Synthesis of molecularly imprinted polymer for removal of effective impurity (benzhydrol) from diphenhydramine hydrochloride drug

Article abstract of DOI:10.1002/jccs.201300494

The present study describes the synthesis and preliminary testing of molecularly imprinted polymers (MIPs) as scavenger resins for removal of the genotoxic impurities (GTI) benzhydrol from active pharmaceutical ingredients (API). A new molecularly imprinted polymer was synthesized using benzhydrol (template molecule), methacrylic acid (functional monomer), ethylene glycol dimethacrylate (cross-linker), 2,2′-azobisisobutironitril (intiator) and chloroform (porogenic solvent). To compare the performance of this polymer, a control polymer or non-imprinted polymer (NIP) was prepared under the same conditions without the use of template molecule. The synthesized polymers were characterized by FT-IR spectroscopy. Selectivity of the molecularly imprinted polymer for absorption benzhydrol impurities through adsorption experiments reviews and the results were compared with the adsorption of impurities by NIP. Various parameters were optimized such as time, pH, type of eluent for elution of impurities from polymer, concentration of sample and saturation of polymer. The proposed method was applied for removal of benzhydrol from Diphenhydramine hydrochloride syrup and passing it through the MIPs led to the quantitative removal of benzhydrol. The present work describes the synthesis and preliminary testing of molecularly imprinted polymers (MIPs) as scavenger resins for removal of the genotoxic impurities (GTI) benzhydrol from active pharmaceutical ingredients (API). Copyright

Full text of DOI:10.1002/jccs.201300494

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Article
Synthesis of Molecularly Imprinted Polymer for Removal of Effective Impurity
Benzhydrol) from Diphenhydramine Hydrochloride Drug
(
Hamid Hashemi-Moghaddam* and Mohammad Reza Alaeian
Department of Chemistry, Damghan Branch, Islamic Azad University, Damghan, Iran
(Received: Sept. 26, 2013; Accepted: Feb. 15, 2014; Published Online: Apr. 1, 2014; DOI: 10.1002/jccs.201300494)
The present study describes the synthesis and preliminary testing of molecularly imprinted polymers
(
MIPs) as scavenger resins for removal of the genotoxic impurities (GTI) benzhydrol from active pharma-
ceutical ingredients (API). Anew molecularly imprinted polymer was synthesized using benzhydrol (tem-
plate molecule), methacrylic acid (functional monomer), ethylene glycol dimethacrylate (cross-linker),
2
,2¢-azobisisobutironitril (intiator) and chloroform (porogenic solvent). To compare the performance of
this polymer, a control polymer or non-imprinted polymer (NIP) was prepared under the same conditions
without the use of template molecule. The synthesized polymers were characterized by FT-IR spectros-
copy. Selectivity of the molecularly imprinted polymer for absorption benzhydrol impurities through ad-
sorption experiments reviews and the results were compared with the adsorption of impurities by NIP.
Various parameters were optimized such as time, pH, type of eluent for elution of impurities from poly-
mer, concentration of sample and saturation of polymer. The proposed method was applied for removal of
benzhydrol from Diphenhydramine hydrochloride syrup and passing it through the MIPs led to the quanti-
tative removal of benzhydrol.
Keywords: Molecularly imprinted polymer; Benzhydrol; Diphenhydramine; Solid phase extraction.
INTRODUCTION
Diphenhydramine hydrochloride (abbreviated DPH,
mized and smaller sample sizes are required. Moreover,
SPE can be easily incorporated into automated analytical
4
-5
sometimes DHM) is a first-generation antihistamine pos-
sessing anticholinergic, antitussive, antiemetic, and seda-
procedures.
Polymeric network materials capable of recognizing
target molecules by molecular imprinting technique are
available. This is a process where functional and cross-
linking monomers are copolymerized in the presence of a
target analyte (the imprint molecule), which acts as a mo-
lecular template. The functional monomers initially form a
complex with the imprint molecule, and following poly-
merization, their functional groups are held in position by
the highly cross-linked polymeric network. Subsequent re-
moval of the imprint molecule reveals binding sites that are
complementary in size and shape to the analyte. In this way,
a molecular memory is introduced into the polymer, which
1
tive properties which is mainly used to treat allergies. This
drug has many impurities, which in this project was work-
ing on benzhydrol. Benzhydrol or Diphenylmethanol is
one of the effective impurities in the drug diphenhydramine
2
hydrochloride.
