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

doi:10.4236/ajac.2011.28106

Paper Menu >>

Journal Menu >>

American Journal of Anal yt ical Chemistry, 2011, 2, 919-928

doi:10.4236/ajac.2011.28106 Published Online December 2011 (http://www.SciRP.org/journal/ajac)

Chemical Fingerprinting of Counterfeits of Viagra and

Cialis Tablets and Analogues via Electrospray Ionization

Mass Spectrometry

Rafael S. Ortiz1,2, Kristiane de Cássia Mariotti2, Wanderson Romão3,4*, Marcos N. Eberlin3,

Renata P. Limberger2, Paulo Mayorga2

1Rio Grande do Sul Technical and Scientifical Division, Brazilian Federal Police,

Porto Alegre, Brazil

2Departme n t of Pharmacy , Federal University of Rio Grande do Sul, Porto Alegre, Brazil

3ThoMSon Mass Spectrome try Laboratory, Institute of Chemistry, University of Campinas -

UNICAMP, Campinas, Brazil

4Department of Chemistry, Federal University of Espírito Santo, Vitória, Brazil.

E-mail: [email protected]

Received October 18, 2011; revised November 25, 2011; accepted December 13, 2011

Abstract

Direct infusion electrospray ionization mass spectrometry (ESI-MS) is shown to provide an efficient tech- nique

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 com-

mercial 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, how-

ever, 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.

Keywords: ESI-MS, Fingerprinting, Counterfeiting, Viagra®, Cialis®

1. 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 formu-

lations containing these active pharmacological ingre-

dients (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 phar-

maceutical market. Therefore, rapid and secure analytical

tools able to control the quality of pharmaceutical for-

mulations are highly desirable.

The identification of SLD or TAD in pure form or in

pharmaceutical formulations―including counterfeits

have been performed by different analytical techniques

such as Raman spectroscopy, nuclear magnetic resonance

spectroscopy, Fourier transform infrared spectroscopy,

liquid chromatography, mass spectrometry using elec-

trospray ionization or fast-atom bombardment, and X-ray

diffractometry [2-8].

Direct infusion electrospray ionization mass spec-

1The symbol ® will be used only in reference to the original medicines.

R. S. ORTIZ ET AL.

920

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 chromato-

graphic 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.

2. 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).

2.1. 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 con-

sists 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 in-

jection of each sample in the module chromatography

(LC) programmed to elute all the soluble components in

a single peak. Mass spectra were acquired and accumu-

lated 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.

2.2. 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

Table 1. Details of seizure samples investigated.

Samples Number of

samples seized

Number of

samples analyzed

of seizure Samples Number of

samples analyzed

Viagra® - 4 - Lazar® 3

132 10 I Potent 75® 2

8 1 II Vimax® 3

3 1 III Maxfil® 3

Counterfeit Viagra

9 7 IV Rigix® 2

Cialis® - 4 - Plenovit® 6

6 1 V Pramil® 8

7 1 VI Pramil® 75 2

40 16 VII V-50® 3

40 10 VIII Libiden® 1

Counterfeit Cialis

40 9 IX

R. S. ORTIZ ET AL.

921

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 contri-

bution of all variables.

3. Results and Discussion

3.1. ESI(+)-MS Fingerprinting

A set of 23 Viagra samples classified as authentic (4) or

counterfeit (19) termed 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 ac-

tive pharmaceutical ingredient present in standard Via-

gra® samples.

To confirm SLD detection, ESI(+)-MS/MS was per-

formed for the ions of m/z 475 and 949 (Figures

2(a)-(b)). The collision-induced dissociation (CID) of

[SLD + H]+ of m/z 475 (Figure 2(a)) agrees well with its

structure and connectivity, and reported spectra [8]. The

ion of m/z 949 [2SLD + H]+ formed, as expected, [SLD +

H]+ of m/z 475 as the main fragment (Figure 2(b)).

Figures 1(b)-(e) show ESI(+)-MS spectra for coun-

terfeit 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 cor-

responding 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 distin-

guished from another possible isomer, vardenafil, by the

absence of an abundant ion of m/z 151 [21]. The pres-

ence of analogue species of SLD can result from inten-

tional 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(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.

