Science and Justice xxx (2014) xxx–xxx

SCIJUS-00430; No of Pages 10

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Analysis of illicit dietary supplements sold in the Italian market:Identification of a sildenafil thioderivative as adulterant usingUPLC–TOF/MS and GC/MS

Fabio Damiano a,⁎, Claudia Silva b, Adolfo Gregori a, Federica Vacondio b, Marco Mor b,Mattia Menozzi c, Domenico Di Giorgio d

a Carabinieri, Department of Scientific Investigation, via Parco Ducale 3, 43126 Parma, Italyb University of Parma, Department of Pharmacy, Parco Area delle Scienze, 27/A, 43124 Parma, Italyc University of Torino, Department of Analytical Chemistry, via Pietro Giuria 5, 11025 Torino, Italyd Italian Medicines Agency, Counterfeit Prevention Unit, via del Tritone 181, 00187 Rome, Italy

⁎ Corresponding author. Tel.: +39 0521537716; fax: +E-mail address: fabio.damiano@carabinieri.it (F. Damia

http://dx.doi.org/10.1016/j.scijus.2014.02.0091355-0306/© 2014 Published by Elsevier Ireland Ltd. on b

Please cite this article as: F. Damiano, et althioderivative as adulterant us..., Sci. Justice

a b s t r a c t

a r t i c l e i n f o

Article history:Received 10 July 2013Received in revised form 2 January 2014Accepted 18 February 2014Available online xxxx

Keywords:PDE-5 inhibitorsDietary supplementSildenafilTadalafilVardenafilThioaildenafilThiohomosildenafil

Identification of pharmaceutical active ingredients sildenafil and tadalafil and the characterization of adimethylated thio-derivative of sildenafil, called thioaildenafil or thiodimethylsildenafil, in illicit dietarysupplements were described.A multi-residual ultra-performance liquid chromatography–time of flight mass spectrometry (UPLC–TOF/MS)method was developed to screen for the presence of the phosphodiesterase-5 (PDE-5) inhibitors sildenafil,tadalafil, and vardenafil and their analogues thioaildenafil and thiohomosildenafil in powders and pharmaceuti-cal dosage forms. The study was developed in connection with an operation supervised by the Italian MedicinesAgency (A.I.F.A.), aimed to monitor dietary supplements in the Italian market. In two of the eleven specimensunder investigation, high-resolution mass spectrometry (HR-MS) allowed the identification of the PDE-5inhibitors sildenafil and tadalafil, while another specimen proved to contain a unapproved dimethylatedthioderivative of sildenafil, thioaildenafil or thiodimethylsildenafil, identified for the first time in Italy as adulter-ant in food supplements.

© 2014 Published by Elsevier Ireland Ltd. on behalf of Forensic Science Society.

1. Introduction

In the recent years, we have witnessed a proliferation in the globalmarket of a number of products which mainly claim to enhance sexualactivities and which are often referred to as natural substances [1–3].Some of them are illegally adulterated with active ingredients such assildenafil (Viagra®, Pfizer), tadalafil (Cialis®, Eli Lilly) and vardenafil(Levitra®, Bayer), phosphodiesterase-5 (PDE-5) enzyme inhibitorsused for the treatment of erectile dysfunction (Fig. 1) [4–9]. Somehave also proved to contain synthetic analogues of approved PDE-5 in-hibitors, such as derivatives of sildenafil modified on the piperazinicportion of the molecule, such as acetildenafil, piperidinoacetildenafil,aildenafil, and homosildenafil [10,11]. Other modifications are relatedto the substitution of the carbonyl group in the pyrazolopyrimidinering of sildenafil with a thiocarbonyl group, as in thiosildenafil andthiohomosildenafil [12–18]. Differently from the parent active pharma-ceutical ingredients, no formal studies have been performed to assure

39 0521537789.no).

ehalf of Forensic Science Society.