In pharmaceutical manufacture, it is used as a funda-
mental component in antihistamines, antihypertensive
agents and antiallergenic agents. Benzhydrol is used as an
intermediate of pharmaceuticals (including antihistamines),
agrochemicals, and other organic compounds. Benzhydryl
is a skeleton for Histamine H1 antagonist which an ethyl-
amine group is attached to a diphenylmethane structure. It
is also used as a fixative in perfumery and as a terminating
group in polymerizations. It is carcinogen and Oral (rat)
6
-9
is now capable of selectively rebinding the analyte.
In this context, it is not surprising that much of the
current research in the molecular imprinting field is fo-
cused on SPE, as here the advantages of MIPs, especially
their low price and their stability in different environments,
come into play, whereas some of the limitations are less im-
portant than other separation techniques. MIPs are not only
more selective than common sample treatment methods us-
ing C18 or ion exchange materials, but are at the same time
more stable than (also very selective) immunoextraction
3
LD50 is 5000 mg/kg.
The need for efficient methods for sample precon-
centration and clean up in medical, food and environmental
analyses is constantly increasing. The advantages of SPE
over liquid-liquid extraction (LLE) are that it is faster and
more reproducible, cleaner extracts are obtained, emulsion
formation is not an issue, solvent consumption is mini-
*
Corresponding author. E-mail: hashemimoghaddam@yahoo.com
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643

Article
Hashemi-Moghaddam and Alaeian
matrices. Since MIPs are compatible with organic solvents,
MIP-SPE can be applied directly after a solvent pre-extrac-
tion step. On the other hand, the low resolution factors are
not an issue since SPE works in the adsorption-desorption
mode. Thus, SPE seems to be one of the most promising ap-
plication niches for MIPs today and at the same time the ap-
plication that is closest to commercialization. This is also
reflected in the comparatively large number of reports deal-
ing with real samples. The need for separation of specific
compounds from complex mixtures, industrial or biologi-
cal, has led to an increase in the synthesis and use of molec-
ularly imprinted polymers (MIPs), which in fact act as
biomimetic materials. MIP-SPE has been used to extract
stretching of aromatic C—H sp2 (ca. 3027.21, and ca.
-
1
-1
3059.97 cm ), stretching of alcohol OH (ca. 3379.80 cm ),
-
1
C—O stretch of alcohols (ca. 1077.13 cm ) are not ob-
served in the MIP spectrum. This difference proves that im-
print molecule (benzhydrol) has been sufficiently leached
from MIP in the soxhlet extraction step.
(2) This feature is clear that absorbances attributed to
-
1
the C—H stretch of methylene group (ca. 2954.48 cm ),
-
1
carbonyl group stretch (ca. 1721.63 cm ), C—O stretch
-
1
2
(ca. 1250.20 cm ) and C—H bend of CH (ca. 1451.32
-
1
cm ) for the molecularly imprinted polymer are relatively
stronger than for non-imprinted polymer. From this com-
parison, it was found that presence of imprint molecule
(benzhydrol) causes incorporation of ethylene glycol di-
methacrylate in the preparation of polymer to be increased.
Optimization of adsorption conditions of impurities
on polymer
1
0-11
the target analyte from blood plasma and serum,
1
2-14
15
urine,
bile, liver extract, chewing gum, environmen-
1
6-18
19-20
21-22
tal water and sediment,
plant tissue,
etc.
In the
present work, benzhydrol-molecularly imprinted polymer
was synthesized using methacrylic acid (MAA) as func-
tional monomer and ethylene glycol dimethacrylate
Effect of time on adsorption of impurities
Six portions of standard or sample solutions (100
mL) containing benzhydrol (0.1 mmol) were transferred
into 250 mL beakers. Then exactly 1 g of MIP adsorbent
were added to each beaker, and the mixtures were shaken
vigorously for 30, 60, 90, 120, 150 and 180 min to facilitate
adsorption of the benzhydrol onto the imprinted polymer
particles. After the solutions were centrifuged, the amount
of unadsorbed benzhydrol in the filtrate solutions was di-
rectly determined by spectrophotometry. Figure 1 shows
that an equilibration time of about 150 min was required for
(
EGDMA) as cross-linking agent.