The structures of the ions of m/z 365, 390 and 779

were investigated by ESI-MS/MS (Figures 4(a)-(b)).

[TAD + H]+ of m/z 390 dissociated as expected from

reported data (Figure 4(a)) [21] whereas the proton-

bonded dimer [2TAD + H]+ of m/z 779 dissociates

mainly to [TAD + H]+ of m/z 390 (Figure 4(b)). For

[lactose + H]+ of m/z 365 (Figure 4(c)) also dissociated

as expected from reported data [17,22] whereas

[2Lactose + Na]+ forms [lactose + Na]+ of m/z 365.

The counterfeit Cialis samples displayed very con-

trasting and variable spectra (Figures 3(b)-(f)). Seizure

V displayed much more abundant TAD ions which indi-

cate the (potentially harmful) use of higher amounts of

the active ingredient. For seizures VI, VIII and IX, how-

ever, instead of TAD ions, abundant SLD ions were de-

tected. 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].

For commercial samples―known to be manufactured

in neighboring countries as Paraguay and Uruguay―two

most typical EASI(+)-MS data were obtained: samples

commercialized under the trademarks Lazar®, Potent 75®,

Vimax®, Maxfil®, Rigix®, Plenovit® and Pramil® (Figure

5(a)) show only SLD ions whereas samples of Pramil

75®, V-50® and Libiden® (Figure 5(b)) showed also the

presence of lactose ions. These drugs are not approved

by the National Health Surveillance Agency (ANVISA)

to be sold in Brazil, but the ESI(+)-MS data show similar

compositions as those of the commercial samples (Fig-

ure 1(a)).

3.2. Chemometry

ESI(+)-MS data were subjected to chemometric treat-

ment via PCA, Figures 6-7. PCA was used to statistic-

cally evaluate the performance of ESI-MS fingerprints in

classifying of sildenafil citrate and tadalafil samples for

quality control purposes. Figures 6 shows PC1 x PC2 x

PC3 scores plots, where the 3 first PCs account for 91%

of total variance. Three groups are observed: authentic

Viagra®, counterfeit Viagra, commercial and some

counterfeit Cialis samples containing the SLD molecule

form group I; counterfeit Cialis samples containing the

TAD molecule form group II; and authentic Cialis® sam-

ples form group III. The ion of m/z 707, 475, 412, 390, s

R. S. ORTIZ ET AL.

922

(a)

(b)

(c)

(d)

(e)

Figure 1. ESI(+)-MS of representative (a) authentic Viagra® and counterfeit Viagra samples: (b) I, (c) II, (d) III and (e) IV.

R. S. ORTIZ ET AL.923

(a)

(b)

(c)

(d)

Figure 2. ESI(+)-MS/MS for some diagnostic ions: (a) [SLD + H]+ of m/z 475; (b) [2SLD + H]+ of m/z 949; (c) [Homosil-

denafil + H]+ of m/z 489; and (d) [Hydroxyhomosildenafil + H]+ of m/z 505.

R. S. ORTIZ ET AL.

924

(a)

(b)

(c)

(d)

R. S. ORTIZ ET AL.925

(e)

(f)

Figure 3. ESI(+)-MS for authentic and counterfeit Cialis® samples.

(a)

(b)

R. S. ORTIZ ET AL.

926

(c)

(d)

Figure 4. ESI(+)-MS/MS for some diagnostic ions: (a) [TAD + H]+ of m/z 390; (b) [2TAD + H]+ of m/z 779; (c) [Lactose + Na]+

of m/z 365; and (d) [2Lactose + Na]+ of m/z 707.

(a)

(b)

Figure 5. ESI(+)-MS spectra of commercial products as (a) Pramil®, Lazar®, Maxfil®, Plenovit®, Potent 75®, Rigix®, Vimax®;

and (b) Pramil 75®, V-50® and Libiden®.

R. S. ORTIZ ET AL.

927

365, 268, 262, and 58 have a greatest influence for the

3PCs loadings.

feit Viagra samples) and group IB (commercial SLD

samples).

