration is increased during pregnancy. The amounts of these
estrogens excreted by pregnant women can be 1000 times greater, de-
pending on the weeks of pregnancy [49]. In addition, both E1 and E3
are the result of E2 oxidation [50].
The results show that in the sample provided by a pregnant vo-
lunteer it was possible to determine the concentration of E3, and also
E1. For E3, by means of the linear equation, it was possible to determine
the concentration of 719.90 ± 0.10 ng mL−1. The mass excreted by
this volunteer (51 mL of urine collected) was 36.72 μg (Fig. S5). For E1,
the concentration determined was 507.01 ± 1.92 ng mL−1. The mass
excreted was 25.86 μg (Fig. S5).
To further validate the developed method, the recoveries were also
evaluated by analyzing spiked human urine samples with three dif-
ferent concentrations of E3 and E1 (Table 2). The recoveries and their
relative errors ranged from 67 ± 2.2% to 70 ± 2.1% for E3, and from
74 ± 3.5% to 84 ± 1.5% for E1. The results indicate that the devel-
oped method was applied efficiently for the selective determination of
estrogenic traits in human urine samples.
4. Concluding remarks
RA-MMIP-HM-BSA was successfully applied in MEPS for determi-
nation of estrogens in human urine samples. MEPS showed to be a
simple, inexpensive and easy to use technique. RA-MMIP-HM-BSA
proved to be more selective for the TM compared to other analytes. The
validation of the method showed results in accordance with the re-
commendations and requirements available in the literature, besides
acceptable LOD and LOQ. This new analytical method was robust and
stable within the limits studied and is in accordance with the resolu-
tions on biological analysis available in the literature. Finally, the
method was satisfactorily applied in a urine sample from a pregnant
volunteer.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgements
The authors would like to thank the Brazilian agencies CNPq
(Conselho Nacional de Desenvolvimento Científico e Tecnológico), and
FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais)
for financial support. H. L. de Oliveira thanks to Prof. Luiz Fernando

9
H.L. de Oliveira, et al.
Cappa de Oliveira and Universidade Federal de Juiz de Fora (UFJF) for
technical support in SEM analysis. Also, this study was financed in part
by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -
Brasil (CAPES) - Finance Code 001, and part of the project involving the
Rede Mineira de Química (RQ-MG) supported by FAPEMIG (Project:
REDE-113/10; Project: CEX-RED-0010-14).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.microc.2019.104162.
References
[1] P.R. Larsen, C.M. Besser, H.M. Kronenberg, S. Melmed, K.S. Polonsky,
M.O. Thorner, Williams Textbook of Endocrinology, Elsevier - Health Sciences
Division, Philadelphia, 2002.
[2] R. Guedes-Alonso, S. Montesdeoca-Esponda, Z. Sosa-Ferrera, J.J. Santana-
Rodríguez, Liquid chromatography methodologies for the determination of steroid
hormones in aquatic environmental systems, Trends Environ. Anal. Chem. 3 (2014)
14–27, https://doi.org/10.1016/j.teac.2014.10.001.
[3] L. Ciofi, D. Fibbi, U. Chiuminatto, E. Coppini, L. Checchini, M. Del Bubba, Fully-
automated on-line solid phase extraction coupled to high-performance liquid
chromatography–tandem mass spectrometric analysis at sub-ng/L levels of selected
estrogens in surface water and wastewater, J. Chromatogr. A 1283 (2013) 53–61,
https://doi.org/10.1016/j.chroma.2013.01.084.
[4] L. Chen, M. Zhang, F. Fu, J. Li, Z. Lin, Facile synthesis of magnetic covalent organic
framework nanobeads and application to magnetic solid-phase extraction of trace
estrogens from human urine, J. Chromatogr. A 1567 (2018) 136–146, https://doi.
org/10.1016/j.chroma.2018.06.066.
[5] M. Abdel-Rehim, Recent advances in microextraction by packed sorbent for bioa-
nalysis, J. Chromatogr. A 1217 (2010) 2569–2580, https://doi.org/10.1016/j.
chroma.2009.09.053.
[6] M. Abdel-Rehim, Microextraction by packed sorbent (MEPS): a tutorial, Anal. Chim.
Acta 701 (2011) 119–128, https://doi.org/10.1016/j.aca.2011.05.037.
[7] E. Soleimani, A. Bahrami, A. Afkhami, F.G. Shahna, Selective determination of
mandelic acid in urine using molecularly imprinted polymer in microextraction by
packed sorbent, Arch. Toxicol. 92 (2018) 213–222 https://doi-org.ez32.periodicos.
capes.gov.br/10.1007/s00204-017-2057-z.
