Developing a phosphodiesterase-5 inhibitor assay to detect counterfeit drugs containing phosphodiesterase-5 inhibitors using spectrophotometry

Materials and equipment

Potassium phosphate monobasic (KH₂PO₄), guanosine 3’,5’-cyclic monophosphate (cGMP), 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 adulterated drug, CobraX (CX1A) was kindly donated by Brunei law enforcement agencies. Biotek^TM ELx808^TM absorbance microplate reader (Thermo Fisher Scientific) was used to read absorbance measured at 630 nm in 96-well microplate-based assays by a spectrophotometric method using MLG as previously described³ was adapted with modifications.

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. Under acidic conditions, the bound MLG-MolyB-Pi produced colour changes from blue to shades of green depending on the concentration of Pi³, based on the observation that high concentrations of Pi resulted in high formation of MLG-MolyB-Pi complexes and high absorbance values.

PDE5i detection assay

A functional PDE5i assay consisted of five steps. Figure 1 gives a schematic overview of the protocol.

Figure 1. Schematic summary protocol of the PDE5i assay.

This developed protocol can be used to detect other types of PDE5i in any drug by substituting the compound in the reaction assay using the specific PDE5i of interest and counterfeit drug.

Step 1: Establishment of standard curve for Pi. A fundamental part of the assay was to establish the limit range of Pi concentrations for the MLG assay. Using n=2 replications, 230 µl of Pi solution (0, 2, 5, 7.5, 10, 12.5, 17.5 and 20 µM) was prepared in 0.5 ml microtubes and 75 µl of stop solution (70% perchloric acid) was added. The solutions were mixed by vortex and 140 µl was aliquoted into each designated well of 96-well microplate for MLG assay.

Step 2: Dual biochemical reaction assays for PDE5-cGMP/GMP-CIAP (PDE5 reaction assay). The optimised PDE5 reaction assay was performed to obtain the total Pi generated per reaction, which was subsequently used to calculate the amount of sildenafil in counterfeit drug samples. For reactions replicated n=3 times, 20 µl of PDE5 solution (2000 unit/µg PDE5) and 0.0001 U/µl CIAP solution were added to a 0.5 ml microfuge tube containing 160 µl reaction buffer (20 mM Tris-HCL at pH 7, 10 mM MgCl₂, 0.10 mM EDTA). The solutions were vortexed followed by addition of 30 µl of 15 mM cGMP and incubation for 30 min at 37˚C. After incubation, 50 µl of stop solution was added to terminate the reaction and vortexed followed by centrifugation at 2000 rpm for 5 min. The solutions were then aliquoted into their designated wells in the 96-well microplate with a total volume of 140 µl per well for the MLG assay.

The percentage yield of Pi was calculated using the following formula:

Percentage yield of Pi (%) $=\frac{No.ofmolesofgeneratedPi}{No.ofmolesofexpectedPi}\times 100$

Step 3: Establishment of standard curve for sildenafil. The effect of different concentrations of sildenafil (1, 3, 10, 30, 100 and 300 nM) upon exposure to PDE5 reaction was investigated and subsequently used to generate a standard curve for pure sildenafil with n=4 repeats. First, 40 µl of PDE5-CIAP mixture was added to a 190 µl of sildenafil-cGMP mixture in a 0.5 ml microtube followed by incubation at 37˚C for 30 min. After incubation, 50 µl of stop solution was added and mixed well using vortex followed by centrifugation at 2000 rpm for 5 min. The solutions were then aliquoted to their designated wells in a 96-well microplate with a total volume of 140 µl per well followed by MLG assay performance. The percentage of PDE5 activity of each concentration of sildenafil was calculated using the formula below:

Percentage of PDE5 Activity (%) = $=\frac{\mathrm{Absorbance}ofsildenafil}{AbsorbanceofoptimumPDE5reaction}\times 100\%$

Step 4: Quantification of sildenafil in CX1A samples via PDE5-CX1A reaction assay. A PDE5-CX1A reaction assay was performed to assess and quantify sildenafil in CX1A samples with n=2 repeats. To a 0.5 ml microtube containing 140 µl of reaction buffer, 20 µl of CX1A solution (0.1 mg/ml and 0.01 mg/ml) and 30 µl of 15 mM cGMP were added and mixed using vortex. For a reaction to start, 40 µl of PDE5-CIAP mixture was added followed by incubation at 37˚C for 30 min. After incubation, 50 µl of stop solution was added to the reaction and mixed well using vortex followed by centrifugation at 2000 rpm for 5 min. The solutions were then transferred to their designated wells in a 96-well microplate with a total volume of 140 µl per well followed by MLG assay.

