Screening of synthetic PDE-5 inhibitors and their analogues as adulterants: Analytical techniques and challenges

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Review

Screening of synthetic PDE-5 inhibitors and their analogues as adulterants: Analytical techniques and challenges Dhavalkumar Narendrabhai Patel a , Lin Li b , Chee-Leong Kee c , Xiaowei Ge c , Min-Yong Low c , Hwee-Ling Koh a,∗ a

Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore Chengdu Branch of National Science Library, Chinese Academy of Sciences, Chengdu 610041, Sichuan Province, China c Applied Sciences, Pharmaceutical Division, Health Sciences Authority, Singapore 169078, Singapore b

a r t i c l e

i n f o

Article history: Received 13 March 2013 Received in revised form 25 April 2013 Available online xxx Keywords: Screening for adulterants PDE-5 inhibitors Designer analogues Analytical techniques Challenges

a b s t r a c t The popularity of phosphodiesterase type 5 (PDE-5) enzyme inhibitors for the treatment of erectile dysfunction has led to the increase in prevalence of illicit sexual performance enhancement products. PDE-5 inhibitors, namely sildenafil, tadalafil and vardenafil, and their unapproved designer analogues are being increasingly used as adulterants in the herbal products and health supplements marketed for sexual performance enhancement. To date, more than 50 unapproved analogues of prescription PDE5 inhibitors were found as adulterants in the literature. To avoid detection of such adulteration by standard screening protocols, the perpetrators of such illegal products are investing time and resources to synthesize exotic analogues and devise novel means for adulteration. A comprehensive review of conventional and advance analytical techniques to detect and characterize the adulterants is presented. The rapid identification and structural elucidation of unknown analogues as adulterants is greatly enhanced by the wide myriad of analytical techniques employed, including high performance liquid chromatography (HPLC), gas chromatography–mass spectrometry (GC–MS), liquid chromatography mass-spectrometry (LC–MS), nuclear magnetic resonance (NMR) spectroscopy, vibrational spectroscopy, liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry (LC–FT-ICR-MS), liquid chromatograph-hybrid triple quadrupole linear ion trap mass spectrometer with information dependent acquisition, ultra high performance liquid chromatography-time of flight-mass spectrometry (UHPLC–TOF-MS), ion mobility spectroscopy (IMS) and immunoassay methods. The many challenges in detecting and characterizing such adulterants, and the need for concerted effort to curb adulteration in order to safe guard public safety and interest are discussed. © 2013 Hwee-Ling Koh. Published by Elsevier B.V. All rights reserved.

Abbreviations: 13 C NMR, carbon-13-nuclear magnetic resonance; 1D NMR, 1 dimensional nuclear magnetic resonance; 1 H NMR, proton nuclear magnetic resonance; 2D NMR, 2 dimensional nuclear magnetic resonance; CART, classification and regression trees; CE-MS, capillary electrophoresis–mass spectrometry; CID, collision induced dissociation; CNLS, constant neutral loss scan; COSY, correlation spectroscopy; DAD, diode array detector; DCBI, desorption corona beam ionization; DEPT, distortionless enhancement by polarization transfer; DOSY–NMR, diffusion ordered spectroscopy–nuclear magnetic resonance; D-PLS, discriminant partial least squares; ED, erectile dysfunction; ESI, electrospray ionization; EU, European Union; FT-ICR-MS, Fourier transform ion cyclotron resonance-mass spectrometry; FT-IR, Fourier transformed infrared; FTIR-ATR, Fourier transformed infrared attenuated total reflectance; GC–MS, gas chromatography–mass spectrometry; HMBC, heteronuclear multiple-bond correlation spectroscopy; HMQC, heteronuclear multiple-quantum correlation spectroscopy; HPLC, high performance liquid chromatography; HPLC-DCBI–MS/MS, high performance liquid chromatography-desorption corona beam ionization–tandem mass spectrometry; HPTLC, high performance thin layer chromatography; HRMS, high resolution mass spectrometry; HSA, Health Sciences Authority Singapore; IDA, information dependent acquisition; IMS, ion mobility spectroscopy; LC–ESI-MSn , liquid chromatography–electrospray ionization-tandem mass spectrometer; LC–FT-ICR-MS, liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry; LC–LTQ Orbitrap XL FTMS, liquid chromatography–linear ion orbitrap XL Fourier transform mass spectrometry; LC–q-TOF-MS/MS, liquid chromatography–quadrupole-time-of-flight-tandem mass spectrometry; LC–MS, liquid chromatography mass-spectrometry; LC–TIS-MS, liquid chromatography–turbo ion spray mass spectrometry; LC–TOF-MS, liquid chromatography–time of flight-mass spectrometry; MDMA, methylenedioxymethamphetamine; MRM, multiple reaction monitoring; MS, mass spectrometry; MS/MS, tandem mass spectrometry; MSn , multi-stage mass spectrometry; NDA, New Drug Application; NIR, near infrared; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; PCA, principal component analysis; PDE-5, phosphodiesterase type 5 enzyme; PIS, product ion scanning; PP, projection pursuit; PREC, precursor ion scanning; SIM, selected ion monitoring; SIMCA, soft independent modelling by class analogy; SRM, selected reaction monitoring; SVM, support vector machines; THMPD, Traditional Herbal Medicinal Products Directive; TIS, turbo ion spray; TLC, thin layer chromatography; TOF, time of flight; TQ-LIT, triple quadruple linear ion trap; UHPLC, ultra high performance liquid chromatography; USFDA, United States Food and Drug Administration; UV, ultraviolet; XRD, X-ray diffraction; XRF, X-ray fluorescence. ∗ Corresponding author. Tel.: +65 65167962; fax: +65 67791554. E-mail address: [email protected] (H.-L. Koh). 0731-7085/$ – see front matter © 2013 Hwee-Ling Koh. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.04.037

