**1. Introduction**

50 Chromatography and Its Applications

Subbiah, V. (1999). *Method Of Isolating Cucurbitacin*.United States Patent. 1999, patent

Sur, S.V. (1991). Isolation of Oxygen-Containing Monoterpenoids of Essential Oils by

Svedström, U.; Vuorela, H.; Kostiainen, R.; Tuominen, J.; Kokkonen, J.; Rauha, J.P.; Laakso, I.

Syu, W.J.; Shen, C.C.; Don, M.J.; Ou, J.C.; Lee, G.H. & Sun, C.M. (1998). Cytotoxicity of

Tiuman, T.S.; Ueda-Nakamura, T.; Garcia Cortez, D.A.; Dias Filho, B.D.; Morgado-Diaz, J.A.;

Umlauf, D.; Zapp, J.; Becker, H. & Adam, K.P. (2004). Biosynthesis of the Irregular

Vortherms, T.A. & Roth BL. Salvinorin A: From natural Product to Human Therapeutics.

Wall, M.E. & Wani, M.C. (1996). Camptothecin and Taxol: From Discovery to Clinic. *Journal* 

Wang, G., Tang, W. & Bidigare, R.R. In Ed: Zhang L., & Demain, A.L., (2005). *Natural* 

Yamada, K.; Murata, T.; Kobayashi, K.; Miyase, T.; & Yoshizaki, F. (2010). A Lipase Inhibitor

Crataegus Leaves and Flowers. *Phytochemistry*, Vol. 60, pp. 821-825.

Preparative Adsorbtion Chromatography with Gradient Elution. *Khimiya* 

& Hiltunen, R. (2002). Isolation and Identification of Oligomeric Procyanidins from

Curcuminoids and Some Novel Compounds from Curcuma zedoaria. *Journal of* 

Souza W. & Nakamura, C.V. (2005). Antileishmanial Activity of Parthenolide, a Sesquiterpene Lactone Isolated from Tanacetum parthenium*. Antimicrobial Agents* 

Monoterpene Artemisia Ketone, the Sesquiterpene Germacrene D and Other Isoprenoids in Tanacetum vulgare L. (Asteraceae). *Phytochemistry*, Vol. 65, pp. 2463-

*Products Drug Discovery and Therapeutic Medicine Terpenoids As Therapeutic Drugs As Pharmaceutical Agents.* Humana Press, New Jersey, United States of Amerika. Watanabe, K.; Mimaki, Y.; Sakagami, H.; & Sashida, Y. (2002). Cycloartane Glycosides from

the Rhizomes of Cimicifuga racemosa and Their Cytotoxic Activities. *Chemical* 

Monoterpene and Monoterpene Glycosides from Monarda punctata.

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*of Ethnopharmacology*, Vol. 51, pp. 239-254.

*Pharmaceutical Bulletin*, Vol. 50, pp. 121-125.

*Phytochemistry*, Vol. 71, pp. 1884-1891.

Herbal medicines are major source of aphrodisiacs and have been used worldwide for thousands of years by different cultures and civilizations. Recently, consumption of dietary supplements has been becoming more popular around the world. Unfortunately, the adulteration of dietary supplements with undeclared synthetic chemical compounds is steadily increasing according to the literature. Some herbal products advertised as "all natural" have in contrast been found to contain synthetic PDE-5 inhibitors. There are currently three PDE5 inhibitors **Sildenafil** (Langtry & Markham, 1999) (Viagra; Pfizer, New York, US), **Tadalafil** (Meuleman, 2003) (Cialis; Eli Lilly, Indianapolis, US), and **Vardenafil** (Keating & Scott, 2003) (Levitra; Bayer Pharmaceuticals Co, Wuppertal, Germany), approved worldwide for the treatment of male erectile dysfunction, further two agents **Udenafil** (Salem et al., 2006) (Zydena; Dong-A PharmTech Co, Korean), **Mirodenafil** (Jung, 2008) (Mvix, Life Science R&D Center of SK chemical, Beijing, Tianjin, Shanghai) were licensed only in Korea. They produce vascular smooth muscle relaxation, promote penile blood flow, and hence, induce erection. These kinds of commercially available herbal aphrodisiac products have been spiked with the above-mentioned legal drugs, but also with their analogues, which have not been subjected to formal pharmacokinetic or other pharmacological testing in either humans or animals.

The practice of self-medication by an increasing number of patients, the incessant aggressive advertising of these herbal aphrodisiacs, the invasion of the medicinal market with uncontrolled dietary supplements and the absence of real directives amplifies the potential health hazards to the community. Since the sildenafil is an chemical, it must not been found in any foodstuffs, but an increasing number of sildenafil analogues have been discovered in dietary and herbal supplements even in soft drinks, this number is steadily increasing and some time their types are unknown and necrosis not observed instead of paracetamol. Hence, it is prudent to test the safety and efficacy thus might have unknown and harmful side-effects. Structural analogues are also synthetic chemicals with slightly altered chemical structures and have similar erectile effects on the body. Nevertheless, it is not uncommon

<sup>\*</sup> Corresponding Author

Chromatographic Separation and Identification of Sildenafil

Cl

HN

N N

Fig. 3. (a) Acetylvardenafil; (b) Desulfovardenafil; (c) Gendenafil.

