Identification and Quantification of an Adulterant in a Dietary Supplement Marketed for Sexual Enhancement

In recent years, the consumption of dietary supplements (DS) has increased worldwide. In Argentina, approximately 14 million DS units were sold between 2015 and 2017. The adulteration of DS with active pharmaceutical ingredients or their analogues has been reported. This represents an alarming emerging risk to public health. The aim of this work was to detect the possible adulteration of a DS marketed in Argentina for the treatment of erectile dysfunction. Initially, thin layer chromatography analysis of the DS capsules content suggested the presence of a major compound. For the isolation and purification of this compound, an easy method consisted of a liquid-liquid extraction (water/CH2Cl2) followed by re-crystallisation from ethanol, is reported. Spectroscopic techniques such as monoand bidimensional nuclear magnetic resonance, Fourier transform infrared spectroscopy and mass spectrometry allowed its identification as tadalafil. A rapid and reliable method was developed for the quantification of tadalafil in this DS by high performance liquid chromatography-mass spectrometry (HPLC-MS/MS). The mean content of tadalafil per capsule was 21.2 mg which represents a slightly higher value than that found in approved products in Argentina (5 or 20 mg per tablet). In addition, an undeclared alga was identified in the DS by microscopic techniques. Flavia Redko, Sabrina Flor, Silvia Lucangioli, Jerónimo Ulloa, Rafael Ricco, Claudio Fernández, Luis Sambrotta, Liliana Muschietti Universidad de Buenos Aires, Cátedra de Farmacognosia, Facultad de Farmacia y Bioquímica (UBA), IQUIMEFA (UBA-CONICET), Buenos Aires, Argentina Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Tecnología Farmacéutica, Buenos Aires, Argentina and Consejo Nacional de Investigaciones Científicas y Tecnológicas, CONICET, Argentina. Universidad de Buenos Aires, Cátedra de Farmacobotánica, Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario (IIDEFAR-CONICET-UNR and Laboratorio Max Planck de Biología Estructural, Química y Biofísica Molecular de Rosario (MPLBIOR-UNR-SOCIEDAD MAX PLANCK) DOI : 10.14302/issn.2328-0182.japst-18-2344 Corresponding author: Liliana V. Muschietti, Cátedra de Farmacognosia, Facultad de Farmacia y Bioquímica (UBA), IQUIMEFA (UBA-CONICET), Buenos Aires, Argentina; +54 11 49648247. Email address: lmusch@ffyb.uba.ar


Introduction
Since 1998, the use of dietary supplements (DS) in Argentina has been regulated in the "Código Alimentario Argentino" (CAA) (Law no. 18284). DS are defined as "products used to increase the regular dietary intake, supplementing the incorporation of nutrients in the diet of healthy people who, in the absence of pathological conditions, present an unmet dietary need".
In terms of composition, DS should provide nutrients such as proteins, vitamins, minerals, lipids, carbohydrates, and fibres. Some botanicals or extracts thereof can also be used [1]. Initially, DS were mainly products based on vitamins, minerals, proteins and fibre, but over the last years, and mainly in response to consumer demands, the profile of these products has changed, and other constituents such as herbal products Internet and, they often take them without obtaining adequate information about the products. An important problem arises from the adulteration of these DS with either synthetic drugs or drug analogues [3,4], which represents an alarming emerging risk to public health. A wide array of DS adulterants has been reported. These substances are added to enhance the properties of the product [5,6].
In Argentina, approximately 14 million DS units were sold between 2015 and 2017 according to IMS [7] and only one adulteration has been reported [8].
According to Vaclavik et al. [9], the products most frequently adulterated with synthetic drugs are those for the treatment of erectile dysfunction (ED), obesity/  [11].
Given that the adulteration of DS is a major problem, we have been screening for the detection of illegal substances in order to discover products that may pose a health risk [8]. In this article we report the isolation and the identification, by spectroscopic

Results and Discussion
Among the many reasons why DS are popular worldwide include their accessibility, since they are readily acquirable in pharmacies as over-the-counter products, in supermarkets, through internet websites, in natural food stores, in herbal shops and at gyms. People believe that these products are 100 % natural, and therefore, innocuous and safer than pharmaceutical

Isolation an Identification of the Major Component of the CH 2 Cl 2 Extract
The isolation consisted of a liquid-liquid extraction (water/CH 2 Cl 2 ) profiting from the acid-base properties of such compounds, followed by re-crystallisation from ethanol, to afford the target compound as white crystals. In our previous work, the isolation of the adulterant was accomplished by a single column chromatography process that yielded the pure compound. In this case, the sample matrix seemed less complex as shown by the TLC and HPLC profiles [8].
Spectroscopic data (Figures 1 and 2) allowed the identification of the major compound as tadalafil

HPLC-MS/MS Quantification of Tadalafil in the DS Capsules
For the quantification of tadalafil in the DS, the HPLC chromatographic conditions needed optimisation.
Several methods using liquid chromatography have been previously described for the identification of tadalafil and related compounds [19,20,21]. Given the chemical characteristics of tadalafil, several additives, such as triethanolamine, trifluoroacetic acid and formic acid have been used to avoid tailing. As shown in figure 3, the tadalafil molecule presents several nitrogen atoms, which interact with the stationary phase resulting in an irregular peak shape. This has been partially improved by the addition of mobile phase modifiers. In our method, we replaced the typical reverse phase by one The content of tadalafil in the DS was determined by HPLC-MS/MS. The mean content per capsule was 21.2 ± 2.6 mg which represents a slightly higher value than that found in approved products in Argentina, i.e., 5 or 20 mg per tablet [22]. Taking these results into account, it can be concluded that, unlike pharmaceuticals, certain DS are not subjected to a complete quality control process. This entails a substantial variability in the tadalafil content among capsules. Even though the amount of tadalafil found is similar to that found in approved marketed products, the presence of this drug in DS poses a serious health risk.
As stated above, most patients consuming DS are unaware of potential adulterations and many consider DS as safe natural products. The common side effects  and numerous simple starch grains ( Figure 5). The presence of Arthrospira was confirmed by comparison of their powder characteristics with those found in the literature [24]. Arthrospira is a genus of cyanobacteria that constitutes dietary supplements commonly known as spirulina. This alga has long been used as food, and although most alga are innocuous, a significant proportion of cyanobacterial species are harmful due to the production of cyanotoxins [25]. The possible presence of these toxins in these DS reinforces the need for a better-quality control [26].

Conflict of Interest
The authors declare they have no conflict of interest. Informed Consent Not applicable