Please cite this article in press as: D.N. Patel, et al., Screening of synthetic PDE-5 inhibitors and their analogues as adulterants: Analytical techniques and challenges, J. Pharm. Biomed. Anal. (2013), http://dx.doi.org/10.1016/j.jpba.2013.04.037

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Contents 1. 2. 3.

4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analogues currently found as adulterants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Techniques used for detection and identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Techniques used for preliminary identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. High performance liquid chromatography (HPLC) methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Mass spectrometry (MS) based hyphenated methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. LC–MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. GC–MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Vibrational spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Nuclear magnetic resonance (NMR) spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. X-ray diffraction (XRD) spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Other methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Synthetic phosphodiesterase type 5 enzyme (PDE-5) inhibitors are used for the treatment of erectile dysfunction (ED) of various aetiology in men. ED is a highly prevalent and important medical condition whereby the patient has trouble getting or keeping an erection [1]. In the USA, it is estimated that around 30 million men are affected by ED [1]. The first three approved PDE-5 inhibitors are sildenafil citrate (Viagra® , Pfizer), tadalafil (Cialis® , Elli Lilly) and vardenafil hydrochloride (Levitra® , Bayer). In addition, other approved PDE-5 inhibitors include udenafil (Zydena® , Dong-A Pharmaceutical Co. Ltd) in South Korea and Malaysia, mirodenafil hydrochloride (Mvix® , SK Chemicals Life Science) in South Korea, and lodenafil carbonate (Helleva® , Cristália Produtos Químicos e Farmacêuticos) in Brazil. Another synthetic PDE-5 inhibitor, avanafil (Stendra® , Vivus Inc.) has also been approved for the treatment of ED in the USA [1]. Fig. 1 shows the chemical structures of approved PDE-5 inhibitors. These PDE-5 inhibitors are also used by men with no ED for recreational use [2]. However, clinically significant side effects, such as headaches, facial flushing, nasal congestion, visual disorders and back pain have been reported [1,3]. Moreover, PDE-5 inhibitors may also cause potentially serious drug–drug interactions [4]. Their co-administration with nitrates or ␣-blockers can result in severe hypotension and syncope [4]. Therefore, it is dangerous for unknowing patients who have cardiac problems to be taking both nitrates and PDE-5 inhibitors. The widespread use and popularity of PDE-5 inhibitors has led to increase in the prevalence of illicit sexual performance enhancement products in many countries [5–9]. In Europe, it was estimated that every year, 6 million products were purchased outside the official health system [10]. Most of such illicit products are herbal dietary supplements and health products [5–11]. These health products are widely perceived as “safe being natural” and increasingly popular as easily affordable alternative for prescription drugs for ED. Numerous websites and advertising campaigns which offer anonymous and cheaper health products for ED can be found. However, some of these products have been found to be adulterated with approved PDE-5 inhibitors and their unapproved synthetic analogues synthesized by minor modifications to the parent structures of approved PDE-5 inhibitors. A previous review reported the number of PDE-5 inhibitors and their analogues found as adulterants in health supplements to be at least 46 [5]. The safety and toxicity profile of these unapproved analogues is often not known and hence consumers of such products are at risk. Fatal cases caused by adulterated dietary supplements have been reported. For example, in Singapore, out of 22 fatalities reported due to the adverse events associated with the use of