O

O

minutes or less) (Bell & Palmer, 2011) Fig 2.

Fig. 2. Avanafil.

N

et al., 2007; Lai et al., 2007b) Fig 4(a).

2011) in Fig 4(c).

N

and Yohimbine Analogues Illegally Added in Herbal Supplements 53

**Avanafil** was discovered by pharmaceutical developer "Vivus" as PDE5 inhibitors, the safety and efficacy for erectile dysfunction, a possible lunch in 2012. It has a particular advantage over its potential competitors, the effects are demonstrable very quickly (in 15

N N

H

N N

O

(a) (b) (c)

Another new analogue of vardenafil, **Desulfovardenafil**, in which the *N*-ethylpiperazine ring and the sulphonyl group were removed from the vardenafil structure, was identified for enhancing erectile function in herbal health product marketed, namely Power58 Platinum (Lai et al., 2007, Lam, 2007) Fig 3(b). New sildenafil analogues **Gendenafil** had an acetyl group instead of sulfonyl-*N*-methylpiperazine moiety was determined as 5-[2-ethoxy-5-acetyl-phenyl]-1-methyl-3-*n*-propyl-1,6-dihydro-*7H*-pyrazolo[4,3-*d*]pyrimidin-7-one (Lin et al., 2008) Fig3(c). Other vardenafil analogue was found to be added illegally into a dietary supplement marketed for male erectile dysfunction (MED). Its structure was determined as 2-(2-ethoxyphenyl)-5-methyl-7-propyl-imidazo[5,1-*f*][1,2,4]triazin-4(*3H*)-one and called "**Piperidenafil** or **Piperidino vardenafil** or trivial name **Pseudovardenafil**" (Park

Very recently, two new analogues of sildenafil in which the piperazine ring and the sulfonyl group were replaced by a piperazinone and a hydroxyethyl structure, respectively were isolated from a herbal product in Germany. Based on the piperazinone structure, the compounds were named **Piperazinonafil** in Fig 4(b) and **Isopiperazinonafil** (Wollein et al.,

O

N H

The one of the last discovered illegal PDE-5 inhibitor is called as "**Acetylvardenafil**" which was found in dieatery supplement known as MEGATON**®** in USA (Lee et al., 2011).

O O N

Sulfonyl group of Vardenafil was substituted by an acetyl group Fig 3(a) .

N

O HN

OH

N

N

N

O

HN

N

O

O

N N

N

for chemicals with similar structures to possess slightly or entirely different before any new chemical is licensed as drug for human use. This testing process is lengthy and costly; on average, it takes 9.5 years and costs US\$800 million to license a new drug. Many drug analogues, without the aforementioned drug testing process, are available for human consumption properties. Phenacetin, structurally similar to paracetamol, has been associated with renal papillary necrosis not observed with paracetamol (Poon, 2007). Many drug analogues, without the aforementioned drug testing process, are avaliable for human comsuption via different channels. Examples include analogues of psychoactive drugs, anabolic steroids, and Sibutramine which was one of the most abused compound as antiobesity drugs.

The most commonly reported side effects of sildenafil are headaches, flushing of the face, upset stomach and nasal congestion. We met a dieatery supplement having combination of sildenafil and paracetamol, probably in order to prevent headeache caused by sildenafil. Other side effects include sensitivity to light, blurred vision, urinary tract infection, diarrhea and dizziness. The main problem with sildenafil and analogues are that they interact with many other medications. They can rapidly decrease blood pressure up to dangerously low. They can interact with nitrates such as nitrogliserin, which are often prescribed to heart patients. Sildenafil has not to be administrated to patients with heart problems taking nitrate medications because of the severe potentiation of vasodilatory effects.

It is well known, the first developed and consequently the most famous phosphodiesterase inhibitor is Sildenafil (Langtry & Markham, 1999) approved by the FDA in early April 1998. Novel PDE5 inhibitor, **Lodenafil carbonate,** breaks down in the body to form two molecules of the active drug lodenafil. This formulation has higher oral bioavailability than the parent drug (Toque et al., 2008) Fig 1.

Lodenafil

Fig. 1. Prodrug lodenafil carbonate.

**Avanafil** was discovered by pharmaceutical developer "Vivus" as PDE5 inhibitors, the safety and efficacy for erectile dysfunction, a possible lunch in 2012. It has a particular advantage over its potential competitors, the effects are demonstrable very quickly (in 15 minutes or less) (Bell & Palmer, 2011) Fig 2.

Fig. 2. Avanafil.

52 Chromatography and Its Applications

for chemicals with similar structures to possess slightly or entirely different before any new chemical is licensed as drug for human use. This testing process is lengthy and costly; on average, it takes 9.5 years and costs US\$800 million to license a new drug. Many drug analogues, without the aforementioned drug testing process, are available for human consumption properties. Phenacetin, structurally similar to paracetamol, has been associated with renal papillary necrosis not observed with paracetamol (Poon, 2007). Many drug analogues, without the aforementioned drug testing process, are avaliable for human comsuption via different channels. Examples include analogues of psychoactive drugs, anabolic steroids, and Sibutramine which was one of the most abused compound as anti-

