**3. Pharmaceutical compounds as templates**

**2. Synthetic approaches**

50 Recent Research in Polymerization

The molecularly imprinted polymerization techniques and methods have been well described in literature [22]. Basically, the imprinting process involves self-assembled of selected functional monomer around a template molecule followed by polymerization in the presence of a cross-linker [23]. The template is then removed from the polymer matrix, thus, leaving behind a cavity complementary in functional group, size and shape, which is available to strongly bind compounds that are closely related to the template molecule. This is demonstrated in **Figure 2** using the synthesis of MIP for fluconazole, an antifungal agent, as an example [24]. In their synthetic reaction [24], they used methacrylic acid, ethylene glycol dimethacrylate and fluconazole as functional monomer, cross-linker and template, respectively. Despite its

**Figure 2.** Synthetic scheme of a MIP for fluconazole adapted from Manzoor et al. [24].

Templates are the most important reagent in the spatial arrangement of functional monomers during polymerization. Many pharmaceutical compounds of different therapeutic classes have been imprinted using the single-template and multi-template synthetic approaches [26–28]. Some of the imprinted compounds are given in **Table 1**. It has been observed that the non-steroidal anti-inflammatory drugs (NSAIDs) and analgesics with the exception of fenoprofen which are frequently detected in environmental waters have been imprinted [29]. NSAIDs are known as the most consumed pharmaceuticals and in most cases their MIPs have been applied as selective SPE sorbents [29]. Most pharmaceutical drugs have hydroxyl and carbonyl functional groups which makes the imprinting process easy by utilizing their ability to form hydrogen bonding with several functional monomers. Also, due to the presence of aromatic rings in some pharmaceutical compounds and functional monomers, it is possible to have electrostatic interactions between the rings of different molecules. In this aspect, Farrington and Regan [30] demonstrated computationally the electrostatic interaction between the aromatic rings of ibuprofen (template) and 4-vinyl pyridine (functional monomer). Currently, MIPs are developed using one pharmaceutical compound as the template (uni-templating and dummy-templating). Also, there are procedures that have been shown in literature where many pharmaceuticals are used as multi-templates [26, 28]. In multi-templating approach, equal amounts of templates are added simultaneously into the polymerization mixture. Once removed at the end polymerization reaction, the resulting MIP can selectively re-bind the removed compounds from the environmental samples.

#### **3.1. Physico-chemical properties of pharmaceutical compounds**

Few pharmaceutical compounds that have been used in molecular imprinting as template molecules are given in **Table 1**. It can be seen from their molecular structures that pharmaceutical compounds compose of a variety of functional groups. The presence of functional groups in the template molecule makes it easy to undergo the molecular interactions such as hydrogen bonding. For example, the presence of carboxylic group in ibuprofen allows for hydrogen bonding in acidic conditions with 2-vinyl pyridine functional monomer [31]. Such hydrogen bonds are easy to break by washing the MIP with acetic acid which is a small molecule that can penetrate the pores of the MIP, thereby, disrupting the molecular interactions. This leads to easy regeneration of the MIP.

**Table 1.** Physico-chemical properties of selected imprinted pharmaceuticals.

The pKa of a drug influences lipophilicity, solubility, protein binding and permeability which in turn directly affects pharmacokinetic (PK) characteristics such as absorption, distribution, metabolism and excretion [32, 33]. It is very important to understand the physico-chemical properties of templates and functional monomers prior to any MIP application. This is highlighted in the work reported by Dai et al. [34]. In their work, the adsorption efficiency of clofibric acid decreased significantly with the increase of pH when the pH was between 6 and 12. This phenomenon could be explained by the ionization of clofibric acid (pK<sup>a</sup> of 3.18) which could occur under strong basic condition. Therefore, clofibric acid was negatively charged. On the other hand, the functional monomer of 2-vinyl pyridine (pK<sup>a</sup>  of 4.98) used in the synthesis of MIP could also be negatively charged. It is known that the ─COOH groups in the selective binding cavity of MIP play a key role in the rebinding of target compounds. The adsorption at basic pH could be due to the hydrophobic interactions [34].

