Separation of aminoantipyrine and its close analogues by molecular imprinting stationary phase

Li Zhiwei, Yang Gengliang1,2, Wang Dexian, Zhou Shengli, Liu ErdongChen Yi2*
College of Chemistry and Environmental Science, Hebei University, Baoding 071002; Center for Molecular Science, Institute of Chemistry, Chinese Academy of Science, Beijing 100080; Hebei JIHENG Pharmacy Co. Ltd., Hengshui 053000, China)

Received Jan.12, 2003; Supported by the National Natural Science Foundation of China (20075005), the Natural Science Foundation of Hebei Province (202096) and Ministry of Science and Technology.

AbstractA synthetic polymer selector for aminoantipyrine is prepared by molecular imprinting technology. Methacrylic acid and ethylene glycol dimethacrylate are copolymerized in the presence of the template aminoantipyrine. The template is extracted from the polymer leaving specific recognition sites, complementary to the template. The polymer is utilized as a stationary phase in HPLC. The mixture of the two close analogues, aminoantipyrine and aminopyrine, can be baseline separated when the mobile solution is composed of methanol: isopropanol = 2: 8. When the concentration of isopropanol is 100%, only aminopyrine is eluted and the aminoantipyrine is completely reserved by the column.
Keywords Molecular Imprinting Stationary Phase, High performance liquid chromatography, Separation, aminoantipyrine, close analogues.

Molecular imprinting polymer (MIP) has drawn increasing attention in recent years[1-4]. Owning to its higher molecular recognition ability and stability than that of biological molecules. MIP has been used in a wide range of applications, such as chiral separartion[5-8], solid phase extraction[9,10], sensor[11,12], immuno-analysis[13] and has become an effective strategy to prepare stationary phase of HPLC having a specific molecular recognition[14]
The first example of molecular imprinting of organic network polymers introduced by Wulff is based on a covalent attachment strategy, i.e., covalent monomer-template, covalent polymer-templates[1]. The most successful non-covalent imprinting systems are based on commodity acrylic or methacrylic monomers, such as methacrylic acid, crosslinked with ethyleneglycol dimethacrylate. The template molecule usually involves relatively polar functional groups, such as carboxylic acid, hydroxyl, amino, and/or aromatic groups, and therefore appropriate host monomers having functional groups that may interact with the functional groups of the template molecule through intermolecular interactions such as ionic interaction are often utilized to compose a more effective imprinted site.
Aminoantipyrine and aminopyrine are the intermediates ofanalginwhich is an alesipyretic and antalgic pharmaceutical. Aminopyrine had also been used for the treatment of heat exposure and ache, but it is replaced by analgin because it will arise the deficiency of leucocyte and granular leudocytes[15]

Fig. 1 The structures of aminoantipyrine and aminopyrine A=aminoantipyrine, B=aminopyrine

In this study, an aminoantipyrine-imprinted polymer is prepared using aminoantipyrine as the template, methacrylic acid as the functional monomer. Aminopyrine is used as a structurally related reference compound for the evaluation of the selectivity of the imprinted polymer. Furthermore, aminopyrine-imprinted polymer is also prepared. Chromatographic analysis showed that the aminoantipyrine-imprinted polymer is capable of recognizing the functional difference of the two molecules, while the aminopyrine-imprinted polymer have no such ability. The structures of the two molecules are shown in Fig.1.

2.1 Reagents and chemical
Aminoantipyrine aminopyrine are provided by Hebei JIHENG Pharmacy Co. Ltd, (Hengshui, China). Methacrylic acid(MAA) is from The First Tianjing Chemical Reagent Company(Tinajing, China). Ethylene glycol dimethacrylate (EDMA) is from Acros(NJ, USA). The compound 2,2′-azobisisobutyronitrile(AIBN) is from the fourth Shanghai chemical and reagent Factory (Shanghai, China). Acetonitrile is purchased from Fisher (USA, HPLC grade).