The most commonly reported side effects of sildenafil are headaches, flushing of the face, upset stomach and nasal congestion. We met a dieatery supplement having combination of sildenafil and paracetamol, probably in order to prevent headeache caused by sildenafil. Other side effects include sensitivity to light, blurred vision, urinary tract infection, diarrhea and dizziness. The main problem with sildenafil and analogues are that they interact with many other medications. They can rapidly decrease blood pressure up to dangerously low. They can interact with nitrates such as nitrogliserin, which are often prescribed to heart patients. Sildenafil has not to be administrated to patients with heart problems taking nitrate

It is well known, the first developed and consequently the most famous phosphodiesterase inhibitor is Sildenafil (Langtry & Markham, 1999) approved by the FDA in early April 1998. Novel PDE5 inhibitor, **Lodenafil carbonate,** breaks down in the body to form two molecules of the active drug lodenafil. This formulation has higher oral bioavailability than the parent

O

O O

O

N

HN <sup>N</sup>

O

N

O O N N N N S S

Lodenafil carbonate

O

Lodenafil

N

HN <sup>N</sup>

O

N

medications because of the severe potentiation of vasodilatory effects.

obesity drugs.

drug (Toque et al., 2008) Fig 1.

O

N

O

N N

NH

O O

N

HO

Fig. 1. Prodrug lodenafil carbonate.

N S O O The one of the last discovered illegal PDE-5 inhibitor is called as "**Acetylvardenafil**" which was found in dieatery supplement known as MEGATON**®** in USA (Lee et al., 2011). Sulfonyl group of Vardenafil was substituted by an acetyl group Fig 3(a) .

Fig. 3. (a) Acetylvardenafil; (b) Desulfovardenafil; (c) Gendenafil.

Another new analogue of vardenafil, **Desulfovardenafil**, in which the *N*-ethylpiperazine ring and the sulphonyl group were removed from the vardenafil structure, was identified for enhancing erectile function in herbal health product marketed, namely Power58 Platinum (Lai et al., 2007, Lam, 2007) Fig 3(b). New sildenafil analogues **Gendenafil** had an acetyl group instead of sulfonyl-*N*-methylpiperazine moiety was determined as 5-[2-ethoxy-5-acetyl-phenyl]-1-methyl-3-*n*-propyl-1,6-dihydro-*7H*-pyrazolo[4,3-*d*]pyrimidin-7-one (Lin et al., 2008) Fig3(c). Other vardenafil analogue was found to be added illegally into a dietary supplement marketed for male erectile dysfunction (MED). Its structure was determined as 2-(2-ethoxyphenyl)-5-methyl-7-propyl-imidazo[5,1-*f*][1,2,4]triazin-4(*3H*)-one and called "**Piperidenafil** or **Piperidino vardenafil** or trivial name **Pseudovardenafil**" (Park et al., 2007; Lai et al., 2007b) Fig 4(a).

Very recently, two new analogues of sildenafil in which the piperazine ring and the sulfonyl group were replaced by a piperazinone and a hydroxyethyl structure, respectively were isolated from a herbal product in Germany. Based on the piperazinone structure, the compounds were named **Piperazinonafil** in Fig 4(b) and **Isopiperazinonafil** (Wollein et al., 2011) in Fig 4(c).

Chromatographic Separation and Identification of Sildenafil

N

Fig. 6. (a) *N*-octylnor-tadalafil; (b) Cyclopentynafil.

N H N

O

Fig. 7. Condensation product of aminotadalafil and hydroxymethylfuraldehyde.

N

<sup>N</sup> <sup>O</sup>

OH

O

O

O

Last flash development for the treatment of MED is discovering of **Zoraxel** (RX-10100) by Rexahn Pharmaceutical company (Albersen et al., 2010). Zoraxel is containing clavulanic acid that is centrally acting in the CNS and may be a more effective MED treatment for patients who are responsive or unresponsive to PDE-5 inhibitors. It is being developed as an orally administered, on-demand tablet to treat sexual dysfunction, and has extensive and well-established safety in humans. For the future, it is being expected, Zoraxel will be on to

O

O

O

N H

al., 2010) Fig 7.

worldwide best-selling drug.

N

O

and Yohimbine Analogues Illegally Added in Herbal Supplements 55

In Taiwan, one of the dietary supplement which was claimed on the treatment of male erectile dysfunction was firstly screened in 2009 and Tadalafil and its doctored version was newly identified. Since it is having amino group instead of methyl in tadalafil it was named as **aminotadalafil** (Zou et al., 2006; Lin et al., 2009) Table-1. This compound has two asymmetric carbons, theoritically two pairs of enantiomers exist. The chromatographic separation of its stereoisomers was reported by using chiral LC-MS (Kurita et al., 2008). Using this method, *RR*-Aminotadalafil and *SR*-Aminotadalafil were detected in some health food. In addition, an interaction product of aminotadalafil was isolated from an illegal health food product. The structure of the interaction product was elucidated and unknown compound was characterized as condensation product of aminotadalafil and hydroxymethylfuraldehyde and is probably the result of a drug-excipient incompatibility (Häberli et

HN

S N

N

(a) (b)

O O

N

O

O

N N

Fig. 4. (a) Piperidenafil; (b) Piperazinonafil; (c) Isopiperazinonafil.

Another sildenafil analogue was detected from a health supplement claimed for human MED, the structure of this new analogue was characterized as **dithiodesmethylcarbodenafil** containing 2 thiocarbonyl groups instead of 2 carbonyl groups, and 4-methyl substitution on the piperazine ring, rather than 4-ethyl substitution when compared to sildenafil (Ge et al., 2011) Fig. 5(a).