Authentic Viagra® and counterfeit Viagra samples

were both grouped together (group I) in Figure 6 There-

fore, a new PCA analysis was performed treating only

the samples from group 1 (Figure 7). Note that this pro-

cedure provides now two subgroups: group IA (counter-

4. Conclusions

Direct infusion ESI(+)-MS of methanolic extrats of tab-

lets provides a relatively simple and fast screening tool

control the quality and to characterize via chemical pro-

files sildenafil and tadalafil tablets for forensic investiga-

tions. This technique requires little sample preparation,

minimizing interferences. Principal component analysis

(PCA) applied to ESI-MS fingerprint data helps to clas-

sify the samples in a more unbiased mode.

5. Acknowledgements

The authors thank the Brazilian Federal Police for pro-

viding the sildenafil citrate and tadalafil samples. This

research has also been generously funded by: FAPESP,

FINEP and CNPq.

6. References

[1] J. G. Hardman, L. E. Limbird and A. G. Gilman, “The

Pharmacological Basis of Therapeutics,” 12th Edition,

McGraw-Hill, New York, 2010.

Figure 6. PCA scores plot for ESI-(+)-MS data. The

samples are classified into three groups: group I ((■)

authentic Viagra®, (●) counterfeit Viagra, (▼) all com-

mercial SLD samples and some () counterfeit Cialis

samples containing the SLD molecule); group II (()

counterfeit Cialis samples containing the TAD molecule);

and group III ((∆) authentic Cialis® samples).

[2] I. Wawer, M. Pisklak and Z. Chilmonczyk, “1H, 13C,

15N NMR Analysis of Sildenafil Base and Citrate (Via-

gra) in Solution, Solid State and Pharmaceutical Dosage

Forms,” Journal of Pharmaceutical Biomedical Analysis,

Vol. 38, No. 5, 2005, pp. 865-870.

doi:10.1016/j.jpba.2005.01.046

[3] M. J. Vredenbregt, L. Blok-Tip, R. Hoogerbrugge, D. M.

Barends and D. de Kaste, “Screening Suspected Counter-

feit Viagra and Imitations of Viagra with Near-Infrared

Spectroscopy,” Journal of Pharmaceutical Biomedical

Analysis, Vol. 40, No. 4, 2006, pp. 840-849.

doi:10.1016/j.jpba.2005.07.048

[4] J. Maurin, F. Plucinski, A. Mazurek and Z. Fijalek, “The

Usefulness of Simple X-Ray Powder Diffraction Analysis

for Counterfeit Control―The Viagra Example,” Journal

of Pharmaceutical Biomedical Analysis, Vol. 43, No. 4,

2007, pp. 1514-1518. doi:10.1016/j.jpba.2006.10.033

[5] M. de Veij, A. Deneckere, P. Vandenabeele, D. de Kaste

and L. Moens, “Detection of Counterfeit Viagra R with

Raman Spectroscopy,” Journal of Pharmaceutical Bio-

medical Analysis, Vol. 46, No. 2, 2008, pp. 303-309.

doi:10.1016/j.jpba.2007.10.021

[6] R. S. Ortiz, M. V. Antunes and R. Linden, “Determinação

de Citrato de Sildenafila e de Tadalafila por Cro-

matografia líquida de Ultraeficiência com Detecção por

Arranjo de Diodos (CLUE-DAD),” Química Nova, Vol.

33, No. 2, 2010, pp. 389-393.

doi:10.1590/S0100-40422010000200028

Figure 7. PCA scores plot for ESI-(+)-MS data presents in

group I. The samples are classified into two groups: group

IA ((●) counterfeit Viagra); and group IB ((▼) authentic

Viagra®; and (◌) all commercial SLD samples).

