Abstract
Background: Phosphodiesterase-5 inhibitors (PDE5i) are the first line of drugs used for the treatment of erectile dysfunction. PDE5i inhibits the activities of phosphodiesterase-5 (PDE5) found in corpus cavernosum reducing production of guanosine monophosphate (GMP) that results in vasodilation of blood vessels thereby prolonging penile erection. Various counterfeit drugs adulterated with unknown levels of PDE5i pose a danger with reported undesirable adverse effects on patients. Methods: We developed an assay to detect counterfeit drugs containing PDE5i using spectrophotometry based on a colour change of malachite green from blue to green when exposed to inorganic phosphate (Pi) measured at 630 nm. Initially, a standard graph of a known Pi concentration was established ranging from 0 to 20 µM followed by a dual biochemical reaction system of converting GMP from cGMP by PDE5, generating Pi from the first reaction’s product by calf intestinal alkaline phosphatase (CIAP), and then measuring Pi using an equation derived from the generated standard curve. Dilutions of CX1A, a counterfeit drug sample adulterated with the PDE5i sildenafil, were prepared via ultrasonication and filtration for assay testing and evaluation. Results: Sil was detected from the two separate samples comprised of low and high dilutions of CX1A quantified as 0.673% and 0.407%, respectively, based on a standard curve of pure sildenafil established from 0.1 nM to 300 µM. Conclusions: This PDE5i assay that we developed has the potential to become an alternative analytical method for detecting PDE5i showing comparable results when evaluated using HPLC.
Highlights
Phosphodiesterase-5 inhibitors (PDE5i) are synthetic drugs for clinical treatment of erectile dysfunction prescribed only by a qualified physician[1]
Materials and equipment Potassium phosphate monobasic (KH PO ), guanosine 3’,5’-cyclic monophosphate, polyvinyl alcohol (PVA), sildenafil citrate salt, calf intestinal alkaline phosphatase (CIAP), human phosphodiesterase 5A1 (PDE5), ammonium molybdate tetrahydrate (MolyB) and malachite green oxalate free base (MLG) were all obtained from Sigma-Aldrich
The MLG assay used to detect the counterfeit PDE5i drugs consisted of complex formation between phosphomolybdate (MolyB) with Pi, which was released from the PDE5-mediated hydrolysed cGMP, to produce MolyB-Pi complex, which was subsequently used to bind with MLG oxalate
Summary
Phosphodiesterase-5 inhibitors (PDE5i) are synthetic drugs for clinical treatment of erectile dysfunction prescribed only by a qualified physician[1]. PDE5i are widely used in the illegal market as an adulterant in counterfeit drugs, especially in herbal supplements claiming the enhancement of male sexual vitality in a natural way[2]. The use of these counterfeit drugs with unknown content has posed significant health risks to patients. A known counterfeit herbal drug, CobraX (CX1A), was investigated to develop a potential analytical assay to detect PDE5i (i.e. sildenafil citrate) by utilizing the by-products (i.e. inorganic phosphate (Pi)) that were released from a reaction among the substrates comprised of cGMP, enzyme PDE5 and PDE5i (often adulterated in counterfeit drugs, i.e. CX1A). Conclusions: This PDE5i assay that we developed has the potential to become an alternative analytical method for detecting PDE5i showing comparable results when evaluated using HPLC
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