Fig. 5. (a) Dithio-desmethylcarbodenafil; (b) **N**itroso-prodenafil.

Other new unapproved analogue of sildenafil was detected in capsules of a herbal dietary supplement promoted as a libido enhancing product. This is the first time a PDE-5 inhibitor and a potential NO donor were identified in one molecule. A hydrolysis experiment showed that the new analogue was a prodrug of aildenafil and was therefore named **nitrosoprodenafil** (Venhuis et al., 2011) Fig 5(b)**.** Both PDE-5 inhibitors and nitrosamines cause vasodilatation by increasing levels of NO. To their coincidental use is warned against because it may cause a fatal drop in blood pressure. In addition, nitrosamines are known carcinogens. The findings indicate the dangerous level of advancement in medicinal chemistry by producers of unapproved drugs.

Tadalafil (Cialis®) was approved in 2003 by the FDA as the third phosphodiesterase type 5 enzyme (PDE-5) inhibitor to treat MED (Meuleman, 2003). Then, different tadalafil analogues have been found as adulterants in illegal products. Hasegawa et al. detected N**octyl-nortadalafil** Fig 6(a) together with cyclopentynafil **Fig** 6(b) in dietary supplement (Hasegawa et al., 2008). Both of them are the first compounds reported to be new tadalafil and sildenafil analogues.

O

N

N

Another sildenafil analogue was detected from a health supplement claimed for human MED, the structure of this new analogue was characterized as **dithiodesmethylcarbodenafil** containing 2 thiocarbonyl groups instead of 2 carbonyl groups, and 4-methyl substitution on the piperazine ring, rather than 4-ethyl substitution when

HN N

OH

O

N N

(b) (c)

HN

O

S O O

HN N

(a) (b)

Other new unapproved analogue of sildenafil was detected in capsules of a herbal dietary supplement promoted as a libido enhancing product. This is the first time a PDE-5 inhibitor and a potential NO donor were identified in one molecule. A hydrolysis experiment showed that the new analogue was a prodrug of aildenafil and was therefore named **nitrosoprodenafil** (Venhuis et al., 2011) Fig 5(b)**.** Both PDE-5 inhibitors and nitrosamines cause vasodilatation by increasing levels of NO. To their coincidental use is warned against because it may cause a fatal drop in blood pressure. In addition, nitrosamines are known carcinogens. The findings indicate the dangerous level of advancement in medicinal

Tadalafil (Cialis®) was approved in 2003 by the FDA as the third phosphodiesterase type 5 enzyme (PDE-5) inhibitor to treat MED (Meuleman, 2003). Then, different tadalafil analogues have been found as adulterants in illegal products. Hasegawa et al. detected N**octyl-nortadalafil** Fig 6(a) together with cyclopentynafil **Fig** 6(b) in dietary supplement (Hasegawa et al., 2008). Both of them are the first compounds reported to be new tadalafil

N

O S N N

> N N

N O

HN N

O

N

HO

N

O

N N

HN

N

O

compared to sildenafil (Ge et al., 2011) Fig. 5(a).

HN

N

chemistry by producers of unapproved drugs.

and sildenafil analogues.

S

O

N

N

S

N N

Fig. 5. (a) Dithio-desmethylcarbodenafil; (b) **N**itroso-prodenafil.

O

Fig. 4. (a) Piperidenafil; (b) Piperazinonafil; (c) Isopiperazinonafil.

(a)

O

<sup>N</sup> <sup>N</sup> <sup>N</sup>

Fig. 6. (a) *N*-octylnor-tadalafil; (b) Cyclopentynafil.

In Taiwan, one of the dietary supplement which was claimed on the treatment of male erectile dysfunction was firstly screened in 2009 and Tadalafil and its doctored version was newly identified. Since it is having amino group instead of methyl in tadalafil it was named as **aminotadalafil** (Zou et al., 2006; Lin et al., 2009) Table-1. This compound has two asymmetric carbons, theoritically two pairs of enantiomers exist. The chromatographic separation of its stereoisomers was reported by using chiral LC-MS (Kurita et al., 2008). Using this method, *RR*-Aminotadalafil and *SR*-Aminotadalafil were detected in some health food. In addition, an interaction product of aminotadalafil was isolated from an illegal health food product. The structure of the interaction product was elucidated and unknown compound was characterized as condensation product of aminotadalafil and hydroxymethylfuraldehyde and is probably the result of a drug-excipient incompatibility (Häberli et al., 2010) Fig 7.

Fig. 7. Condensation product of aminotadalafil and hydroxymethylfuraldehyde.

Last flash development for the treatment of MED is discovering of **Zoraxel** (RX-10100) by Rexahn Pharmaceutical company (Albersen et al., 2010). Zoraxel is containing clavulanic acid that is centrally acting in the CNS and may be a more effective MED treatment for patients who are responsive or unresponsive to PDE-5 inhibitors. It is being developed as an orally administered, on-demand tablet to treat sexual dysfunction, and has extensive and well-established safety in humans. For the future, it is being expected, Zoraxel will be on to worldwide best-selling drug.

Chromatographic Separation and Identification of Sildenafil

\*Detected in our lab. for the first time by us.