[7] D. Zhong, J. Xing, S. Zhang and L. Sun, “Study of the

Electrospray Ionization Tandem Mass Spectrometry of

R. S. ORTIZ ET AL.

928

Sildenafil Derivatives,” Rapid Communication Mass

Spectrometry, Vol. 16, No. 19, 2002, pp. 1836-1843.

doi:10.1002/rcm.794

[8] S. R. Gratza, M. Zeller, D. W. Mincey and C. L. Flurer,

“Structural Characterization of Sulfoaildenafil, an Analog

of Sildenafil,” Journal of Pharmaceutical Biomedical

Analysis, Vol. 50, No. 2, 2009, pp. 228-231.

doi:10.1016/j.jpba.2009.04.003

[9] P. P. de Souza, L. C. A. de Oliveira, R. R. Catharino, M.

N. Eberlin, D. V. Augusti, H. G. L. Siebald and R. Au-

gusti, “Brazilian Cachaça: ‘Single Shot’ Typification of

Fresh Alembic and Industrial Samples via Electrospray

Ionization Mass Spectrometry Fingerprinting,” Food

Chemistry, Vol. 115, No. 3, 2009, pp. 1064-1068.

doi:10.1016/j.foodchem.2008.12.026

[10] A. P. B. Gollücke, R. R. Catharino, J. C. de Souza, M. N.

Eberlin and D. Q. Tavares, “Evolution of Major Phenolic

Components and Radical Scavenging Activity of Grape

Juices through Concentration Process and Storage,” Food

Chemistry, Vol. 112, No. 4, 2009, pp. 868-873.

doi:10.1016/j.foodchem.2008.06.060

[11] R. R. Catharino, H. M. S. Milagre, S. A. Saraiva, C. M.

Garcia, U. Schuchardt and M. N. Eberlin, “Biodiesel

Typification and Quality Control by Direct Infusion

Electrospray Ionization Mass Spectrometry Fingerprint-

ing,” Energy Fuels, Vol. 21, 2007, pp. 3698-3701.

doi:10.1021/ef7003078

[12] A. C. L. Amorim, A. M. C. Hovell, A. C. Pinto, M. N.

Eberlin, N. P. Arruda, E. J. Pereira, H. R. Bizzo, R. R.

Catharino, Z. B. M. Filho and C. M. Rezende, “Green and

Roasted Arabica Coffees Differentiated by Ripeness,

Process and Cup Quality via Electrospray Ionization

Mass Spectrometry Fingerprinting,” Journal of Brazilian

Chemistry Society, Vol. 20, No. 2, 2009, pp. 313-321.

doi:10.1590/S0103-50532009000200017

[13] L. A. Marques, R. R. Catharino, R. E. Bruns and M. N.

Eberlin, “Electrospray Ionization Mass Spectrometry Fin-

gerprinting of Perfumes: Rapid Classification and Coun-

terfeit Detection,” Rapid Communication Mass Spec-

trometry, Vol. 20, No. 24, 2006, pp. 3654-3658.

doi:10.1002/rcm.2779

[14] R. R. Catharino, R. Haddad, L. G. Cabrini, I. B. S. Cunha,

A. C. H. F. Sawaya and M. N. Eberlin, “Characterization

of Vegetable Oils by Electrospray Ionization Mass Spec-

trometry Fingerprinting: Classification, Quality, Adul-

teration, and Aging,” Analytical Chemistry, Vol. 77, No.

22, 2005, pp. 7429-7433. doi:10.1021/ac0512507

[15] A. C. H. F. Sawaya, D. M. Tomazela, I. B. S. Cunha, V.

S. Bankova, M. C. Marcucci, A. R. Custodio and M. N.

Eberlin, “Electrospray Ionization Mass Spectrometry Fin-

gerprinting of Propolis,” Analyst, Vol. 129, No. 8, 2004,

pp. 739-744. doi:10.1039/b403873h

[16] W. Romão, P. M. Lalli, M. F. Franco, G. Sanvido, N. V.

Schwab, R. Lanaro, J. L. Costa, B. D. Sabino, M. I. M. S.

Bueno, G. F. de Sa, R. J. Daroda, V. de Souza and M. N.

