Chemical Fingerprinting of Counterfeits of Viagra and Cialis Tablets and Analogues via Electrospray Ionization Mass Spectrometry

Direct infusion electrospray ionization mass spectrometry (ESI-MS) is shown to provide an efficient technique for the investigation of polar composition of forensic tablets for male erectile dysfunction. ESI-MS fingerprinting of 41 commercial sildenafil samples (Viagra, Cialis, Lazar, Libiden, Maxfil, Plenovit, Potent 75, Rigix, V-50, Vimax, Pramil 75 and Pramil) and 56 counterfeit samples (Viagra and Cialis) were obtained. The spectra for the authentic Viagra tablets showed abundant ions exclusively corresponding to the sildenafil (SLD) molecule: [SLD + H] of m/z 475; [SLD + Na] of m/z 497; and [2SLD + H] of m/z 949. The spectra for commercial sildenafil samples also showed predominat SLD ions. Tablets of authentic Cialis showed mainly ions of m/z 343, 365 and 707 from the lactose molecule (the excipient); as well as a minor ion of m/z 390 corresponding to the active ingredient tadalafil (TAD) in its protonated form [TAD + H]. For counterfeit Cialis samples, however, normally TAD ions of much high abundances was observed, together with ions corresponding to sildenafil analogues such as those of m/z 489 (homosildenafil) and 505 (hydroxyhomosildenafil). Principal component analysis was applied to ESI-MS fingerprint data, placing samples according to their contents of active ingredients hence authentic and counterfeit samples are easily recognized.


Introduction
Sildenafil (SLD) and tadalafil (TAD) are selective inhibitors of phosphodiesterase type-5 (PDE-5) [1] acting therefore as efficient drugs to treat male erectile dysfunction.The two most common commercial formulations containing these active pharmacological ingredients (API) are Viagra ® (sildenafil citrate, Pfizer) and Cialis ® (tadalafil, Eli Lilly) 1 and the analysis of their tablets is currently a common practice for many forensic laboratories.Besides the criminal aspect, the illegal trade of these drugs can cause serious health problems due to uncontrolled amounts, low quality and diverse com-positions.The counterfeiting of inhibitors of PDE-5 has become an important and dangerous problem for pharmaceutical market.Therefore, rapid and secure analytical tools able to control the quality of pharmaceutical formulations are highly desirable.
Direct infusion electrospray ionization mass spec- 1 The symbol ® will be used only in reference to the original medicines.
trometry (ESI-MS) has been shown to provide a fast and robust technique for quality control of complex chemical mixtures such as those of alcoholic [9] and non alcoholic [10] beverages, biodiesel [11], coffees [12], perfumes [13], vegetable oils [14], propolis [15] and drugs of abuse [16].The use of direct infusion ESI-MS in the chemical characterization of forensic samples of PDE-5 inhibitors seems therefore advantageous, since it would eliminate the need for isolation and prior chromatographic separation, minimizing the time of analysis and interferences.Chemometrics methods applied to ESI-MS data [17,18] could also improve selectivity for secure counterfeit screening.
In this study, ESI-MS in the positive ion mode was shown to provide fast (less than a minute) and secure technique of the forensic analysis of samples of PDE-5 inhibitors.To test this ability, forty one commercial samples (Viagra ® , Cialis ® , Lazar ® , Libiden ® , Maxfil ® , Plenovit ® , Potent 75 ® , Rigix ® , V-50 ® , Vimax ® , Pramil 75 ® and Pramil ® ) and fifty seven counterfeit samples (Viagra and Cialis) were analyzed, and the data subjected to principal component analysis, PCA.

Materials and Methods
HPLC-grade methanol and formic acid solutions were purchased from Sigma-Aldrich and used with no further purification.Eight authentic samples of Viagra ® and Cialis ® containing 50 mg of SLD (4) and 20 mg of TAD (4), respectively, were supplied by Pfizer Ltda and Eli Lilly do Brasil Ltda laboratories.The thirty three tablets of different trademarks of foreign origin and fifty six counterfeit samples (Table 1) were provided by the Bra-zilian Federal Police forensic laboratory (Rio Grande do Sul, Brazil).

ESI-MS
An API 2000 Q-Trap LC-MS/MS mass spectrometer from MDS Sciex/Applied Biosystems (Foster City, CA, USA) coupled to a Alliance 2695 Separations Module LC (Waters, Milford Massachusetts, USA) was used.Data acquisition and processing were performed using the Analyst software from Applied Biosystems (Foster City, CA, USA).General conditions were as follows: source temperature of 100˚C, capillary voltage of 3.0 kV and cone voltage of 40 V.The sample preparation consists in dissolution of a small fraction-about 10 mg-of the core of each tablet in a solution containing 40 mL of methanol and 40 μL of concentrated formic acid aqueous solution.ESI-MS was performed with the automatic injection of each sample in the module chromatography (LC) programmed to elute all the soluble components in a single peak.Mass spectra were acquired and accumulated over 60 s on the range of m/z 50 to 1000.Structural analysis of some diagnostic ions was performed by ESI-MS/MS using collision energies varying from 15 to 45 eV.The collision gas pressure (argon) was optimized to produce extensive fragmentation of the ions under investigation.