Table 1. Some synthetic PDE-5 inhibitors as analogues sildenafil and tadalafil were isolated by us.

and Yohimbine Analogues Illegally Added in Herbal Supplements 57

In our central instrumental analysis laboratory, we also try to detect these commercially available supplements (which are sent by the Ministry of Food Agriculture and Livestock of Turkey before it grants a license for import to Turkey) whether they possess new or old sildenafil analogues by using high-performance liquid chromatography with diode array detection and mass spectrometry (HPLC-DAD–MS) and nuclear magnetic resonance (NMR) analyses, NMR is the only analytical technique which provides full structural information from novel compounds, acquisition of MS data or comparison of retention times may not be sufficient, in this case, LC-MS/NMR allowed to identify the adulterants without any need for references (Kesting et al., 2010) this means that these kind of analogues are not easy to detect by ordinary laboratory methods, we have identified the listed analogues are given in Table 1-2 up to date. With the aim of evaluating the potential risks of commercialized aphrodisiac products on consumer health, the aim of present work is to investigate simple HPLC-MS method and NMR data of synthetic and natural analogues of aphrodisiacs.

**Table-1** shows the determination of some sildenafil and tadalafil analogues and Dapoxetin HCl, in commercially available health supplements in Turkey. Their formulas, 1H-13C-NMR spectra, ESI(+) m/e values and their LC chromatograms are given in Table 1 and Fig 8, 9, respectively. Fig 9 shows the good separation of caffeine, some sildenafil analogues and

Fig. 8. HPLC chromatogram of *4.75* (Sildenafil), *4.80* (Vardenafil), *3.84* (Tadalafil), *3.62* (Aminotadalafil), *4.39* (Nor-carbodenafil) (*RT* : Retention times as min).

In our central instrumental analysis laboratory, we also try to detect these commercially available supplements (which are sent by the Ministry of Food Agriculture and Livestock of Turkey before it grants a license for import to Turkey) whether they possess new or old sildenafil analogues by using high-performance liquid chromatography with diode array detection and mass spectrometry (HPLC-DAD–MS) and nuclear magnetic resonance (NMR) analyses, NMR is the only analytical technique which provides full structural information from novel compounds, acquisition of MS data or comparison of retention times may not be sufficient, in this case, LC-MS/NMR allowed to identify the adulterants without any need for references (Kesting et al., 2010) this means that these kind of analogues are not easy to detect by ordinary laboratory methods, we have identified the listed analogues are given in Table 1-2 up to date. With the aim of evaluating the potential risks of commercialized aphrodisiac products on consumer health, the aim of present work is to investigate simple HPLC-MS method and NMR data of synthetic and

**Table-1** shows the determination of some sildenafil and tadalafil analogues and Dapoxetin HCl, in commercially available health supplements in Turkey. Their formulas, 1H-13C-NMR spectra, ESI(+) m/e values and their LC chromatograms are given in Table 1 and Fig 8, 9, respectively. Fig 9 shows the good separation of caffeine, some sildenafil analogues and

Sildenafil-T 2: Diode Array

Vardenafil-T 2: Diode Array

Tadalafil-T 2: Diode Array

Aminotadalafil-T 2: Diode Array

Nor-Carbodenafil-T 2: Diode Array

4.75

4.80

3.84

3.62

TIC 2.71e4

TIC 2.86e4

TIC 2.59e4

TIC 4.93e4

TIC 2.89e4

Fig. 8. HPLC chromatogram of *4.75* (Sildenafil), *4.80* (Vardenafil), *3.84* (Tadalafil), *3.62*

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 <sup>0</sup> Time

(Aminotadalafil), *4.39* (Nor-carbodenafil) (*RT* : Retention times as min).

4.39

natural analogues of aphrodisiacs.

**Default file**

100 %

100 %

0

100 %

0

100 %

0

100 %

0


\*Detected in our lab. for the first time by us.

Table 1. Some synthetic PDE-5 inhibitors as analogues sildenafil and tadalafil were isolated by us.

Chromatographic Separation and Identification of Sildenafil

140.8 142.5 144.6 158.6 171.3 168.5

3.19

4.30

4.24

4.36

2.72

2.73 0.90 1.01 1.14 2.10 1.55 2.86 3.33 3.87

3.65 5.04 6.58 9.15 11.37 11.92 12.92

**Default file**

2.69

2.81 2.96 3.04 3.18

100

%

**Default file**

100

%

**Default file**

100

100

0

%

%

129.7 118.2

1.40 0.74

and Yohimbine Analogues Illegally Added in Herbal Supplements 59

L-ArgininHCl-T 1: Scan ES+

TIC 7.65e8

TIC 4.48e4

TIC 9.58e8

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 <sup>0</sup> Time

L-ArgininHCl-T 285 (2.855) Cn (Cen,2, 80.00, Ht) 1: Scan ES+

175.5 2.57e7

212.8 222.5 238.5 207.8 177.7 187.8 189.5 197.4 236.9 251.5252.9 257.2

8.64 7.18

8.40 7.597.91

8.62

8.97

9.239.63 10.15 10.57 10.81 11.18

120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 <sup>0</sup> m/z

Natural 2: Diode Array

Natural 1: Scan ES+

7.18 4.28

4.584.80 5.13 5.51 6.49 5.92

176.4

Fig. 10. HPLC-MS ion chromatogram of *2.69* (L-Arginin HCl), *2.72* (Inosine), *3.19* (Icariin),

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 <sup>0</sup> Time

*4.3* (Yohimbin), *7.18* (Imperatonin), *8.64* (Osthole). (*RT* : Retention times as min).