This study was performed to ascertain the optimum
conditions for maximum recovery of benzhydrol from drug
diphenhydramine hydrochloride using a molecularly im-
printed polymer as solid phase extraction adsorbent and the
MISPE-eluate fractions were analyzed by spectrophotom-
etry. Different experimental conditions, e.g. adsorption
time, the type of eluting solvent, the effect of pH and con-
centration of sample have been investigated.
9
1% sorption. The amount of benzhydrol bound to the
RESULTS AND DISCUSSION
polymer was calculated by subtracting the amount of
unadsorbed substrate from the initial amount of template.
Effect of sample pH on adsorption of impurities
The effect of varying pH values on impurities uptake
was investigated using the batch procedure. Six portions of
The aim of this work was to evaluate the feasibility of
using an imprinted polymer as scavenger resins for re-
moval of the genotoxic impurities (GTI) benzhydrol from
active pharmaceutical ingredients (API).
FT-IR spectra
Synthesized molecularly imprinted and control poly-
mers were subjected to characterization by FT-IR. Both
polymers have similar IR spectra indicating the similarity
in the backbone structure.
In the IR spectra, the absorptions due to carboxyl OH
-
1
stretch (ca. 3500 cm ), carbonyl group stretch (ca. 1730
-
1
-1
cm ), C—O stretch (ca. 1260 cm ) and C—H vibrations
-
1
(
ca. 756, ca. 1390, ca. 1460, and ca. 2956 cm ) were ob-
served. In addition to backbone similarity concluding, two
important results were also acquired from spectra that are:
(
1) The absorbances pertaining to benzhydrol, stretch-
Fig. 1. Influence of adsorbing time on the Extraction
of impurities.
-
1
ing of aromatic C=C (ca. 1493.92, and ca. 1597.70 cm ),
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JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Synthesis of Molecularly Imprinted Polymer for Removal of Effective Impurity
standard or sample solutions (100 mL) containing benz-
Table 1. Comparison of MIP and NIP
hydrol (0.1 mmol) were transferred into 250 mL beakers,
and the pH value was adjusted in range 3-8 with 0.01
Polymer
Type
Initial
Final
Extraction
a
a
K
d
concentration concentration
(%)
-
1
mol.L , HCl or NaOH. Then exactly 1 g of MIP adsorbent
was added to each beaker, and the mixtures were shaken
vigorously for 150 min to facilitate adsorption of the benz-
hydrol onto the imprinted polymer particles. According to
the results shown in Figure 2 was found the adsorption
quantity of impurities increases with the pH value increases
and polymer does maximum adsorption at pH = 5. So pH
MIP
NIP
1000
1000
47
962
0.2
3.9 × 10
95.3
3.8
-
4
-
1
[a] µmol L
-
3
-1
1
5
0 mol L of impurities and the pH value was adjusted to
-
1
.0 with 0.01 mol.L , HCl or NaOH. Then were added 1 g
of MIP to a solution and to another 1 g NIP and the mixtures
were shaken vigorously for 150 min to facilitate adsorption
of the benzhydrol onto the imprinted polymer particles.
Then Both filtrate and adsorption the filter solution was
measured with a spectrophotometer. The results in Table 1
show that MIP performs better adsorption than the NIP and
this confirms the accuracy of the molecular format.
Efficient eluent for removal of the retained benzhy-
drol
5
.0 is chosen for this experiment and after pH 5.0 the ad-
sorption capacity of the polymer decreases. The decrease
of adsorption of bezhydrol at the pH values other than 5
may be due to formation of ionic compounds, which those
are more soluble in water.
Adsorption capacity of impurities by MIP
Adsorption of impurities from sample solution was
investigated in batch experiments. At this stage, the effect
of sample concentration on the adsorption impurities was
studied to obtain the best concentration for the sample solu-
tion. Solution with 0.4, 0.6, 0.8, 0.9, 1.0, 1.2 1.5 and 2.0
In order to choose a proper eluent for the retained
benzhydrol, after the extraction of 0.1 mmol benzhydrol
from 100 mL of aqueous sample solution, the benzhydrol
was stripped with 5 mL of various concentrations of differ-
ent organic and mineral acids. Benzhydrol was stripped
with 5 mL of eluents. In order to choose the most efficient
eluent, different organic solvents and various concentra-
tions of different acids in organic solvents were tested. As it
is shown in Table 2, acidic eluents are more effective for
stripping of benzhydrol from polymer. The lipophilicity of
prepared particles assists that by the protonation of MAA
-
1
mmol L concentrations of impurities were prepared and
-
1
the pH value was adjusted to 5.0 with 0.01 mol.L , HCl or
NaOH. Then exactly 1 g of MIP adsorbent were added to
each beaker, and the mixtures were shaken vigorously for
1
50 min to facilitate adsorption of the benzhydrol onto the
imprinted polymer particles. In order to reach the Satura-
tion the initial impurities concentrations were increased un-
til the plateau values (adsorption capacity values) were ob-
tained. The data were shown in Figure 3. The average max-
+
-
1
carboxylic groups in binding sites with the help of H ions,
imum adsorption capacity was 98.3 µmol g for three
replicate measurements.