Fig.2 Preparation of imprinted polymers

2.2 Apparatus
The chromatography system consisted of JASCO (JASCO, Japan) PU-1580 and PU-1586 pumps, an UV-VIS wavelength UV-1570 detector. Data processing is carried out with a JASCO LC-1500 chromatography workstation. A CS501-SP thermostat is from Sida Experimentation Apparatus Factory (Chongqing, China). The eluent used for the separation studies is methanol-isopropyl alcohol. The flow rate is 0.3ml/min, the detector is at 280 nm and the separation is performed at room temperature.
2.3 Preparation of the stationary phase and packing of the column
Aminoantipyrine-imprinted and aminopyrine-imprinted polymers are prepared identically in the meantime with MAA as functional monomer. The polymer is synthesized following the general imprinting protocol shown in Fig.2. 1.0 mmol aminoantipyrine, 6.0 mmol MAA are dissolved in acetonitrile in a glass polymerization test tube, then 30 mmol EDMA and 0.065g AIBN are added into the solution. The test tube is purged with nitrogen for 10 min and sealed under vacuum. After that, polymerization is carried out in a water bath with the temperature maintained at 60ºCfor 24 h. The polymerized monolith is ground into particles and by passing through a 35m sieve and by repeated suspending in water to remove the small particles, the size fraction of 25-35m is obtained and it is packed in a 150×4.6mm stainless steel HPLC column. Non-imprinted blank polymers in the absence of template are prepared and treated in the same manner. Then a solution of methanol-acetic acid(9:1) is used to remove the template. In the experiments, three different polymers are prepared, which are the imprinted polymer made up with aminoantipyrine as the template(P(1)), the imprinted polymer with aminopyrine as the template( P(2)) and the blank polymer (P(3)).
The separation factor () is determined by
where k’ is the capacity factor of the aminoantipyrine and k’ is the capacity factor of the aminopyrine.
The capacity factors are determined by
k’= (t-t)/t0,
where t is the retention time of the solute and t is void time of the column, which is determined by NaNO as the void marker. All the procedures are carried out at room temperature.

3.1 Effect of templates
Methacrylic acid (MAA) is chosen as the functional monomer because MAA could interact with the amino groups(see Fig.1) of the compounds. The molecular recognition ability of the imprinted polymers is evaluated by comparing the retention time of aminoantipyrine and aminopyrine on the columns packed with the polymers. As shown in Table 1,it can be seen that P(2) and P(3) polymers had no selectivity for aminoantipyrine and aminopyrine, while the retention and the selectivity of aminoantipyrine-imprinting polymer P(1) for the two molecules are greatly strengthened by molecular imprinting. This showed that the amino group in aminoantipyrine could form hydrogen bond with the monomer and produce specific sites in the polymer. Aminopyrine, on the other hand, as no amino group exists in the molecular structure and the amide group could only form so weak hydrogen bond with the monomer that it is hard to produce effectively specific sites.
3.2 Chromatographic application
P(1) is employed as the stationary phase of the chromatographic column. Different factors, such as, the composition of the organic solvents, flow rate, temperature, are investigated to study the influence on the separation of the two analogues.

Table 1
The capacity factors of the analytes on the imprinted polymers

Polymers k’ k’
P (1) 1.044 2.925 2.802 1.2
P (2) 1.892 1.892
P (3) 1.248 1.248


time (min)                      time (min)
Fig.4 Chromatograms of the separation of the two analogues. Run conditions: column,150×4.6mm ; flow rate, 0.3mL/min; wavelength, 280nm; (a)Eluent is 100% isopropanol (b)Eluent is methanol:isopropanol=2:8.

3.3 Effect of the composition of the organic solvents
Experiment showed that polarity of the organic solvents had significant effect on the retention behavior of the template. In Fig.3, it can be seen that the k’value of antipyrine changes slightly, the k’value of aminoantipyrine changes quickly with the change of concentration of the solvent. On the other hand, it increases with the increase of isopropyl alcohol concentration in the methanol-isopropyl alcohol system. When the isopropanol concentration is 100%, only aminopyrine is eluted and the aminoantipyrine is completely reserved by the column (see Fig.4a).
3.4 Effect of the flow rate and temperature
The migration times of both aminoantipyrine and aminopyrine decrease with the increase of flow rate, but the resolution of the analogues become poor. This may be that the interaction of the temperate molecule with the polymer becomes weak at high flow rate conditions.
Various temperatures changing from 25ºC to 55ºC are also investigated to study the influence on the retention of the two analogues(Fig.5). Under higher temperature, both the template and aminopyrine migrate fast, but the template changes faster than the aminopyrine. Specific interaction of the template and the specific sites showed stronger dependence on temperature.

Fig.3 Effect of the composition of the organic solvents      Fig.5 Effect of the different Temperatures

From the above results, aminoantipyrine imprinted polymer showed better selectivity on the close analogues (Fig.4b). In HPLC application, under given condition, such as, when 100% isopropanol is used as the mobile phase, aminopyrine is not reserved whereas aminoantipyrine is completely reserved. This is very important in separation or purification of the drugs.

Education Press, 1999, 360.