Unapproved ED medications – a dangerous market

Before Viagra® lost its patent in 2013 [19], competing products which had not been approved for medical use nor investigated for consumer safety were starting to appear on the market – including synthetic analogs of sildenafil. From 2002 to 2014, 39 illegal compounds were identified [16], most frequently: homosildenafil; hydroxyhomosildenafil; hongdenafil; hydroxyhongdenafil; aminotadalafil; piperidenafil; and methiosildenafil.[20]

These compounds can be found in plant-based food supplements, drugs of unknown origin, and bulk powders [20, 21]. For example, homosildenafil and hydroxyhomosildenafil have been found in herbal, anti-fatigue tablets that are sold as “herbal Viagra”.[22] Zou et al. [17] identified sildenafil, homosildenafil, hydroxyhomosildenafil, hongdenafil, vardenafil, and tadalafil in bulk powder samples, whereas tadalafil and sildenafil were found in 31 commercial products analyzed by Do et al. [23]. Another sildenafil analog – isosildenafil – has been very little studied, and has not been approved for medical use.[19]

The truth is that counterfeit and other illegal substances masquerading as legitimate, approved medications carry a significant health risk. Impurities found in illegal ED medicines include glibenclamide, talcum powder, amphetamines, and dyes. Both illegal and pharmaceutical PDE-5 inhibitor usage can lead to headaches, muscle pain, nausea, vomiting, hot flashes, and vision problems. And, given unregulated concentration levels, hypotension and similar effects may occur. This is especially dangerous when taken together with other vasodilating agents such as allyl nitrites, a.k.a. “poppers” – and can even lead to death.

Applying UHPLC-MS/MS to forensic investigations of fake pharma

For a court case resulting from fatal intoxication, judicial authorities asked the Institute of Toxicology Research in Borowa, Poland, for assistance in a toxicological analysis of a suspected counterfeit compound. The Institute’s researchers did this, and simultaneously developed a useful new method for use in identifying illegal ED medications.

Here’s a brief look at how they did that. The Institute had previously developed a screening method for the simultaneous determination of many groups of substances, including PDE-5 inhibitors and their metabolites. This method was developed and fully validated with the use of spiked blood samples and a quantification method using an internal standard.

Using that method, the sample preparation procedure was based on quick and simple liquid-liquid extraction (pH 9 with ethyl acetate). The identification of unknown substances was achieved with the use of an ultra-high-performance liquid chromatograph (UHPLC) Shimadzu Nexera X2 coupled with a tandem mass spectrometry (QqQ) Shimadzu LCMS-8050.

Due to the high sensitivity of this equipment, the sample volume could be reduced to only 200 µL. For the screening analysis of the court-provided blood sample, the Q3 scan mode was applied, while confirmation of the chemical compounds was done with the use of product ion scan experiments. Precursors were fragmentated at three collision energies (10, 20 and 35 V).

The results of this toxicological analysis with detailed chromatographic and mass spectrometry conditions as well as characteristic fragments are presented in figures. Five analogs belonging to the phosphodiesterase inhibitors group (including two metabolites) were identified by the aforementioned method. The substances detected were sildenafil and its metabolites (N-desmethyl sildenafil and UK-331, 849), as well as two synthetic analogs of PDE-5 inhibitors (isosildenafil and homosildenafil), whose presence in the blood may indicate that the medication was manufactured by and/or purchased from illegal sources.

The danger associated with sildenafil analogs is the lack of data regarding toxicity and safety. Due to the similarities of their structures with legal phosphodiesterase-5 inhibitors such as sildenafil, the analogs may indeed act similarly. On the other hand, they may exhibit more or less potent action – indeed, the reaction to their use is entirely unpredictable. This unpredictability, and the fact that sildenafil, other phosphodiesterase-5 inhibitors, and their analogs may be present in a number of widely available products, creates a great demand for quick, simple, and reliable methods for substance identification.

Conclusion

By adapting a previously developed method for biological analysis, researchers were able to identify and quantify the presence of hazardous substances – PDE-5 inhibitors – in unregulated medications for the treatment of ED.

The method they developed provides a solidly replicable approach of interest to toxicologists and clinical or analytical chemists wishing to detect and identify new drugs emerging on the illicit pharmaceutical market. The method highlights the high sensitivity and selectivity of the Shimadzu Nexera X2, as well as how its high-speed MS/MS detection makes it possible to operate the Q1/Q3 in a scanning mode: something unique for this type of detector. In addition, the method minimizes the use of biological sample material, resulting in the need for much lower organic solvent volumes in the sample preparation process.

Chromatographic and spectrometric conditions

Analyses were performed using a UHPLC, Shimadzu Nexera X2 (Kyoto, Japan). The separation was done using a Kinetex XB-C18 column (150 x 2.1 mm i.d., particle size 2.6 µm; Phenomenex, Torrance, CA, USA) at a column temperature of 40 °C. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient elution was carried out at a constant flow of 0.4 mL/min. The gradient applied was as follows: 0 min, 5% B; 12 min, 98% B; 14 min, 98% B; and 15 min, 5% B. Return to the initial gradient compositions (95% A/5% B) was performed for 5 min.

Detection of the investigated compounds was achieved using a QqQ-MS/MS, Shimadzu LCMS-8050 (Kyoto, Japan). The spectrometer was equipped with an electrospray ionization (ESI) source; identification of the investigated substances was carried out in the product ion scan mode. The following MS parameters were fixed: nebulizing gas flow, 3 L/min; heating gas flow, 10 L/min; interface temperature, 250 °C; desolvation line temperature, 200 °C; heat block temperature, 350 °C; and drying gas flow, 10 L/min.

Detailed procedure is described below:

0.2 mL of blood was transferred into 10 mL plastic vials. Next, 0.2 mL 0.5 M ammonium carbonate buffer (pH 9) was added. Liquid-liquid extraction with 2 mL of ethyl acetate was carried out for 10 min. The samples were centrifuged at 2,540 x g at 4 °C for 10 min. The organic phase was transferred into 2 mL Eppendorf tubes and evaporated to dryness under a stream of inert nitrogen gas at 40 °C. The dry residues were dissolved in 0.05 mL of methanol. The solution was then transferred into glass inserts of autosampler vials and analyzed by ultra-high-performance liquid chromatography-triple quadrupole-tandem mass spectrometry (UHPLC-QqQ-MS/MS). The injection volume was 2.0 μL. We utilize small amounts of environmentally unhealthy organic solvents and low biological sample volume compared to conventional methods.

The biggest advantage of this method is that while being fast and simple, it is selective, sensitive, and, thanks to UHPLC-MS/MS usage, enables simultaneous detection of different PDE-5 inhibitors, and hence characteristic fragments in product ion spectra may be helpful in identification of such substances in forensic cases.