Fig. 9. HPLC chromotogram of *3.13* (Caffeine), *3.37* (Dimethylacetildenafil), *3.67* (Udenafil), *4.02* (Noracetildenafil), *4.43* (Dimethylsildenafil), *5.02* (Dimethylhomosildenafil), *6.43* (Hydroxythiohomosildenafil), *8.14* (Thiomethisosildenafil), *8.69* (Thiosildenafil), *9.24* (Dapoxetine HCl), *10.14* (Thiohomomethisosildenafil) (*RT* : Retention times as min).

Dapoxetin HCl. **Dapoxetine HCl**, is reported to be a selective serotonin reuptake inhibitor under investigation for the treatment of premature ejaculation (PE) (Li et al., 2009a). Since we have met this compound in one of the herbal drinks for adults, we have added it to our list. As it is well known, caffeine is very common for this kind of health supplements, it was added to chromatogram as well.

#### **1.1 Natural aphrodisiacs**

Formulas, 1H-13C-NMR spectra, ESI(+) m/e values and LC-MS ion chromatograms of natural aphrodisiacs are given in Table 2 and Fig. 10.

L- **Arginine** is not herb, but a nonessential amino acid, it is found naturally in foods such as meat, dairy, poultry and fish, it also may be synthesized in the laboratory, in spite of there is insufficient evidence to rate effectiveness for male fertility and female sexual problem, but it is possible to see the ingredients of many aphrodisiacs, recently. It is also available as oral Larginine supplements, which some product manufacturers market as a "natural Viagra" (Stanislavov & Nikolova, 2003).

1-9+Dap-Caff- 2: Diode Array

6.43 5.02 8.69

8.14

10.14

9.24

3.67

4.43 4.02

3.13

3.37

TIC 4.60e3

Fig. 9. HPLC chromotogram of *3.13* (Caffeine), *3.37* (Dimethylacetildenafil), *3.67* (Udenafil), *4.02* (Noracetildenafil), *4.43* (Dimethylsildenafil), *5.02* (Dimethylhomosildenafil), *6.43* (Hydroxythiohomosildenafil), *8.14* (Thiomethisosildenafil), *8.69* (Thiosildenafil), *9.24* (Dapoxetine HCl), *10.14* (Thiohomomethisosildenafil) (*RT* : Retention times as min).

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 <sup>0</sup> Time

Dapoxetin HCl. **Dapoxetine HCl**, is reported to be a selective serotonin reuptake inhibitor under investigation for the treatment of premature ejaculation (PE) (Li et al., 2009a). Since we have met this compound in one of the herbal drinks for adults, we have added it to our list. As it is well known, caffeine is very common for this kind of health supplements, it was

Formulas, 1H-13C-NMR spectra, ESI(+) m/e values and LC-MS ion chromatograms of

L- **Arginine** is not herb, but a nonessential amino acid, it is found naturally in foods such as meat, dairy, poultry and fish, it also may be synthesized in the laboratory, in spite of there is insufficient evidence to rate effectiveness for male fertility and female sexual problem, but it is possible to see the ingredients of many aphrodisiacs, recently. It is also available as oral Larginine supplements, which some product manufacturers market as a "natural Viagra"

added to chromatogram as well.

(Stanislavov & Nikolova, 2003).

natural aphrodisiacs are given in Table 2 and Fig. 10.

**1.1 Natural aphrodisiacs** 

**Default file**

100

%

Fig. 10. HPLC-MS ion chromatogram of *2.69* (L-Arginin HCl), *2.72* (Inosine), *3.19* (Icariin), *4.3* (Yohimbin), *7.18* (Imperatonin), *8.64* (Osthole). (*RT* : Retention times as min).

Chromatographic Separation and Identification of Sildenafil

currently recommended (Albersen et al., 2010).

and neurotoxicity in an Iranian male patient.

**2. Material and methods** 

**2.1 Equipments** 

supplements.

and Yohimbine Analogues Illegally Added in Herbal Supplements 61

**Yohimbine** is an alkaloid which are found naturally in *Pausinystalia yohimbe*, a classical aphrodisiac which has been recently revalued for its pro-sexual properties and extensively commercialized without control in some countries (Saini et al., 2010, Melnyk et al., 2011) . However, there is little evidence on its efficacy in the treatment of ED and it is, therefore, not

**Inosine** is a nucleoside consisting of ribose and hypoxanthine, most commonly found in supplements that claim to be "energy promoters", naturally it is found in brewer's yeast and organ meats, however it can be synthesized in laboratory (Deuster & Simmons, 2004). Their nerve-stimulating action probably enhances sexual functions or help to remedy sexual

**Icariin** is one of the primary active component of *Epimedium* extracts, which has been used to treat impotence and improve sexual function by acting as PDE5 inhibitor as nafil derivatives (Liu et al., 2005; Dell'Agli, 2008). Since the illegally using of synthetic nafil derivatives have been prohibited, recently icariin has become very attractive by the drug manufacturers and suppliers, because of its natural character. Nowadays, the percentage ratio of Icariin in the commercial *Epimedium* extracts has been increased over 80-90. However there is no sufficient information about its pharmacological profiles and safety. Further investigation needs to be done to examine any benefits that could occur from