00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00

complementary medicine and health supplements, there were nine deaths due to the consumption of sexual performance enhancement products adulterated with sildenafil and glibenclamide [11]. The patients experienced severe hypoglycaemia and unconsciousness before they died [11]. In Hong Kong, a 28-year-old previously healthy man suffered unsteady gait and frequent falls due to the consumption of a health product adulterated with acetildenafil [12]. The problem is more complex as the number of such adulterants is steadily increasing and now spreading into functional foods and energy drinks [5,13,14]. The manufacturers of such illicit and adulterated sexual performance enhancement products are now using new and exotic analogues which are difficult to be detected by routine screening and inspection methods. Therefore, advanced analytical methods and strategies are crucial to screen for adulteration with analogues of PDE-5 inhibitors. Some previous articles have described screening strategies, analytical methods and origin of known analogues [5,9]. The purpose of this paper is to comprehensively review the literature on PDE-5 inhibitors and their analogues as adulterants, the analytical methods for their detection and the challenges faced in screening for such adulterants. The structural similarities and differences between different analogues are also presented. This information could be very useful in detection of such adulteration. 2. Analogues currently found as adulterants Herbal dietary supplements and health products aimed at increasing sexual performance are sometimes adulterated with PDE-5 inhibitors and their unapproved analogues. Singh et al. have previously collated information on 18 PDE-5 inhibitors and analogues [9]. Venhuis et al. reviewed 43 analogues of PDE-5 inhibitors known till 2011 and described their intellectual origin from patents filed by various pharmaceutical companies [5]. However, important information such as chemical formulae, accurate masses and the structural relationship among all currently known analogues of PDE-5 inhibitors has not been described. Table 1 shows a list of synthetic analogues in chronological order of their first detection as an adulterant. Table 2 lists the various currently known analogues of PDE-5 inhibitors, their chemical formulae, accurate masses and UV max . To date, more than 50 unapproved structurally modified analogues of PDE-5 inhibitors have been reported as adulterants [15–63]. Analogues of sildenafil were more frequently detected than those of vardenafil and tadalafil (Tables 1 and 2). This could be attributed to the readily available and low cost raw materials required for the synthesis of sildenafil analogues [5,6]. In contrast, the raw material piperonal required for the synthesis of tadalafil

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Fig. 1. Chemical structures of approved PDE-5 inhibitors.

analogues is heavily monitored by various drug regulatory agencies [5,64]. The reason is that piperonal is also used in the production of 3,4-methylenedioxymethamphetamine (MDMA), popularly known as ecstasy or XTC [5,64]. Therefore, despite less synthetic steps in manufacturing [5] and having substantially longer duration of action (36 h) compared to sildenafil (4 h), only nine analogues of tadalafil have been reported so far in adulterated products (Table 2). Similarly, only seven structural analogues of vardenafil have been reported as adulterants (Table 2). Although vardenafil has the quickest onset of action, its duration of action is shortest compared to sildenafil and tadalafil. This could be a possible reason deterring dishonest manufacturers to employ analogues of vardenafil as adulterants [5]. Schemes 1–3 show the chemical structures of the different analogues of sildenafil and related compounds. Homosildenafil was the first reported analogue of sildenafil in a food beverage in 2003 [15]. Since then, several structural analogues of sildenafil have been reported, with newer and alluring analogues being steadily reported. These include depiperazinothiosildenafil [54], descarbosildenafil [14], propoxyphenyl aildenafil [55], propoxyphenyl thioaildenafil [55], propoxyphenyl thiohydroxyhomosildenafil (PPTHHS) [56] and propoxyphenyl hydroxyhomosildenafil [59]. Unknown exposure to such analogues could place consumers at high risk as the safety profile and pharmacokinetics of these analogues are often not known. Compared to sildenafil, PP-THHS is at least ten-fold more potent in inhibiting isolated PDE-5 enzyme [56,65] and two hundred times more potent in inhibiting PDE-1 enzyme in vitro [65]. An adverse event associated with a health supplement adulterated with PP-THHS resulted in giddiness, headache, shortness of breath and backache [66]. Scheme 4 shows the chemical structures of the different analogues of tadalafil. All the analogues of tadalafil found as adulterants differ from each other at the N-atom of the pyrazine ring [36,43,53]. Such N-alkyl analogues including tadalafil were previously reported by GlaxoSmithKline as a part of their drug discovery efforts [67]. However, the first analogue of tadalafil, aminotadalafil, identified as an adulterant by our laboratory in collaboration with the Health Sciences Authority (HSA) of Singapore in 2006 is not described in patents and scientific literature [18,32]. In addition, there are also reports of adulteration with optical isomers of tadalafil [47,68]. It was proposed that the use of substandard reagents and poor quality control could be responsible for their presence [47,68]. Scheme 5 shows the different analogues of vardenafil. The analogues of vardenafil have variations at the substituents attached to the 2-ethoxyphenyl ring. The latest identified analogues of