[8] C.F. Silva, K.B. Borges, C.S. do Nascimento Jr., Rational design of a molecular
imprinting polymer for dinotefuran: theoretical and experimental studies aiming
the development of an efficient adsorbent for microextraction by packed sorbent,
Analyst 143 (2018) 141–149, https://doi.org/10.1039/C7AN01324H.
[9] T.F.D. Pereira, A.T.M. da Silva, K.B. Borges, C.S. Nascimento Jr., Theoretical in-
vestigation on the pre-polymerization rational design of carvedilol-imprinted
polymer and selectivity studies, J. Mol. Struct. 1177 (2019) 101–106, https://doi.
org/10.1016/j.molstruc.2018.09.043.
[10] A.T.M. da Silva, H.L. de Oliveira, C.F. Silva, M.C. Fonseca, T.F.D. Pereira,
C.S. Nascimento Jr., E.C. Figueiredo, K.B. Borges, Efficient molecularly imprinted
polymer as a pipette-tip solid-phase sorbent for determination of carvedilol en-
antiomers in human urine, J. Chromatogr. B 1061–1062 (2017) 399–410, https://
doi.org/10.1016/j.jchromb.2017.07.056.
[11] M.C. Fonseca, C.S. Nascimento Jr., K.B. Borges, Theoretical investigation on func-
tional monomer and solvent selection for molecular imprinting of tramadol, Chem.
Phys. Lett. 645 (2016) 174–179, https://doi.org/10.1016/j.cplett.2015.12.061.
[12] C.S. Magalhães, J.S. Garcia, A.S. Lopes, E.C. Figueiredo, M.A.Z. Arruda,
M.A.Z. Arruda (Ed.), Trends in Sample Preparation, Nova Science Publishers, New
York, 2007.
[13] C.J. Allender, C. Richardson, B. Woodhouse, C.M. Heard, K.R. Brain,
Pharmaceutical applications for molecularly imprinted polymers, Int. J. Pharm. 195
(2000) 39–43, https://doi.org/10.1016/S0378-5173(99)00355-5.
[14] Y.W. Chien, S. Lin, Optimisation of treatment by applying programmable rate-
controlled drug delivery technology, Clin. Pharmacokinet. 41 (2002) 1267–1300
https://doi-org.ez32.periodicos.capes.gov.br/10.2165/00003088-200241150-
00003.
[15] J.Z. Hilt, M.E. Byrne, Configurational biomimesis in drug delivery: molecular im-
printing of biologically significant molecules, Adv. Drug Deliv. Rev. 56 (2004)
1599–1620, https://doi.org/10.1016/j.addr.2004.04.002.
[16] B. Buszewski, M. Szultka, Past, present, and future of solid phase extraction: a re-
view, Crit. Rev. Anal. Chem. 42 (2012) 198–213, https://doi.org/10.1080/
07373937.2011.645413.
[17] E.C. Figueiredo, G.O.I. Moraes, L.M.R. Silva, A.J. Santos-Neto, F.H. Florenzano,
Restricted Access Molecularly Imprinted Polymer Capped With Albumin (RAM-
MIP-BSA). BR Filed Patent, PI1020120153394, filed in June 22 (2012).
[18] G.O.I. Moraes, L.M.R. Silva, A.J. Santos-Neto, F.H. Florenzano, E.C. Figueiredo, A
new restricted access molecularly imprinted polymer capped with albumin for di-
rect extraction of drugs from biological matrices: the case of chlorpromazine in
human plasma, Anal. Bioanal. Chem. 405 (24) (2013) 7687–7696 https://doi-org.
ez32.periodicos.capes.gov.br/10.1007/s00216-013-7275-5.
10
Microchemical Journal 150 (2019) 104162
[19] M. Marć, A. Panuszko, J. Namieśnik, P.P. Wieczorek, Preparation and character-
ization of dummy-template molecularly imprinted polymers as potential sorbents
for the recognition of selected polybrominated diphenyl ethers, Anal. Chim. Acta
1030 (2018) 77–95, https://doi.org/10.1016/j.aca.2018.05.022.
[20] A. Poliwoda, M. Mościpan, P.P. Wieczorek, Application of molecular imprinted
polymers for selective solid phase extraction of bisphenol a, Ecol. Chem. Eng. S 23
(4) (2016) 651–664, https://doi.org/10.1515/eces-2016-0046.
[21] A.M. Chrzanowska, A. Poliwoda, P.P. Wieczorek, Characterization of particle
morphology of biochanin A molecularly imprinted polymers and their properties as
a potential sorbent for solid-phase extraction, Mater. Sci. Eng., C 49 (2015)
793–798, https://doi.org/10.1016/j.msec.2015.01.069.
[22] The United States Pharmacopeia (USP), The United States Pharmacopeial
Convention, 36th Revision, USA, (2013).