., Analysis of illicit dietary s(2014), http://dx.doi.org/10.1

the efficacy and safety of these analogues, therefore the phenomenoncan pose a severe health risk [19,20]. According to the data publishedby the Italian Medicines Agency (A.I.F.A.), Italian regulatory authority,the market seems to be very profitable [21,22]. A.I.F.A., in cooperationwith other authorities, is responsible in controlling the spread of unau-thorized analogues of active pharmaceutical ingredients in the marketand is making great efforts to fight the phenomenon of pharmaceuticalcounterfeiting. For instance, A.I.F.A. is a member of I.M.P.A.C.T., the“International Medical Products Anti-Counterfeiting Taskforce”. Thetaskforce, proposed byW.H.O. (World Health Organization) at an inter-national conference in Rome in February 2006, is active in reinforcinginternational collaboration to seek solutions to this global challengeand in raising awareness of the dangers of counterfeit medical products.

Recently, Interpol has signed an agreement with pharmaceuticalcompanies to strengthen the fight and controls on counterfeit drugs;major pharmaceutical companies involved in the problem have startedlarge investment plans [23].

Due to a taboo associated with erectile dysfunction, PDE-5 inhibitorsare widely sold on internet as counterfeit medicines or as dietary supple-ments [24]. Various analytical techniques (NMR, LC–UV, LC–MS, GC–MS,IR) have been employed and different analytical methods have beendeveloped to identify this class of molecules in illegal products [25–33].

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Fig. 1. Chemical structures of compounds 1-2-3.

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This article describes the identification of a sildenafil thioderivativein illicit dietary supplement, carried out at the Department of ScientificInvestigation of Carabinieri (R.I.S.) in collaborationwith theDepartmentof Pharmacy of the University of Parma, in connectionwith a joint oper-ation A.I.F.A.–Carabinieri Health Protection (N.A.S.), aimed to monitordietary supplements in the Italian market.

The Italian Medicines Agency — Counterfeit Unit provided 11 speci-mens, in particular food supplements that were sold in Italian shops.The specimens were powders or pharmaceutical dosage forms(capsules and tablets); three of them were adulterated with PDE-5inhibitors. In particular, in two specimens, the analyticalmethod allowedto determine the presence of sildenafil and tadalafil, while one specimencontained an unapproved PDE-5 inhibitor analogue of sildenafil. Thecharacterization of this unknown compound was carried out combininginformation coming from the UPLC–TOF/MS and GC/MS analyses. Theuse of both analytical techniques allowed firstly the identification of

Fig. 2. Photo samples u

Please cite this article as: F. Damiano, et al., Analysis of illicit dietary sthioderivative as adulterant us..., Sci. Justice (2014), http://dx.doi.org/10.1

the substituted piperazine portion of the molecule and secondly tonarrow the search and focus the investigation on two thioderivatives ofsildenafil, called thiohomosildenafil and thioaildenafil, of identical mo-lecular weight. In comparison to purchased reference standards, it waspossible to establish that the unknown derivative was thioaildenafil,also called thiodimethylsildenafil. To our knowledge, this is the firsttime that this derivative of sildenafil has been found as an adulterant inillicit food supplements sold in the Italian market.

A multi-residual ultra-performance liquid chromatography–time offlight mass spectrometry (UPLC–TOF/MS) method to screen PDE-5 in-hibitors sildenafil, tadalafil, and vardenafil and their thioanaloguesthioaildenafil and thiohomosildenafil in pharmaceutical dosage formshas been set-up and validated according to the ICH guideline Q2A“Test on validation of analytical procedures” for precision, accuracy,linearity and sensitivity [34].