**Osthole** and **imperatorin**, coumarin compounds have been reported to exhibit various biological activities (Liao et al., 2010). It was reported that, both of them were found to help relax the corpus cavernosa of the penis, which would potentially help with blood flow, in phenylephrine-precontracted endothelium-intact rabbit corpus cavernosum (Chen et al.,

Herbal medications are being progressively utilized all over the world and it is believed that herbal remedies are not hazards, however some adverse reactions have been increased. Tribulus terrestris is frequently used because of its aphrodisiac effect. But there is an article (Talasaz, 2010), which reports a case of *T-terrestris*-induced hepatotoxicity, nephrotoxicity

In this text, we also present a new sildenafil analogue was found to have been added illegally to a herbal drinks marketed for the enhancement of sexual function. This analogue has never been found. Therefore, it prompted us to elucidate its structure. The structure was determined as 5-[5-[[(3,5-dimethyl-1-piperazinyl]sulfonyl]-2-propoxyphenyl]-1,6-dihydro-1 methyl-3-propyl-7*H*-pyrazolo [4,3-d]pyrimidin-7-one. Owing to the inclusion of a methylene group in dimethylsildenafil, the detected compound was called **Dimethyhomosildenafil**. The sample was purified with column chromatography. The IR, HPLC-/MS (ESI+), and

Uncorrected melting points were measured on an Büchi B-540 capillary melting point apparatus. 1H (400 MHz) and 13C(100 MHz) NMR spectra were recorded employing a

completely assigned NMR data of **dimethylhomosildenafil** have been observed.

functions when these are reduced due to degeneration of nerve tissue.

2000; Chiou et al., 2001), however there is no information for human uses.


Table 2. Natural aphrodisiacs were isolated by us from some herbal supplements.

N H

N

H

OH

NH NH2

 **Formulas 1H-NMR** δ ppm

**(CD3OD**): 1.27 (m, J=7.2Hz, 2H), 1.85 (q, J=7.2Hz, 2H), 3.21 (m, J=2.8Hz, 1H),

**(CD3OD**): 1.16 (q, 12Hz, 1H), 1.35-1.42 (m, 1H), 1.46-1.56 (m, 2H), 1.65-1.69 (m, 1H), 1.90 (qd, 1H), 1.99 (dq, J=10Hz, 1H), 2.23 (t, J=11.2Hz, 1H), 2.34 (dd, J=12.0, 2.8Hz, 1H), 2.46 (dt, 1H), 2.62 (td, J=11.2, 4.4, 1H), 2.72 (dd, J=15.6, 4.8Hz, 1H), 2.90 (dd, J=11.2, 3Hz, 1H), 2.93-3.02 (m, 1H), 3.1 (dd, J=11.2, 4.8Hz, 1H), 3.4 (br.d, J=11.8, 1H), 3.76 (s, 3H), 4.23 (m, 1H), 6.95 (t, J=7.6Hz, 1H), 7.02 (t, 7.6Hz, 1H), 7.27 (d, J=7.6Hz, 1H),

**13C-NMR(CDCl3)**: 21.7, 23.3, 31.4, 34.3, 36.7, 40.7, 51.9, 52.3, 52.9, 59.8, 61.3, 66.9, 108.3, 110.7, 118.1, 119.4, 121.4, 127.4,

**(DMSO-d6)**: 3.51 & 3.62 (m, 2H), 3.91 (q, J=4Hz, 1H), 4.09 (t, J=4Hz, 1H), 4.46 (t, J=5Hz, 1H), 5.1 (br.s, 1H), 5.15 (br.s, 1H), 5.4 (br.s, 1H), 5.83 (d, J=4.4Hz, 1H), 8.04 (s, 1H), 8.30 (s, 1H), 12.35 (br.s, 1H).

**(CD3OD**): 0.9 (d, J=6Hz), 1.63 (s, 3H), 1.72 (s, 3H), 3.15-3.75 (benzylic, ethylenic and sugar protons), 3.89 (s, 3H), 3.9-4.22 (sugar protons), 5.06 (d, J=7.2 Hz, 1H), 5.24 (t, 1H), 5.41 (d, J=1.6Hz, H), 6.65 (s, 1H), 7.01 (d, J=8.8Hz, 2H), 7.88 (d, J=8.8Hz, 2H)

**(CDCl3):** 1.67 (s, 3H), 1.84 (s, 3H), 3.53 (d, J=7.2Hz, 2H), 3.92 (s, 3H), 5.22 (t, J=7.2Hz, 1H), 6.23 (d, J=9.2Hz, 1H), 6.83 (d, J=8.6Hz, 1H), 7.29 (d, J=8.7Hz, 1H),

**(CDCl3)**: 1.72 (s, 3H), 1.74 (s, 3H), 5.01 (d, 2H), 5.61 (t, J=7Hz, 1H), 6.37 (d, J=9.2Hz, 1H), 6.81 (d, J=2.4Hz, 1H), 7.36 (s, 1H), 7.69 (d, J=2.4Hz, 1H), 7.76

7.61 (d, J=9.2 Hz, 1H)

(d,J=9.2Hz, 1H)

3.57 (t, J=6.4Hz, 1H), 4.6(s, 1H).

