Elsevier

Journal of Chromatography A

Volume 1218, Issue 37, 16 September 2011, Pages 6439-6447
Journal of Chromatography A

Development and validation of a ultra-high-performance liquid chromatography-UV method for the detection and quantification of erectile dysfunction drugs and some of their analogues found in counterfeit medicines

https://doi.org/10.1016/j.chroma.2011.07.029Get rights and content

Abstract

Pharmaceutical counterfeiting is a permanently growing problem. Control laboratories are constantly analysing counterfeit medicines. In industrialised countries, one of the main counterfeited class of medicines are erectile dysfunction drugs. This paper describes the development and validation of a fast method to detect and quantify the three authorised phosphodiesterase type 5 inhibitors and five analogues. The method is based on the use of a sub-2 microns polar-embedded column with a gradient using acetonitrile as organic modifier and 10 mM ammonium formate buffer (pH 3.5) as aqueous component of the mobile phase. The separation was achieved in less than 4.5 min. The method has also been compared to the registered HPLC method for the assay of Viagra® which was considered as the reference method. The method is also compatible with on-line coupling mass spectrometry and will significantly reduce analysis times and solvent consumption.

Introduction

The number of cases of pharmaceutical counterfeiting is constantly growing since the first cases were detected in the early 90s [1]. In industrialised countries, one of the most counterfeited classes of medicines is the phosphodiesterase type 5 inhibitors (PDE5-i) [2]. Among them only three drugs are approved and marketed: sildenafil citrate (Pfizer), tadalafil (Eli Lilly) and vardenafil hydrochloride (Bayer). These drugs are used in erectile dysfunction disorders (Viagra®, Cialis® and Levitra®). Sildenafil citrate is also used in pulmonary arterial hypertension (Revatio®).

Due to the taboo associated with erectile dysfunction, PDE5-inhibitors are widely sold over the internet as both counterfeited medicines and illegal adulterants in herbal dietary supplements. In the latter the biggest diversity of analogues was found [2], [3], [4]. For this study, three analogues of sildenafil (acetildenafil, hydroxyacetildenafil and dimethylsildenafil), one of vardenafil (pseudovardenafil), one of tadalafil (aminotadalafil) and the bioactive diastereoisomer of tadalafil (trans-tadalafil) have been chosen. Their chemical structures are shown in Fig. 1. These compounds are representative of what is commonly found in illegal preparations.

All of these analogues have already been found in illegal preparations. These preparations have been analysed using different analytical systems (LC-UV, LC-MS, IR, NMR, X-ray diffraction, etc.) [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28]. The presented validated method allows a fast separation and quantification of the three authorised PDE5-i and five of their analogues. This method may constitute a good basis for the analysis of illegal erectile dysfunction medicines by official control laboratories.

The present paper describes a method enabling the separation and quantification of nine PDE5 inhibitors in a single run: sildenafil, tadalafil, vardenafil and some of their analogues and impurities (trans-tadalafil [16]). A full validation using spiked placebo validation samples has been performed using the “total error” approach [31], [32], [33], [34], [35], [36], [37], [38]. The robustness of the method has also been investigated. The precision and accuracy for the quantification of sildenafil citrate in Viagra® tablets has been compared to the HPLC method from the Viagra® registration dossier set as reference method. The method described here can be used as routine method for the analysis of PDE5-inhibitors and can be coupled in principle to a mass spectrometer for identity confirmation or structure elucidation.

The proposed method allows a faster and more environmental friendly high throughput analysis of both illegal and legal preparations containing PDE5-inhibitors.

Section snippets

Standards

The reference standards of sildenafil citrate (batch 904958), tadalafil (batch RS0480) and vardenafil dihydrochloride trihydrate (batch BXR3835 R-1013-02B) were kindly provided by Pfizer SA/NV (Belgium), Eli Lilly SA/NV (Benelux) and Bayer SA/NV (Belgium), respectively.

Reference standards of hydroxyacetildenafil (batches 1068-005A2 and 1068-013A2), acetildenafil (batch 1046-011A2), dimethylsildenafil (batch 1035-122A1), aminotadalafil (batch 1034-001A1) and pseudovardenafil (batch 1070-002A2)

Initial conditions selection

The separation method has been developed in HPLC with UV detection in order to be applicable by a large amount of control laboratories. Acetonitrile was chosen as organic modifier as it causes less back pressure and better baseline stability than methanol. A 0.1% formic acid aqueous solution (pH 2.8) was used as aqueous component of the mobile phase to be compatible with on-line mass spectrometry.

Initial HPLC conditions were a linear gradient starting from 5% acetonitrile to 100% in 27 min. The

Conclusion

This paper describes for the first time a fully validated method which enables the detection and the quantification of authorised phosphodiesterase type 5 inhibitors and some of their analogues in less than 4.5 min. This rapidity associated to a low flow rate permits the analysis of a large number of samples with a reduced cost and associated solvent consumption.

The main problem with counterfeit medicines is that their chemical composition is unknown. This is why they represent a real danger for

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