2. Materials and methods

2.1. Standards

Sildenafil citrate reference standard 1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)phenylsulfonyl]-4-methylpiperazine (batch 82354-01) wasobtained from Pfizer GQOC Scientist & Laboratory Service, Analyti-cal Sciences (Sandwich, Kent, UK). Tadalafil (6R-trans)-6-(1,3-benzodioxol-5-yl)- 2,3,6,7,12,12a-hexahydro-2-methyl-pyrazino[1′,2′:1,6] pyrido[3,4-b]indole-1,4-dione (batch RS0534) wasobtained from Eli Lilly and Company Lilly Corporate Center(Indianapolis, IN, USA). Vardenafil HCl reference standard 2-[2-ethoxy-5-(4-ethylpiperazin-1-yl)sulfonylphenyl]-5-methyl-7-propyl-1H-imidazo[5,1-f] [1,2,4] triazin-4-one (batchR-1013-0D)was obtainedfrom Bayer Health Care Quality Unit Elberfeld (Wuppertal, Germany).Thioaildenafil (thiodimethylsildenafil) reference standard (3R, 5S)-rel-1-[[3-(4,7-Dihydro-1-methyl-3-propyl-7-thioxo-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-4-ethoxyphnenyl]sulfonyl]-3,5-dimethyl-piperazine(batch 1-JMS-114-1) was purchased from TRC (Toronto ResearchChemicals Inc., Canada). Thiohomosildenafil reference standard 5-[2-ethoxy-5-(4-ethylpiperazin-1-yl)sulfonylphenyl]-1-methyl-3-propyl-4H-pyrazolo[4,3-d]pyrimidine-7-thione (batch 1220-033A10) waspurchased from TLC Pharmachem (Ontario, Canada).

nder investigation.

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Table 1Chromatographic gradient applied.

Time (min) Flow (ml/min) % A % B

Start 0.4 87 1310 0.4 50 5010.75 0.4 95 512.25 0.4 95 512.50 0.4 87 1315.00 0.4 87 13

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2.2. Samples

The samples under investigation were dietary supplements, com-mercially available in retail Italian shops (not including pharmaciesand herbalist shops). These dietary supplements claimed to containherbal extracts, such as Panax Ginseng, Tribulus terrestris, Rhodiolarosea, and Lepidium meyenii whose active pharmaceutical ingredi-ents, according to the labels, were related to an improvement inthe sexual performance. No label indication on the presence ofPDE-5 inhibitors was present.

Available pharmaceutical dosage forms were (Fig. 2):

- tablets (weight of about 1.0–1.4 g);- capsules (containing about 400–500 mg of powder);- soft capsules (weight of about 600–800 mg);- powders (packets containing about 1 g of substance).

Some samples showed a heterogeneity of matrix, revealing animproper mixing of the pharmaceutical ingredients and the lack ofGood Laboratory Practise (GLP) protocols in the preparation phase.

2.3. Reagents

Optima LC/MS grade acetonitrile (99.9%) and methanol (99.9%),formic acid and ammonium formate were purchased from FischerChemical (Geel, Belgium).

Fig. 3. UPLC–TOF chromatogram of die

Please cite this article as: F. Damiano, et al., Analysis of illicit dietary sthioderivative as adulterant us..., Sci. Justice (2014), http://dx.doi.org/10.1

2.4. Preparation of calibration standards (CS) and quality controls (QC)

Stock solutions of each compound were prepared weighting 4.0mg of pure substance and diluting it with 4.0 ml of methanol(final concentration: 1 mg/ml). A 5 μg/ml stock solution of eachcompound in methanol was then prepared by serial dilution.Calibration standards (CS) were set up mixing equal volumesof the corresponding 5 μg/ml stock solutions to reach thefinal levels of concentrations: 0.1 μg/ml, 0.2 μg/ml, 0.3 μg/ml,0.5 μg/ml, 0.7 μg/ml and 1 μg/ml. Stock solutions for CS and QCwere prepared separately in methanol. Quality control standardswere prepared at the three concentration levels of 0.15 μg/ml,0.4 μg/ml, and 0.6 μg/ml.

2.5. Preparation of samples

The isolation of active pharmaceutical ingredients from pharma-ceutical dosage forms required the set up of a general extraction pro-tocol starting from the heterogeneous and unknown drug andexcipient mixtures in our hands. We firstly evaluated the solubilityof the pure pharmaceutical standards in a set of known organic ex-traction solvents. Preliminary solubility assays showed that stan-dards were completely soluble in methanol in the 0.1–1.0 mg/mlconcentration range. Methanol was therefore the extraction solventof choice. Before the solid/liquid extraction phase, all samples werefirstly subjected to a homogenization phase in a mortar. In the caseof coated tablets, the coating was first removed and only the contentof the tablet was analyzed. The volume of extraction solvent waschosen in order to guarantee the complete solubility of the activepharmaceutical ingredient and to avoid excessive dilution of thesample, which could lead to the lack of sensitivity. In the final extrac-tion protocol, about 5–7 mg of sample were dissolved in 10 ml ofmethanol and sonicated for 15 min. 20 μl of this solution was dilutedup to 700 μl (dilution of 35 times) with methanol, filtered with a0.2 μm PTFE filter into autosampler vials and directly analyzed byUPLC–TOF/MS.

tary supplement of study (no. 1).

