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

Li Zhiwei¹, Yang Gengliang^1,2, Wang Dexian¹, Zhou Shengli³, Liu Erdong¹, Chen Yi^2*
(¹ College of Chemistry and Environmental Science, Hebei University, Baoding 071002; ²Center for Molecular Science, Institute of Chemistry, Chinese Academy of Science, Beijing 100080; ³Hebei JIHENG Pharmacy Co. Ltd., Hengshui 053000, China)

1 INTRODUCTION      
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 of analgin which 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].
　
          A                                 B
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. EXPERIMENTAL
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

3 RESULTS AND DISCUSSION
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

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.

4. CONCLUSIONS
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.

REFERENCES
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分子烙印固定相用于分离氨基安替比林及其结构类似物
李志伟¹ 杨更亮 ¹，2 王德先¹ 周胜利³ 刘二东¹ 陈义²
(¹ 河北大学化学与环境科学学院, 保定 071002; ²中国科学院化学所分子科学中心, 北京 100080; ³河北冀衡药业有限公司., 衡水 053000)
摘要 本文以甲基丙烯酸为功能单体,乙二醇二甲基丙烯酸酯为交联剂，合成了一种氨基安替比林分子烙印聚合物。将所得聚合物用作高效液相色谱固定相，研究了对结构类似物氨基安替比林和氨基比林的拆分能力。分析结果表明，在以甲醇：异丙醇＝2:8条件下两种结构类似物可以得到基线分离，实验还发现在以100％异丙醇为流动相时只有氨基比林被洗脱出来，而模板分子氨基安替比林完全被保留在色谱柱上。
关键词 分子烙印固定相，高效液相色谱，分离，结构类似物。

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