#### **3.2. Uni-templating**

In most cases, uni-templating is done in order to extract one target analyte based on one attractive attribute such as bioactivity or ease excretion from human metabolism or the negative effect of that analyte such as toxicity or widespread in the environment. In the synthesis of MIPs, the target compounds are usually used as the template molecules. A diversity of pharmaceutical compounds have been detected in environmental waters [17, 35], therefore, most pharmaceuticals have been imprinted for the purpose of developing selective analytical methods. Conventionally, a single-template is imprinted which subsequently lead to the isolation of one pharmaceutical compound from water matrix [36]. Many pharmaceuticals that include ketoprofen [3], indomethacin [25], 17β-estradiol [27] and diclofenac [37] have been imprinted using the single templates. MIPs that have been synthesized using this approach usually possess high selectivity towards the compound that was used as template molecule. High selectivity maybe due to molecular recognition that could be strongly influenced by functional groups, shape and size of the target compound. In this case, selectivity is usually evaluated by extracting a mixture of organic compounds that consist of the target compound and other structurally related compounds. Zunngu et al. [3] tested selectivity of ketoprofen MIP for its ability to extract similar compounds (triclosan, gemfibrozil and fenoprofen) along the target compound from spiked deionized water. Their results showed accepted recovery (104%) for ketoprofen, and for competitors the recoveries did not exceed 20%. In a different study, Ming et al. [27] demonstrated the competitive adsorption ability of 17β-estradiol MIP towards the target compound in the presence of estriol, estrone, bisphenol A and hexestrol in aqueous solutions. Single-template MIPs usually lead to superior sample clean-up that subsequently results in cleaner chromatograms, however, this approach do not allow for a simultaneous multi-compound analysis. Multi-compound analysis can only be performed using uni-templating procedure after physical mixing a number of MIP for individual compounds. However, this approach is not financial feasible as it is expensive to synthesize a number of MIPs whose mixtures will be used to target a number of pharmaceuticals.

#### **3.3. Multi-templating**

The pKa

of a drug influences lipophilicity, solubility, protein binding and permeability which

H CH3

OH OH

in turn directly affects pharmacokinetic (PK) characteristics such as absorption, distribution, metabolism and excretion [32, 33]. It is very important to understand the physico-chemical properties of templates and functional monomers prior to any MIP application. This is highlighted in the work reported by Dai et al. [34]. In their work, the adsorption efficiency of clofibric acid decreased significantly with the increase of pH when the pH was between 6 and 12.

N

<sup>N</sup> <sup>O</sup>

H

**Table 1.** Physico-chemical properties of selected imprinted pharmaceuticals.

H

**Therapeutic class Compound Chemical structure**

H3C

H3C N

O

F

HO

O

F F NH

CH3

N H

CH NH <sup>3</sup>

N

H

NH2

Cl

OH

O O O

CH3

O

OH

NH2

OH

N H

H3C CH3

NSAID Ibuprofen

52 Recent Research in Polymerization

Antidiabetic Metformin

ARV Efavirenz

Antibiotic Tetracycline

Anti-epileptic Carbamazepine

One of the advantages of MIPs is the selective extraction of analytes in complex matrices. However, at times it is desired that a class/many compounds are removed or extracted from the environmental or real samples simultaneously. To achieve this, researchers have explored the possibility of imprinting multiple templates all at once for the

**Dummy template**

Diphenylamine

H

N

**Target compounds**

Diclofenac

Mefenamic acid

Selective in the

100–112

[44]

54 Recent Research in Polymerization

presence of

ibuprofen,

naproxen,

ketoprofen and

salicyclic acid

H

Cl

N

C

O

OH

H

C

HO

O

CH3

> N

CH3

Cl

Phenothiazine

H

CH

2CHN(CH3)2

CH3

(C

H2)3N(CH3)2

Selective in

93–98

[42]

the presence

of tetracycline,

naphthalene and

anthracine

N

COCH3

S

(C

H2)3N(CH3)2

N

Cl

N

N

S

S

2-chlorophenothiazine

H

N

Cl

(C

CH2

N

S

N (C

H2)2OH

H2)

2

N

Cl

S

S

Salbutamol

Ractopamine

Selectivity was done

—

[41]

chromatographically

using clenbuterol,

terbutaline and

adrenaline as

competitors.

HO

OH

OH

OH

H

N

OH

OH

NH

**Selectivity**

**Recovery** 

**Reference**

**(%)**

lomefloxacin, enrofloxacin and gatifloxacin).