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Fig. 4. Time of flight spectra mass of active compound sildenafil found.

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2.6. Ultra-performance liquid chromatography/time of flight massspectrometry (UPLC–TOF/MS)

UPLC–TOF/MS analysis was performed employing a Waters AcquityUPLC system interfaced with a Waters Xevo G2 Time of flight (TOF)mass analyzer.

The column employed for gradient separation was a Waters ClassHSS C18 column (150mm, 2.1mm i.d., 1.7 μmparticle size) thermostat-ed at 50 °C for all the analyses. The UPLC gradient conditions wereoptimized in order to maximize the responses of the pharmaceutical

Fig. 5. UPLC–TOF chromatogram of dieta

Please cite this article as: F. Damiano, et al., Analysis of illicit dietary sthioderivative as adulterant us..., Sci. Justice (2014), http://dx.doi.org/10.1

standards in positive ion mode and to obtain a good separation fromthe other components of the pharmaceutical dosage form. In particular,in order to maximize the response in electrospray, several modifiersof the aqueous phase were tested among volatile acids and massspectrometry-compatible buffers. Finally, chosen mobile phase eluentswere as follows: 5 mM ammonium formate buffer solution, pH 3.0(mobile phase A) and acetonitrile with 0.1% formic acid (mobile phaseB). UPLC gradient conditions are reported in Table 1. The back pressureat the beginning of gradient was around 600 bar, and it fluctuated be-tween 600 and 400 bar during thewhole elution. The overall acquisition

ry supplement under study (no. 2).

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Fig. 6. Time of flight spectra mass of active compound tadalafil found.

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timewas 11minwith 4min (from11 to 15min) of reequilibration time.Sampleswere kept thermostated at 8 °C in the autosampler during anal-ysis. These chromatographic conditions allowed the separation of hy-drophilic peaks with lower retention times, related to excipients or toother components such as caffeine, while PDE-5 inhibitors showed agood separation in the last 5 min of run.

Data were acquired by the Waters Xevo G2 TOF mass spectrometermerging information coming from different channels. In channel 1 thecollision cell was turned off and the analyte didn't fragment, in channel

Fig. 7. UPLC–TOF chromatogram of dieta

Please cite this article as: F. Damiano, et al., Analysis of illicit dietary sthioderivative as adulterant us..., Sci. Justice (2014), http://dx.doi.org/10.1

2 the collision cell was activated and the analyte could be fragmentedwith its own characteristic pattern. In channel 3, the calibration of theTOF during all the measures was checked and corrected every 20 s dur-ing the analysis by direct injection into the MS system of a solution ofLeukine-Enkephaline standard. ChosenMS parameterswere as follows:Electrospray (ESI) ion source working in positive ion mode (ESI+) witha scan time of 0.1 s; capillary voltage: 0.8 kV; cone voltage: 20 V; range:m/z 50–1000; collision energy: 6 eV for channel 1; and from 10 eV to40 eV for channel 2.

ry supplement under study (no.3).

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Fig. 8. GC–MS chromatogram and spectra mass (EI) of dietary supplement under study (no. 3).

Fig. 9. Chemical structures of thiohomosildenafil and thioaildenafil.

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2.7. Gas chromatography/mass spectrometry (GC/MS)

GC was performed on a 25 m·0.2 mm i.d.·0.33 μm film Agilent HP5-MS190915-102 capillary column, using anAgilent 6890Ngas chromato-graph coupled to an Agilent 5975B mass spectrometer. GC conditionswere as follows: injection volume: 1 μl; pulsed split injection 60.0:1 at260 °C; temperature set to 80 °C for 3 min, then ramped to 280 °C at10 °C/min (held for 15 min), and ramped to 300 °C at 10 °C/min (heldfor 40 min), giving a total run time of 80 min. Helium gas carrier washeld at a constant flow rate of 1 ml/min. An electron-impact ion sourcecoupled to a single quadrupole mass analyzer was employed operatingin full-scan mode (40–600 m/z) with a sampling rate of 0.68 scans/s;MS-Source temperature: 230 °C; MS-Quad temperature: 150 °C.