7.36 (d, J=7.4Hz, 1H).

134.4, 135.9, 175.6.

**Compound name** 

**Yohimbine**  (Saini et al., 2010; Melnyk et al.,

2011) C21H26N2O3

**Inosine**  (Deuster & Simmons, 2004) C10H12N4O5

**Icariin** 

**Osthole**  (Liao et al., 2010) C15H16O3

**Imperatorin**  (Liao et al., 2010) C16H14O4

(Liu et al., 2005; Dell'Agli, 2008) C33H40O15

**L-Arginin HCl**  (Stanislavov & Nikolova, 2003) C6H14N4O2

**M.w.**  *ESI(+)m/z*

174 *175(M+H)* <sup>2</sup>NH

355 *356(M+H)*

268

676 *677(M+H)*

244 *245(M+H)*

270

*269(M+H)* O

OH

OH

O

OH

N H

O

H

O

N N

O

OH

O O O

O

Table 2. Natural aphrodisiacs were isolated by us from some herbal supplements.

OH OH

O O

OH OH OH

*271(M+H)* <sup>O</sup> <sup>O</sup> <sup>O</sup>

N

O O

O OH

O

NH

O

OH OH

H

**Yohimbine** is an alkaloid which are found naturally in *Pausinystalia yohimbe*, a classical aphrodisiac which has been recently revalued for its pro-sexual properties and extensively commercialized without control in some countries (Saini et al., 2010, Melnyk et al., 2011) . However, there is little evidence on its efficacy in the treatment of ED and it is, therefore, not currently recommended (Albersen et al., 2010).

**Inosine** is a nucleoside consisting of ribose and hypoxanthine, most commonly found in supplements that claim to be "energy promoters", naturally it is found in brewer's yeast and organ meats, however it can be synthesized in laboratory (Deuster & Simmons, 2004). Their nerve-stimulating action probably enhances sexual functions or help to remedy sexual functions when these are reduced due to degeneration of nerve tissue.

**Icariin** is one of the primary active component of *Epimedium* extracts, which has been used to treat impotence and improve sexual function by acting as PDE5 inhibitor as nafil derivatives (Liu et al., 2005; Dell'Agli, 2008). Since the illegally using of synthetic nafil derivatives have been prohibited, recently icariin has become very attractive by the drug manufacturers and suppliers, because of its natural character. Nowadays, the percentage ratio of Icariin in the commercial *Epimedium* extracts has been increased over 80-90. However there is no sufficient information about its pharmacological profiles and safety. Further investigation needs to be done to examine any benefits that could occur from supplements.

**Osthole** and **imperatorin**, coumarin compounds have been reported to exhibit various biological activities (Liao et al., 2010). It was reported that, both of them were found to help relax the corpus cavernosa of the penis, which would potentially help with blood flow, in phenylephrine-precontracted endothelium-intact rabbit corpus cavernosum (Chen et al., 2000; Chiou et al., 2001), however there is no information for human uses.

Herbal medications are being progressively utilized all over the world and it is believed that herbal remedies are not hazards, however some adverse reactions have been increased. Tribulus terrestris is frequently used because of its aphrodisiac effect. But there is an article (Talasaz, 2010), which reports a case of *T-terrestris*-induced hepatotoxicity, nephrotoxicity and neurotoxicity in an Iranian male patient.

In this text, we also present a new sildenafil analogue was found to have been added illegally to a herbal drinks marketed for the enhancement of sexual function. This analogue has never been found. Therefore, it prompted us to elucidate its structure. The structure was determined as 5-[5-[[(3,5-dimethyl-1-piperazinyl]sulfonyl]-2-propoxyphenyl]-1,6-dihydro-1 methyl-3-propyl-7*H*-pyrazolo [4,3-d]pyrimidin-7-one. Owing to the inclusion of a methylene group in dimethylsildenafil, the detected compound was called **Dimethyhomosildenafil**. The sample was purified with column chromatography. The IR, HPLC-/MS (ESI+), and completely assigned NMR data of **dimethylhomosildenafil** have been observed.

#### **2. Material and methods**

#### **2.1 Equipments**

Uncorrected melting points were measured on an Büchi B-540 capillary melting point apparatus. 1H (400 MHz) and 13C(100 MHz) NMR spectra were recorded employing a

Chromatographic Separation and Identification of Sildenafil

Fig. 11. IR spectrum of Dimethylhomosildenafil.

toxicity profile.

and Yohimbine Analogues Illegally Added in Herbal Supplements 63

methisosildenafil (Reepmeyer & Avignon, 2009). The IR spectrum of dimethylhomosildenafil is given in Fig 11. This compound also must be put on the inspection list for illegal health-related substances because of the unknown safety and

VARIAN MERCURY 400 MHz FT spectrometer, with CDCl3 as solvent. Chemical shifts (δ) are in ppm relative to TMS. The LC/MS were taken on a Waters Micromass ZQ connected with Waters Alliance HPLC, using ESI(+) method, with C-18 column. Elemental analyses were performed by Leco CHNS-932. The infrared spectrum was recorded in the 600-3600 cm-1 range using a Jasco FT-IR-420 spectrometer and KBr pellets.