acidic eluent interacts, via hydrogen bonds, with the poly-
Comparison of MIP and NIP adsorption
Two solutions were prepared at a concentration of
Fig. 3. The effect of benzhydrol initial concentration
on the adsorption quantity of synthesized poly-
mer. Other conditions: 1 g of synthesized poly-
mer, pH 5.0, shaking time 150 min, temperature
25 °C.
Fig. 2. Effect of pH of sample solution on impurities
uptake.
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Hashemi-Moghaddam and Alaeian
Table 2. Effect of type of eluent on extraction efficiency
minutes, the results show that the intensity benzhydrol
peak reduces apparently after purification. In addition, it
demonstrates the selectivity of the polymer to the target
molecule rather than diphenhydramine and these proves
the performance of molecularly imprinted polymer as scav-
enger resins for removal of the genotoxic impurity.
Eluent
Recovery %
-
1
0
0
0
0
.2 mol L H SO
79.26
80.11
46.01
74.46
56.89
51.89
5.76
2
4
-
1
.4 mol L H SO
2
4
-
1
.4 mol L HCl
-
1
.4 mol L CH COOH
3
Methanol
Ethanol
0
0
-
1
1
EXPERIMENTAL
.2 mol L KOH in methanol
-
.2 mol L HCl in methanol
92.11
Reagents: All materials that were required during the pro-
ject include, Methacrylic acid (MAA), 2,2¢-azobisisobutyro-
nitrile (AIBN), ethylene glycol dimethacrylate (EGDMA), chlo-
roform, methanol, hydrochloric acid, sodium hydroxide, sulfuric
acid, ethanol, acetic acid, potassium hydroxide, potassium dihy-
drogen phosphate, phosphoric acid and acetonitrile were obtained
from Merck. Diphenhydramine hydrochloride and benzhydrol
were obtained from Sigma-Aldrich. All chemicals and reagents
were of analytical grade and used without any further purifica-
tion. A stock solution of benzhydrol was prepared by dissolving
an appropriate amount of benzhydrol in ethanol and pH was ad-
justed with HCl or NaOH.
mer to disrupt the interaction between benzhydrol and
polymer. From the data given in Table 2, it is obvious that 5
-
1
mL of 0.2 mol L HCl in methanol can strip the retained
benzhydrol almost quantitatively. Thus, this eluting solvent
was chosen for further studies.
Analysis of real samples
For evaluate the performance of synthesized MIP for
absorption of impurity from the real sample, diphenhydra-
mine syrup was chosen as a real sample and these were ana-
lyzed before and after of purification by high performance
liquid chromatography. Thus, 12 mL of the syrup was di-
luted to 50 mL with water and its pH was adjusted to five.
Apparatus: HPLC measurements were carried out on
Kenauer D-14163 (Berlin, Germany). The high-performance liq-
uid chromatography (HPLC) system was composed of a D-14163
pump (Knauer Co., Germany), a reversed C18 column (4.6 × 150
mmi.d.; Knauer, Germany), a D-14163-detector (Knauer, Ger-
many) set at 220 nm, and a reversed C18 guard column (4.6 × 15
mm; Knauer, Germany). The mobile phase consisted of acetani-
2
0 mL form this solution was mixed with 0.5 gr of polymer
for 150 min and its supernatant and solution of syrup with-
out purification were injected to HPLC column separately.
The condition for HPLC separation was mentioned in ap-
paratus section.
-
1
lide: 0.1 mole L of phosphate buffer, pH = 3 (35:65, v:v), the
-
1
The obtained results were shown in Figure 4. The
peak corresponding to the benzhydrol appeared after 4
flow rate was 1.2 mL min at 30 °C, and the injection volume was
0 ml. Quantification was carried out by the external standard
2
method using a commercial standard sample of benzhydrol. Inte-
gration of the chromatograms was made with the Knauer EZChrom
software package.