3. Results and discussion

3.1. Identification and quantification of sildenafil and tadalafil intwo specimens

The developed method was employed to analyze the eleven speci-mens obtained by A.I.F.A. and N.A.S. to check the presence of the threePDE-5 inhibitors sildenafil, tadalafil and vardenafil. In two of the elevensamples, the UPLC/MS chromatographic run revealed the presence ofsildenafil and tadalafil as adulterants. The high-resolution mass spectraacquired, their UPLC retention times and the relative differences to theo-retical values for molecular and daughter ionsm/z values, confirmed thepossibility to attribute the identity of the two PDE-5 inhibitors based onthe accurate mass measurements and fragmentation fingerprint.

Figs. 3–4 exhibit the chromatogram of a sample under study withthe chromatographic peak of sildenafil (peak 1 in Fig. 3) and high

Please cite this article as: F. Damiano, et al., Analysis of illicit dietary sthioderivative as adulterant us..., Sci. Justice (2014), http://dx.doi.org/10.1

resolution mass spectra acquired. Molecular ion peak for sildenafilshows a value of m/z = 475.2133 with a calculated relative differencewith respect to the theoretical value Δm/z=+1.3 ppm. Fragmentationpattern of sildenafil is already known and was here confirmed withfragment ions at m/z = 283.1229 (fragment corresponding to thepartial cleavage of pyrazole–pyrimidine ring; Δm/z = +6.4 ppm) andm/z = 100.0990 (refer to piperazinic ring; Δm/z = −9.9 ppm).

Figs. 5–6 exhibit the chromatogram of a sample under study withthe chromatographic peak of tadalafil (peak 1 in Fig. 5) and high resolu-tion mass spectra acquired. Molecular ion peak for tadalafil shows avalue of m/z = 390.1464 with a calculated relative difference withrespect to the theoretical value Δm/z = +2.6 ppm. Fragmentationpattern of tadalafil is already known and was here confirmed withfragment ions at m/z = 268.1082 (fragment corresponding to thecleavage of benzodioxolane and of the 1-methylpiperazine-2,5-dionering; Δm/z = −1.5 ppm); m/z = 169.0766 (cleavage of thebenzodioxolane ring; Δm/z = −3.0 ppm); and m/z = 135.0446(benzodioxolane ring; Δm/z = −3.0 ppm).

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Fig. 10. Time of flight spectra mass of thiohomosildenafil and thioaildenafil— difference between main fragments (UPLC–TOF/MS channel 1 above and zoom of channel 2 spectra below,see Section 2.6).

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3.2. Identification of an unknown compound

UPLC–TOF/MS chromatogram of a specimen represented by n = 3capsules of orange color containing about 450mg of not homogeneous-ly mixed brown powder revealed a peak with a retention time and

Fig. 11. Time of flight spectra mass of thioderi

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molecular weight (Fig. 7) different from those of the already analyzedPDE-5 inhibitors sildenafil, tadalafil and vardenafil.

Exploiting the high resolution of the TOF detector, we were able tohypothesize with a high degree of confidence the molecular formulasable to generate those accurate m/z values and isotopic patterns. This

vative of sildenafil found — thioaildenafil.

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Fig. 12. Typical UPLC–TOF chromatographic of a standard solution, for the simultaneous quantitation: vardenafil (1), sildenafil (2), tadalafil (3), thiohomosildenafil (4) and thioaildenafil(5).

Table 2Linear regression equations for 5 PDE-5 inhibitors investigated simultaneously detectedby UPLC–TOF and values of LOD/LOQ found.

Compound Regression equation R2 LOD(μg/ml)

LOQ(μg/ml)

Sildenafil y = 0.855x + 13.163 0.998 0.003 0.009Tadalafil y = 0.314x − 2.457 0.997 0.001 0.004Vardenafil y = 2.104x + 15.558 0.998 0.001 0.004Thiohomosildenafil y = 2.101x + 17.574 0.998 0.001 0.005Thiodimethylsildenafil y = 2.115x + 44.702 0.999 0.001 0.005

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allowed the subsequent search on internet and marketed databases ofactive pharmaceutical ingredients related to approved PDE-5 inhibitorssharing that molecular formula.

