Development of a group-specific antibody-based immunoassay method for simultaneously detecting sildenafil-like adulterants in herbal spirit drinks

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.

Graphical abstract

KEYWORDS:

• Sildenafil-like adulterants
• polyclonal antibody
• enzyme-linked immunosorbent assay

Disclosure statement

The authors declare no competing financial interest.

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Additional information

Funding

This work was supported by the National Key Research and Development of China (2018YFC1602904), the National Natural Science Foundation of China (31871887), the Science and Technology Planning Project of Guangzhou (201704020082), the Science and Technology Planning Project of the Guangxi (AB18126048), the Guangdong Basic and Applied Basic Research Foundation (2019B1515210025, 2020A1515011238 and 2018B030314005), the Key Scientific Research Projects of Guangdong Provincial Universities and Colleges (2018KZDXM011).