[23] S. Kollipara, G. Bende, N. Agarwal, B. Varshney, J. Paliwal, International guidelines
for bioanalytical method validation: a comparison and discussion on current sce-
nario, Chromatographia 73 (2011) 201–217 https://doi-org.ez32.periodicos.capes.
gov.br/10.1007/s10337-010-1869-2.
[24] N. Kadian, K.S.R. Raju, M. Rashid, M.Y. Malik, I. Taneja, M. Wahajuddin,
Comparative assessment of bioanalytical method validation guidelines for phar-
maceutical industry, J. Pharm. Biomed. Anal. 126 (2016) 83–97, https://doi.org/
10.1016/j.jpba.2016.03.052.
[25] Guidance for Industry, Bioanalytical Method Validation, US. Department of Health
and Human Services Food and Drug Administration, Center for Drug Evaluation and
Research (CDER), Rockville, USA, 2001.
[26] R.C.S. da Silva, M.N. Santos, B.C. Pires, L.F. Dinali, F.A.C. Suquila, C.R.T. Tarley,
K.B. Borges, Assessment of surfactants on performance of molecularly imprinted
polymer toward adsorption of pharmaceutical, J. Environ. Chem. Eng. 7 (2019)
103037, https://doi.org/10.1016/j.jece.2019.103037.
[27] R.C.S. da Silva, B.C. Pires, K.B. Borges, Double-imprinted polymer based on cross-
linked poly(vinylimidazole–trimethylolpropane trimethacrylate) in solid phase ex-
traction for determination of lead from wastewater samples by UV–vis spectro-
photometry, Int. J. Environ. Anal. Chem. 99 (2019) 949–967, https://doi.org/10.
1080/03067319.2019.1616717.
[28] P.A.G. Cormack, A.Z. Elorza, Molecularly imprinted polymers: synthesis and char-
acterization, J. Chromatogr. B 804 (2004) 173–182, https://doi.org/10.1016/j.
jchromb.2004.02.013.
[29] E. Turiel, A. Martín-Esteban, Molecularly imprinted polymers for sample prepara-
tion: a review, Anal. Chim. Acta 668 (2010) 87–99, https://doi.org/10.1016/j.aca.
2010.04.019.
[30] B. Sellergren, Molecularly Imprinted Polymers: Man-Made Mimics of Antibodies
and their Application in Analytical Chemistry, Elsevier Science BV, Amsterdam,
2001.
[31] A. Martín-Esteban, Molecularly imprinted polymers: new molecular recognition
materials for selective solid-phase extraction of organic compounds, Fresenius J.
Anal. Chem. 370 (2001) 795–802 https://doi-org.ez32.periodicos.capes.gov.br/10.
1007/s002160100854.
[32] F. Qiao, H. Sun, H. Yan, K.H. Row, Molecularly imprinted polymers for solid phase
extraction, Chromatographia 64 (2006) 625–634 https://doi-org.ez32.periodicos.
capes.gov.br/10.1365/s10337-006-0097-2.
[33] L.L.G. de Oliveira, F.A.C. Suquila, F.M. de Oliveira, G.L. Scheel, C.R.T. Tarley,
Synthesis and application of restricted access material-ion imprinted poly(al-
lylthiourea) for selective separation of Cd2+ and humic acid exclusion, React.
Funct. Polym. 134 (2019) 93–103, https://doi.org/10.1016/j.reactfunctpolym.
2018.11.007.
[34] R.H. McMenamy, V.M. Rosenoer, M. Oratz, M.A. Rothschild (Eds.), Albumin
Structure, Function and Uses, Pergamon Press Inc, Oxford, 1977.
[35] F.A.C. Suquila, L.L.G. de Oliveira, C.R.T. Tarley, Restricted access copper imprinted
poly(allylthiourea): the role of hydroxyethyl methacrylate (HEMA) and bovine
serum albumin (BSA) on the sorptive performance of imprinted polymer, Chem.
Eng. J. 350 (2018) 714–728, https://doi.org/10.1016/j.cej.2018.05.176.
[36] Y.-L. Zhang, J. Zhang, C.-M. Dai, X.-F. Zhou, S.-G. Liu, Sorption of carbamazepine
from water by magnetic molecularly imprinted polymers based on chitosan-Fe3O4,
Carbohydr. Polym. 97 (2013) 809–816, https://doi.org/10.1016/j.carbpol.2013.
05.072.
[37] Z. Lu, A. Hanif, C. Lu, G. Sun, Y. Cheng, Z. Li, Thermal, mechanical, and surface
properties of poly(vinyl alcohol) (PVA) polymer modified cementitious composites
for sustainable development, J. Appl. Polym. Sci. 135 (2018) 46177–46184,
https://doi.org/10.1002/app.46177.