Information from molecular ion at m/z = 505.2060 [M + H]+ wasnot sufficient to determine unequivocally the structure of molecule, asit generated too many potential entries.

Interesting information came from the peak at m/z = 327.1280[M + H]+, which corresponds to the pyrazolopyrimidin-7-one frag-ment of sildenafil, substituted on the C-7 of the ring with a sulphuratom instead of an oxygen (Fig. 11).

Gas chromatography–mass spectrometry analysis did not allow thedetermination of the molecular weight of the molecule, due to the lowvolatility of this compound. Fig. 8 shows the peak of analyte to be iden-tified (peak 1 in Fig. 8) and the corresponding mass spectra. Neverthe-less, further information could be obtained from the mass fingerprint:in particular a peak atm/z = 113 was attributed to the piperazine ringwith two methyl groups or an ethyl group bound.

Thisfinding helped focus the subsequent research on two compounds,thioaildenafil (also called thiodimethylsildenafil) and thiohomosildenafilthat show the samemolecularweight, but differ in the piperazine portionof the molecule, being a 2,6-dimethylpiperazino or a 1-ethylpiperazinoderivative, respectively (Fig. 9).

Proper reference standards were purchased to confirm the chemicalinvestigation and to quantify the illicit active pharmaceutical ingredientsin the capsules. UPLC–TOF/MS analysis revealed a different mass finger-print for reference compounds thiohomosildenafil (4) and thioaildenafil(5); in particular, for 5 a characteristic fragment at m/z = 71.0735[M + H]+ corresponding to a portion of 2,6-dimethylpiperazinering was detected; for 4 a corresponding fragment at m/z = 72.0805[M + H]+, corresponding to a portion of the 1-ethylpiperazine ring aswell as a fragment at m/z = 355.1577 related to a larger portion ofthe molecule (Fig. 10) were observed.

The comparison of high resolution mass spectra, mass fingerprints,and chromatographic peak retention times of standard 5 with thoseof the unknown compound allowed to confirm the presence ofthioaildenafil in the pharmaceutical dosage forms (capsules) understudy (Fig. 11).

Please cite this article as: F. Damiano, et al., Analysis of illicit dietary sthioderivative as adulterant us..., Sci. Justice (2014), http://dx.doi.org/10.1

3.3. Screening of PDE-5 inhibitors — analytical method validation

The finding of a described adulterant in the pharmaceutical dosageforms under study prompted us to enlarge the applicability of the ana-lytical method previously developed to the simultaneous detection ofthe five PDE-5 inhibitors sildenafil, tadalafil, and vardenafil, and theirthio-derivatives thiohomosildenafil and thioaildenafil. The analyticalmethod was validated according to the ICH guideline ICH Q2A to quan-titate the five compounds in food supplements. The selectivity of themethod was tested checking the retention time of each compoundand their separation during the chromatographic run. Fig. 12 shows rep-resentative chromatograms obtainedwith the indicated gradient condi-tions. No interfering peak was observed in the samples at them/z ratiosand retention times of each analyte.

Linear regression equations for each PDE-5 inhibitor and relative co-efficients of determination (R2) are reported in Table 2. The LOD andLOQ for each compound were determined as the lowest concentrationyielding signal-to-noise ratios of at least 3:1 and 10:1, respectively.

Precision and accuracy of the method have been calculated by ana-lyzing the QC levels within analyte linear range (0.15 μg/ml, 0.4 μg/ml,0.6 μg/ml). The intra-day and inter-day precision was expressed bythe coefficient of variation percentage (CV%), whereas intra-day andinter-day accuracy was expressed by bias percentage (bias%). Table 3shows the obtained results that are within the values required by theICH guideline Q2A (precision≤ 15% for all concentrations and accuracynot exceeding 15% for all concentrations).

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Table 3Intra-day and inter-day precision and accuracy values of three QC concentrations.