Chemometric Treatment
Multivariate analysis by PCA was performed by running the software STATISTICA, version 7 (StatSoft ® ).The ESI-MS data were compiled to generate a final matrix of 97 cases (samples) and 99 variables (m/z values with their respective relative intensities).Only ions with a relative abundance higher than 10% were considered for the final matrix data.These data were previously mean-centered and auto scaled to variance 1, ensuring an equal contribution of all variables.
Figures 1(b)-(e) show ESI(+)-MS spectra for counterfeit Viagra samples (seizures I-IV, Table 1).SDL ions also predominate but for seizures I and II (Figures 1(b)-(c)), the ESI(+)-MS detects an ion of m/z 489 corresponding probably to the protonated molecule of an immediate analogue of SLD.The ESI(+)-MS/MS data for this ion (Figure 2(c)) display fragments similar or the same as those produced by [SDL + H] + : those of m/z 58, 70, 84, 99, 254, 283, 297, 313 and 325.This molecule can be attributed to homosildenafil (HSD), being distinguished from another possible isomer, vardenafil, by the absence of an abundant ion of m/z 151 [21].The presence of analogue species of SLD can result from intentional addition or from inadequate purification.
Figure 1(e) shows the presence of low abundant but unique ions of m/z of 221, 237, 355, 429 and 445.For sample III, however, a very similar ESI(+)-MS data as that observed for authentic Viagra ® samples (Figure 1(d)) was acquired.
Authentic and counterfeit Cialis samples were also analyzed by ESI(+)-MS (Figures 3(a)-(f)).The spectrum for the authentic Cialis ® sample (Figure 3 The structures of the ions of m/z 365, 390 and 779 were investigated by ESI-MS/MS (Figures 4(a)-(b)).
The counterfeit Cialis samples displayed very contrasting and variable spectra (Figures 3(b)-(f)).Seizure V displayed much more abundant TAD ions which indicate the (potentially harmful) use of higher amounts of the active ingredient.For seizures VI, VIII and IX, however, instead of TAD ions, abundant SLD ions were detected.For seizure VII, both TAD and SDL ions were detected.An ion of quite low abundance (m/z 505) was also observed in Figure 3(b) corresponding to another analogue of SLD, namely hydroxyhomosildenafil (OH-HSD) [23], as confirmed by ESI(+)-MS/MS (Figure 2(d)) [21].The HSD and OH-HSD analogues display higher pharmacological activity than SLD [24].
feit Viagra samples) and group IB (commercial SLD samples).Authentic Viagra ® and counterfeit Viagra samples were both grouped together (group I) in Figure 6 Therefore, a new PCA analysis was performed treating only the samples from group 1 (Figure 7).Note that this procedure provides now two subgroups: group IA (counter-

Conclusions
Direct infusion ESI(+)-MS of methanolic extrats of tablets provides a relatively simple and fast screening tool control the quality and to characterize via chemical profiles sildenafil and tadalafil tablets for forensic investigations.This technique requires little sample preparation, minimizing interferences.Principal component analysis (PCA) applied to ESI-MS fingerprint data helps to classify the samples in a more unbiased mode.
(a)) shows a low abundant but diagnostic [TAD + H] + ion of m/z of 390.More abundant ions were attributed to the lactose excipient , that is: [lactose + H] + of m/z 343; [lactose + Na] + of m/z 365; and [2lactose + Na] + of m/z 707 [25,26].Cialis tablet are known to contain 20 mg of TAD and 245 mg of lactose.

Table 1 . Details of seizure samples investigated. Samples Number of samples seized Number of samples analyzed Register of seizure Samples Number of samples analyzed
[19,20]as I, II, III and IV (Table 1), were analyzed by ESI(+)-MS.Figure 1(a) shows typical ESI(+)-MS obtained for authentic Viagra ® tablets.The main ions correspond to the SLD molecule detected in different forms such as [SLD + H] + of m/z 475; [SLD + Na] + of m/z 497; [2SLD + H] + of m/z 949, and fragment ions of m/z 377, 311, 283 and 99[19,20].Therefore, ESI(+)-MS provides, for the methanolic extract, a typical fingerprint data showing mainly SLD ions from the active pharmaceutical ingredient present in standard Viagra ® samples.