Percentage of PDE5 activity (%) $=\frac{AbsorbanceofCX1Adrugsolution}{AbsorbanceofoptimumPDE5reaction}\times 100$

The concentration of sildenafil was calculated using the following formula, which was derived from the standard curve of sildenafil:

Y = Bottom + $\frac{Top-Bottom}{1+{10}^{(logIC50-X)slope}}$

Where Y = percentage of PDE5 activity, X = Concentration of sildenafil, top value: 99.99, bottom value: -1.230, logIC₅₀ (half-maximal inhibitory concentration): -0.505 and slope: -0.722

Step 5: MLG assay. After the desired solutions were aliquoted into their designated well in the 96-well microplate, the MLG assay was performed, where 14 µl of MolyB reagent (50 mM MolyB in 3.4 M sulphuric acid) was added to each well containing the mixture, mixed thoroughly and incubated for 5 min. Then, 26 µl of MLG reagent (1.0 mM MLG, 6.0 mM sulphuric acid, 0.16% PVA) was added, mixed well and incubated for 10 min. After incubation, the absorbance was measured using Biotek^TM ELx808^TM absorbance microplate reader at 630 nm.

Statistical analysis

Statistical comparisons were made by means of two-way repeated measures Analysis of Variance (ANOVA). The data were analysed with Prism software (Version 6, Graph Pad Inc.). The results were shown as mean ± S.E.M and n represents the number of repeats in each experiment. P values of 0.05 or lower was considered to be statistically significant.

Establishment of a standard curve for Pi

Pi concentrations ranging from 0 µM to 20 µM were used to generate a standard curve for Pi. Table 1 and Figure 2 show the relative absorbance values for Pi (0 µM to 20 µM) and the linear relationship between absorbance and concentration of Pi, respectively. The equation derived from the standard curve was used to quantify the amount of Pi generated from PDE5 reaction. Raw results of this assay, alongside each of the assays performed in this study, are available as Underlying data⁴.

Figure 2. The linear relationship between absorbance and Pi concentration from 0 to 20 µM.

The correlation allows the establishment of standard curve of Pi.

Dual biochemical reaction assays for PDE5-cGMP/GMP-CIAP (PDE5 reaction assay)

Using an optimized PDE5 reaction, 1.842 moles of Pi was generated as shown in Table 2.

Establishment of standard curve of sildenafil

Table 3 shows the percentage of PDE5 activity at each concentration of sildenafil. Using the values obtained, a sigmoidal curve was established between PDE5 activity and log concentration of sildenafil, with an IC₅₀ of 0.3124 µM (Figure 3).

Figure 3. The standard curve of sildenafil (Sil) from 0.1 nM to 300 μM with IC₅₀: 0.3124 µM.

Sigmoidal relationship was established for standard curve of pure Sil.

Quantification of sildenafil in CX1A samples

From the PDE5-CX1A reaction assay, the percentage values of PDE5 activity using low (0.1 mg/ml) and high dilution (0.01 mg/ml) samples of CX1A were 31.33% and 78%, respectively. Using the equation derived from the sigmoidal curve of sildenafil, the concentration of sildenafil was determined as the percentage of sildenafil in low and high dilutions of CX1A samples, i.e. 0.673% and 0.407%, respectively. The mass of sildenafil in CX1A tablet is 3.149 mg. The calculated values were as tabulated in Table 4 and Table 5.

Underlying data

Open Science Framework: PDE5i Assay. https://doi.org/10.17605/OSF.IO/PV59F⁴.

This project contains ‘Aziemah et al. 2019 Raw_data for developed PDE5i assay.xlsx’, which includes raw data for all experiments performed in this study.

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).