\*Fluoroquinolones—only two chemical structures are given, however, there were eight target compounds (fleroxacin, ofloxacin, norfloxacin, pefloxacin, ciprofloxacin,

**Table 2.** Chemical structures of dummy template molecules, their relative extracted compounds and analytical performance. pre-concentration and extraction of a certain group of compounds where a cocktail of pharmaceutical compounds is used in the polymerization set-up. Practical examples for this include the work of Duan et al. [26] where five acidic pharmaceuticals which are ibuprofen, naproxen, ketoprofen, diclofenac and clofibric acid, were used as multi-templates in a synthesized MIP that showed selective recognition and ability to extract these target compounds from lake water and WWTP effluent using molecularly imprinted solid-phase extraction (MISPE) technique. In their work, Duan et al. [26] obtained the recoveries that were greater than 95% for all five acidic pharmaceuticals in lake water and wastewater spiked with 1 μg.L−1 of each compound. Dai et al. [38] have also reported the selective removal of the same group of pharmaceutical compounds using a multitemplate MIP from contaminated water. These researchers demonstrated the selectivity of the multi-template MIP in the presence of fenoprofen and carbamazepine (both pharmaceutical compounds). In their study, the removal efficiency for the five target pharmaceuticals in water was greater than 80%, whereas, less than 40% was reported for fenoprofen and carbamazepine used as competitors. In the same aspect, a dual template (naproxen and ketoprofen) MIP has been reported [28], where the ability to recognize the template molecules was tested chromatographically in the presence of structural analogues (ibuprofen, fenbufen, fenoprofen and flurbiprofen). Also, Dai et al. [34] prepared a novel double-template MIP by precipitation polymerization using carbamazepine and clofibric acid as the double templates.

For a multi-template (naproxen, ibuprofen and diclofenac) MIP, it was observed that the selectivity collapses easily during the extraction of target compounds from aqueous phase [31]. This could be strongly influenced by bigger cavities that are created due to the usage of multi-templates. This could allow the easy access of many presumably smaller molecules into the binding sites. It has been demonstrated that the untargeted compounds can be selectively washed off from the multi-template MIP surface due to their weaker non-specific interactions with the polymer [31]. Contrarily, some researchers have indicated that the use of a dummy template during polymerization increased the selectivity of the final MIPs that target more that target one compound [39, 40].

#### **3.4. Dummy-templating**

The usage of target compounds as template molecules could have negative impact on the analysis of real samples due to their bleeding upon application into the sample matrix. This could be severe in the cases of incomplete template removal. To avoid this problem, the use of dummy templates for the synthesis of MIPs has been proposed in many studies [41–43]. In certain instances, the selected dummy templates exhibit the properties of more than one compound, and its chemical structure is closely related or similar to more than one pharmaceutical drug [42, 44]. In such cases, the prepared MIP is able to selectively extract more than one compound. This has been demonstrated by the synthesis of a MIP using diphenylamine as the template whose chemical structure closely resembles that of both diclofenac and mefenamic acid (**Table 2**) [44]. As shown in **Table 2**, the dummy molecule (diphenylamine) for both diclofenac and mefenamic acid can be characterized by two phenyl groups which are both attached to the amine group, such groups also appear on the structures of the target compounds. Similarly, the approach has also been reported in the synthesis of MIP required for SPE of phenothiazines from meat samples, where phenothiazine and 2-chlorophenothiazine were used as dummy templates, thereby taking the advantage of their core chemical structures that compose of phenothiazine [42]. Both MIPs synthesized with phenothiazine and 2-chlorophenothiazine dummy templates were able to capture four different phenothiazines (acepromazine, promethazine, perphenazine and chlorpromazine), simultaneously. In a different work, a compound, daidzein, was used as a non-poisonous dummy template in the synthesis of MIP for fluoroquinolone antibiotics [43]. Their synthesized MIP was successfully applied for matrix solid-phase dispersion extraction of eight fluoroquinolones from fish samples. Such work gave high recoveries and selectivity for target compounds (**Table 2**). Similarly, the analysis of single pharmaceutical drug, ractopamine, commonly used for the treatment of asthma has been performed using dummy template MIP which has been synthesized using salbutamol as a dummy template [41]. The drawback that could be associated with the use of dummy templates in molecular imprinting could rise during the applications in real samples. Selectivity can be reduced greatly due to the differences in the physico-chemical properties of the dummy template and the targeted compound(s). For instance, in **Table 2**, it can be seen that diphenylamine (dummy template) is relatively smaller, in terms of size, as compared to the diclofenac (target compound). The same can be observed in the case of phenothiazine presented in **Table 2**. This could lead to easy binding of smaller molecules with similar functional groups into MIPs.