Nominal Conc. (μg/ml) Intra-day (n = 5) Inter-day (n = 15)

Conc. Found (μg/ml) (mean ± S.D.) Accuracy (% Er) Precision (% RSD) Conc. Found (μg/ml) (mean ± S.D.) Accuracy (% Er) Precision (% RSD)

Sildenafil0.150 0.140 ± 0,018 93.0 13.2 0.139 ± 0.007 92.6 5.00.400 0.379 ± 0,018 94.8 4.9 0.389 ± 0.025 97.2 6.40.600 0.622 ± 0,014 103.8 2.3 0.595 ± 0.025 99.1 4.2

Tadalafil0.150 0.142 ± 0.007 94.4 4.8 0.154 ± 0.012 102.9 7.90.400 0.392 ± 0.009 98.0 2.4 0.417 ± 0.031 104.3 7.60.600 0.622 ± 0.024 103.7 4.0 0.634 ± 0.025 105.7 3.9

Vardenafil0.150 0.143 ± 0.006 95.7 4.1 0.148 ± 0.011 98.4 5.00.400 0.387 ± 0.011 96.8 2.8 0.388 ± 0.016 97.1 4.20.600 0.560 ± 0.009 93.3 1.5 0.562 ± 0.013 93.7 2.3

Thiohomosildenafil0.150 0.157 ± 0.007 104.4 4.9 0.153 ± 0.010 101.7 6.60.400 0.391 ± 0.011 97.7 2.8 0.411 ± 0.023 102.7 5.60.600 0.624 ± 0.013 104.1 2.0 0.640 ± 0.023 106.8 3.6

Thioaildenafil0.150 0.138 ± 0.003 92.1 1.9 0.131 ± 0.010 87.6 6.60.400 0.385 ± 0.008 96.4 2.1 0.386 ± 0.011 96.4 3.00.600 0.582 ± 0.010 96.7 1.6 0.598 ± 0.023 99.6 3.9

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The validated method has been successfully employed to determinethe dosage of PDE-5 inhibitors under study in specimens obtainedby A.I.F.A. and N.A.S. and currently available in the Italian market. Themethod allowed the quantification, in three samples, of sildenafil(88mg in 450mg dosage capsules, corresponding to a 19.6%), of tadalafil(7 mg in a 1 g of powder, equal to a 0.7%) and thioaildenafil (84 mg in450 mg dosage capsules, corresponding to a 18.6%).

4. Conclusions

In this present study, in connection with a survey aimed to monitordietary supplements claiming to contain natural ingredients in the Ital-ianmarket, identification of pharmaceutical active ingredients sildenafiland tadalafil and the characterization of thioaildenafil, dimethylatedthio-derivative of sildenafil, was described.

These findings confirm the phenomenon, increasing in recent years,of adulteration of food supplements in the Europeanmarket. This repre-sents a serious risk to public health considering that the syntheticanalogues of approved PDE-5 inhibitors are often not characterized fortheir efficacy and toxicity and that their mechanism of action mightinvolve other receptors.

About fifty analogues of approved PDE-5 inhibitors have been foundas adulterants. Their presence in the market represents an importantchallenge for the scientific investigation and for the subsequent judicialprocess.

Synthetic analogues of PDE-5 inhibitors can be considered as“designer drugs”, as, almost daily, new products with different namesand packaging appear, containing research chemicals not often listedas controlled substances. These compounds cannot be quickly identi-fied, because often they are not included in spectrum libraries becauseof the lack of reference standards.

In this context, it is therefore important for the chemical toxicologylaboratory to develop and validate accurate analytical protocols to iden-tify molecules that could remain undetected or unidentified by thesimple routine analysis.

Acknowledgements

The authors would like to thank Eli Lilly (Indianapolis, IN, USA),Bayer Health Care (Wuppertal, Germany) and Pfizer (Sandwich, Kent,

Please cite this article as: F. Damiano, et al., Analysis of illicit dietary sthioderivative as adulterant us..., Sci. Justice (2014), http://dx.doi.org/10.1

UK), in particular their Italian Regulatory Offices for supplying theauthors with reference standards.

Special thanks also to Carabinieri Health Protection (N.A.S.) for theircooperation and for their investigative skills.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.scijus.2014.02.009.

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