ABSTRACT

Phosphodiesterase type 5 (PDE-5) inhibitors are commonly used to treat erectile dysfunction. There is a problem with synthesis and illegal use of a wide range of analogues of the licenced drugs and a simple class-wide analytical method is required. In this work, based on structural modelling, we developed an immunological method using norneovardenafil as a hapten as it contains only the general sub-structure and the common features of sildenafil-like adulterants, such as hydrophobic centres, hydrogen-bond donor atoms and hydrogen-bond acceptor atoms. Thus theoretically it could induce production of antibody which could recognise multiple sildenafil-like adulterants. By immunising rabbits, a group-specific polyclonal antibody was obtained with the desired broad-spectrum molecular recognition performance against sildenafil-like adulterants. Then, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed for the detection of sildenafil-like adulterants in herbal spirit drinks. Under the optimised conditions, the icELISA method showed broad linear ranges for acetildenafil, sildenafil and vardenafil respectively of 0.7 to 27.7 μg/kg, 1.0 to 70.7 μg/kg and 1.5 to 22.7 μg/kg, with half-maximal inhibition concentration (IC₅₀) values of 4.5 μg/kg, 8.3 μg/kg and 5.7 μg/kg, respectively. For eleven herbal spirit drinks, there was good agreement between total levels of sildenafil-like adulterants measured by icELISA and levels of each of four individual adulterants determined by LC-MS/MS. In short, the developed icELISA can be employed for rapid and simple screening for adulteration of herbal spirit drinks with sildenafil-like compounds.

摘要

5 型磷酸二酯酶 (PDE-5) 抑制剂通常用于治疗勃起功能障碍。大量许可药物类似物的合成和非法使用存在问题，因此需要一种简单的全类别分析方法。在这项工作中，基于结构模型，我们开发了一种使用去甲新伐地那非作为半抗原的免疫学方法，因为它只包含西地那非样掺杂物的一般子结构和共同特征，例如疏水中心、氢键供体原子和氢-键受体原子。因此理论上它可以诱导产生可以识别多种西地那非样掺假物的抗体。通过对家兔进行免疫接种，获得了对西地那非样掺假物具有所需广谱分子识别性能的群特异性多克隆抗体。然后，开发了一种间接竞争性酶联免疫吸附试验 (icELISA)，用于检测草本酒精饮料中的西地那非样掺杂物。在优化条件下，icELISA方法显示出乙酰地那非、西地那非和伐地那非的广泛线性范围，分别为0.7～27.7 μg/kg、1.0～70.7 μg/kg和1.5～22.7 μg/kg，半数抑制浓度（IC₅₀ ) 值分别为 4.5 μg/kg、8.3 μg/kg 和 5.7 μg/kg。对于 11 种草本酒精饮料，通过 icELISA 测量的西地那非样掺杂物的总水平与通过 LC-MS/MS 确定的四种单独掺杂物的水平之间存在良好的一致性。简而言之，开发的 icELISA 可用于快速、简单地筛查含有西地那非类化合物的草本烈性饮料掺假。