UV-VIS spectrophotometer (Varian-Cary100, Australia)
was used for measuring impurities in Standard solutions after
contact with polymer. Soxhlet extraction apparatus was used for
removing the target molecule of the polymer network. A model
3
510 Jenway pH-meter was used for the pH adjustments. The
Fourier transform infrared (FTIR) spectra of template molecule
benzhydrol), non-imprinted and molecularly imprinted poly-
mers were obtained using a 6700 Thermo Nicolet FTIR spectrom-
(
-
1
eter which IR spectra were recorded in the range 400-4000 cm .
Preparation of a benzhydrol-MIP and the correspond-
ing non-imprinted polymer: An MIP and a non-imprinted poly-
mer (NIP) should be prepared in parallel and with identical com-
positions (except that template is to be omitted from the NIP). The
procedure for the polymer synthesis was as follows: To a three-
Fig. 4. Chromatogram of diphenhydramine syrup be-
fore and after of purification.
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JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Synthesis of Molecularly Imprinted Polymer for Removal of Effective Impurity
necked round-bottom flask were added template (benzhydrol: 0.5
mmol or 0.46 g), functional monomer (MAA: 10 mmol or 0.85
mL), cross-linker (EGDMA: 50 mmol or 9.45 mL) and initiator
C -C
V
i
f
K =
´
(1)
(2)
d
C f
W
(C
-C )V
f
i
Q =
(
AIBN: 1.25 mmol or 0.21 g) in chloroform (100 mL). The mix-
W
ture was sparged with nitrogen for 10 min to remove dissolved ox-
ygen, which can inhibit free radical polymerization. The poly-
merization was allowed to continue in a water bath at 60 °C for 18
h. After polymerization, a hard polymer monolith was obtained,
which was crushed and ground into a fine powder with a mortar
and pestle. Soxhlet extraction was performed to remove the tem-
plate with 70/30 (V/V) methanol/acetic acid overnight. Then, the
polymer was washed several times with pure methanol to remove
the acetic acid and facilitate drying. The dried polymer is ready
for testing. The method for preparation of NIP was exactly similar
to procedure for the synthesis of MIP with an exception that
benzhydrol (imprint molecule) was omitted in the preparation of
NIP. Schematic illustration of the process of preparing the benz-
hydrol-MIP is shown in Figure 5.
-
1
where Q represents the adsorption capacity (µmol L ), C
i f
and C
represent the initial and equilibrium concentration of metoprolol
-
1
in the aqueous phase (µmol L ), W is the weight of the polymer
g) and V is the volume of the aqueous phase (L). The percent ex-
traction, E, was calculated using the following equation:
(
C -C
i f
E =
´ 100
(3)
Ci
CONCLUSIONS
Novel molecularly imprinted polymers have been de-
signed which can recognize benzhydrol as a pharmaceuti-
cal impurity. These MIPs have potentials to purify APIs as
selective adsorbents due to high affinity binding sites. Re-
binding tests indicates that high-affinity interactions are
present between the binding sites of the hosts and the target
impurities, which results in high efficiency purification and
it is suitable for repeated use without considerable loss of
adsorption capacity. The new MIPs developed answer to
the industrial call for systems that provide a high binding
between impurities and MIPs, as high recovery of the APIs
is achieved.
Batch procedure: In 100 mL polyethylene bottles, previ-
ously cleaned with detergent, DDI water, dilute nitric acid and
DDI water in sequence, added buffer solution and the benzhydrol
solution, and immersed imprinted polymer with shaking at 25 °C.
At pre-fixed time, an aliquot of the supernatant was separated and
the benzhydrol were determined by UV-Vis spectrophotometer at
2
20 nm. The adsorbed benzhydrol was eluted with 0.2 M HCl in
methanol and the desorbed benzhydrol was measured with UV-
Vis spectrophotometer. The phase distribution ratio (Kd) and ad-
sorption capacity (Q) were calculated using the following equa-
tions:
ACKNOWLEDGEMENTS
We are grateful to the Damghan Islamic Azad Univer-
sity, Laboratory Research Complex and Pars material re-
search and testing (PMRT) for valuable technical assis-
tance.
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