Eberlin, “Chemical Profile of Meta-Chlorophenylpipera-

Zine (m-CPP) in Ecstasy Tablets by Easy Ambient Sonic-

spray Ionization, X-Ray Fluorescence, Ion Mobility Mass

Spectrometry and NMR,” Analytical and Bioanalytical

Chemistry, Vol. 400, No. 9, 2011, pp. 3053-3064.

doi:10.1007/s00216-011-4883-9

[17] A. C. H. F. Sawaya, P. V. Abdelnur, M. N. Eberlin, S.

Kumazawa, M. R. Ahn, K. S. Bang, N. Nagaraja, V. S.

Bankova and H. Afrouzan, “Fingerprinting of Propolis by

Easy Ambient Sonic-Spray Ionization Mass Spectrome-

try,” Talanta, Vol. 81, No. 1-2, 2010, pp. 100-108.

doi:10.1016/j.talanta.2009.11.043

[18] A. S. Araujo, L. L. da Rocha, D. M. Tomazela, A. C. H. F.

Sawaya, R. R. Almeida, R. R. Catharino and M. N. Eber-

lin, “Electrospray Ionization Mass Spectrometry Finger-

printing of Beer,” Analyst, Vol. 130, No. 6, 2005, pp.

884-889. doi:10.1039/b415252b

[19] P. Zou, S. S. Oh, P. Hou, M. Low and H. Koh, “Simulta-

neous Determination of Synthetic Phosphodiesterase-5

Inhibitors Found in a Dietary Supplement and Pre-Mixed

Bulk Powders for Dietary Supplements Using High-Per-

formance Liquid Chromatography with Diode Array De-

tection and Liquid Chromatography-Electrospray Ioniza-

tion Tandem Mass Spectrometry,” Journal of Chroma-

tography A, Vol. 1104, No. 1-2, 2006, pp. 113-122.

doi:10.1016/j.chroma.2005.11.103

[20] L. Blok-Tip, B. Zomer, F. Bakker, K. D. Hartog, M.

Hamzink, J. Ten Hove, M. Vredenbregt and D. de Kaste,

“Structure Elucidation of Sildenafil Analogues in Herbal

Products,” Food Additive and Contamination, Vol. 21,

No. 8, 2004, pp. 737-748.

doi:10.1080/02652030412331272467

[21] S. Ahn, J. Y. Hong, M. K. Hong, Y. P. Jang, M. S. Oh, J.

H. Jung and J. Hong, “Structural Determination of Silde-

nafil and Its Analogues in Dietary Supplements by Fasta-

tom Bombardment Collision-Induced Dissociation Tan-

dem Mass Spectrometry,” Rapid Communication Mass

Spectrometry, Vol. 23, No. 19, 2009, pp. 3158-3166.

doi:10.1002/rcm.4238

[22] L Nyadong, G. A. Harris, S. Balayssac, A. S. Galhena, M.

Malet-Martino, R. Martino, R. M. Parry, M. D. Wang, F.

M. Fernández and V. Gilard, “Combining Two-Dimen-

sional Diffusion-Ordered Nuclear Magnetic Resonance

Spectroscopy, Imaging Desorption Electrospray Ioniza-

tion Mass Spectrometry, and Direct Analysis in Real-

Time Mass Spectrometry for the Integral Investigation of

Counterfeit Pharmaceuticals,” Analytical Chemistry, Vol.

81, No. 12, 2009, pp. 4803-4812. doi:10.1021/ac900384j

[23] S. Singh, B. Prasad, A. Savaliya, R. Shah, V. Gohil and A.

Kaur, “Strategies for Characterizing Sildenafil, Vardenafil,

Tadalafil and Their Analogues in Herbal Dietary Supple-

ments, and Detecting Counterfeit Products Containing

These Drugs,” Trends Analytical Chemistry, Vol. 28, No. 1,

2009, pp. 13-28. doi:10.1016/j.trac.2008.09.004

[24] B. J. Venhuis, D. M. Barends, M. E. Zwaagstra and D. de

Kaste, “Recent Developments in Counterfeits and Imita-

tions of Viagra, Cialis and Levitra: A 2005-2006 Up-

date,” National Institute for Public Health and the Envi-

ronment (RIVM), Bilthoven, 2007.