[38] Chemicalize, estriol and estrone pH's, https://chemicalize.com/, (2018) , Accessed
date: December 2018.
[39] L.R. Nacano, M.G. Segatelli, C.R.T. Tarley, Selective sorbent enrichment of nickel
ions from aqueous solutions using a hierarchically hybrid organic-inorganic
polymer based on double imprinting concept, J. Braz. Chem. Soc. 21 (2010)
419–430, https://doi.org/10.1590/S0103-50532010000300004.
[48] K. Ikehata, N.J. Naghashkar, M.G. El-Din, Degradation of aqueous pharmaceuticals
by ozonation and advanced oxidation processes: a review, Ozone Sci. Eng. 28
(2006) 353–414, https://doi.org/10.1080/01919510600985937.
[49] J. Lintelmann, A. Katayama, N. Kurihara, L. Shore, A. Wenzel, Endocrine disruptors
in the environment (IUPAC technical report), Pure Appl. Chem. 75 (2003) 631–681
https://doi-org.ez32.periodicos.capes.gov.br/10.1351/pac200375050631.
[50] J.R. Prater, R. Horton, M.L. Thompson, Reduction of estrone to 17 β-estradiol in the
presence of swine manure colloids, Chemosphere 119 (2015) 642–645, https://doi.
org/10.1016/j.chemosphere.2014.07.072.
H.L. de Oliveira, et al.
Further Reading
[40] P. Wang, X. Qiu, Y. Yang, Determination of estrogens in human urine by vortex-
assisted dispersive liquid–liquid microextraction based on floating organic acid
droplet combined with high-performance liquid chromatography-fluorescence de-
tection, J. Liq. Chromatogr. Relat. Technol. 38 (2015) 640–646, https://doi.org/10.
1080/10826076.2014.913522.
[41] K. Liao, M. Mei, H. Li, X. Huang, C. Wu, Multiple monolithic fiber solid-phase
microextraction based on a polymeric ionic liquid with high-performance liquid
chromatography for the determination of steroid sex hormones in water and urine,
J. Sep. Sci. 39 (2016) 566–575 https://doi-org.ez32.periodicos.capes.gov.br/10.
1002/jssc.201501156.
[42] A.C. Naldi, P.B. Fayad, M. Prévost, S. Sauvé, Analysis of steroid hormones and their
conjugated forms in water and urine by on-line solid-phase extraction coupled to
liquid chromatography tandem mass spectrometry, Chem. Cent. J. 10 (2016) 1–17
https://doi-org.ez32.periodicos.capes.gov.br/10.1186/s13065-016-0174-z.
[43] Q. Zhang, L. Han, J. Wang, H. Lin, P. Ke, J. Zhuang, X. Huang, Simultaneous
quantitation of endogenous estrone, 17β-estradiol, and estriol in human serum by
isotope-dilution liquid chromatography–tandem mass spectrometry for clinical la-
boratory applications, Anal. Bioanal. Chem. 409 (2017) 2627–2638 https://doi-org.
11
Microchemical Journal 150 (2019) 104162
ez32.periodicos.capes.gov.br/10.1007/s00216-017-0207-z.
[44] X. An, W. Chai, X. Deng, H. Chen, G. Ding, A bioinspired polydopamine approach
toward the preparation of gold-modified magnetic nanoparticles for the magnetic
solid-phase extraction of steroids in multiple samples, J. Sep. Sci. 41 (2018)
2697–2864, https://doi.org/10.1002/jssc.201800080.
[45] G. Gao, S. Li, S. Li, Y. Wang, P. Zhao, X. Zhang, X. Hou, A combination of com-
putational−experimental study on metal-organic frameworks MIL-53(Al) as sor-
bent for simultaneous determination of estrogens and glucocorticoids in water and
urine samples by dispersive micro-solid-phase extraction coupled to UPLC-MS/MS,
Talanta 180 (2018) 358–367, https://doi.org/10.1016/j.talanta.2017.12.071.
[46] G. Gao, S. Li, S. Li, L. Zhao, T. Wang, X. Hou, Development and application of
vortex-assisted membrane extraction based on metal–organic framework mixed-
matrix membrane for the analysis of estrogens in human urine, Anal. Chim. Acta
1023 (2018) 35–43, https://doi.org/10.1016/j.aca.2018.04.013.
[47] J. Merib, D.A. Spudeit, G. Corazza, E. Carasek, J.L. Anderson, Magnetic ionic liquids
as versatile extraction phases for the rapid determination of estrogens in human
urine by dispersive liquid-liquid microextraction coupled with high-performance
liquid chromatography-diode array detection, Anal. Bioanal. Chem. 410 (19)
(2018) 4689–4699 https://doi-org.ez32.periodicos.capes.gov.br/10.1007/s00216-
017-0823-7.