vardenafil as adulterants were acetyl vardenafil [52] and hydroxythiovardenafil [58]. 3. Techniques used for detection and identification The persistent supply and unlimited possibilities of new analogues of PDE-5 inhibitors make their detection and identification challenging. The complex matrix of herbal products containing multiple ingredients presents an additional challenge. Over the years, many different analytical techniques have been used for the detection and characterization of synthetic PDE-5 inhibitors and their analogues as adulterants. Some of the techniques have been previously reviewed [9]. In this paper, we have critically reviewed up to date literature on different screening methods and recent development in such methods used for the detection of adulteration with PDE-5 inhibitors. Table 1 shows the different techniques used in the identification of each of the unapproved analogue while List 1 shows a list of techniques in alphabetical order for easy reference. The following section gives an overview of the application of these methods in the identification and characterization of PDE-5 inhibitors and their analogues as adulterants. 3.1. Techniques used for preliminary identification Thin Layer Chromatography (TLC) using silica gel plates GF 254 has long been used to characterize the crude plant drug and pharmacologically active components of standardized extracts and their formulations [69–73]. It is also used in the identification and purity check of drug substances and pharmaceutical formulations [74]. TLC is simple and cheap, and it can be used for preliminary identification of known PDE-5 inhibitors for which the reference standards are available [9,69]. However, sensitivity of TLC is low. Established TLC procedures can be used for rapid screening of herbal dietary supplements and health products for adulteration with known PDE-5 inhibitors. Cai et al. have reported simple TLC method using chloroform/ethyl acetate/methanol/water solvent mixture as a mobile phase for the rapid identification of eight PDE-5 inhibitors as adulterants using UV detection [69]. As PDE-5 inhibitors are nitrogen containing compounds, their spots on the TLC plate can be detected by spraying with reagents such as the Dragendorff reagent [70,71] and potentially with iodoplatinate and other non-specific reagents (e.g. sulphuric acid). Furthermore, preparative TLC has also been used for the isolation of unknown PDE-5 inhibitors [36]. The use of high performance TLC (HPTLC) in screening for PDE5 inhibitors has also been reported [72,73]. It has improved

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Table 1 List of unapproved synthetic analogues of PDE-5 inhibitors in chronological order of their first detection and analytical techniques used for their identification. No.

Analogue

Year of first report

Type of product

Techniques used

Reference

1 2 3 4 5

Homosildenafil Hydroxyhomosildenafil Acetildenafil, hongdenafil Aminotadalafil Hydroxyacetildenafil

2003 2004 2004 2006 2006

UV spectroscopy, HPLC-DAD, IR, FABMS, NMR – LC–ESI-MSn , direct- infusion ESI-MSn , IR, NMR HPLC-DAD, ESI-MS/MS, HRMS, IR, NMR HPLC-DAD, LC–ESI-MSn , HRMS, FTIR, NMR

[15] [16,17] [17] [18,19] [20]

6

Piperidenafil, piperidinovardenafil, pseudovardenafil Piperidino acetildenafil, piperidino hongdenafil Norneosildenafil, piperidino sildenafil Nor-acetildenafil, desmethylhongdenafil Methisosildenafil, Aildenafil Carbodenafil Benzylsildenafil Thioaildenafil, thio- methisosildenafil, dimethyl sildenafil thione, sulfodimethyl sildenafil KF31327, thioquinapiperifil

2006

Beverage Beverage Capsule Capsule Pre-mixed bulk powder Capsule

LC-DAD-MS, GC–MS, Direct infusion-MSn

[21]

2006 2006 2007 2007 2007 2007 2007

– – Capsules Capsules – – –

FTICRMS, LC–ESI-MSn – LC–DAD–MS, GC–MS, Direct infusion–MSn LC–DAD–MS, CID–MS, GC–MS, NMR – – HPLC–DAD, LC–ESI–MS, CID–MS, GC–MS, NMR

[22,23] [24] [25] [26] [24] [24] [27,28]

2008

HPLC–DAD, LC–MSn , LC–TOF–MS, FTIR, NMR

[29,30]

2008

Tablets, capsules Capsules

HPLC–DAD, LC–ESI–MS/MS, HR–MS, FTIR, NMR

[31]

2008

Capsules

HPLC–DAD, LC–ESI–MS/MS, HR–MS, FTIR, NMR

[31,32]

2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2009 2009 2009 2009 2010

Capsules – Capsules Capsules Functional food – – – – Capsules Capsules Capsules Tablets Tablets

HPLC–DAD, LC–ESI–MS/MS, HR–MS, FTIR, NMR HPLC–DAD, LC–ESI–MS/MS, NMR Preparative TLC, HPLC, LC–ESI–MS, FT-ICR-MS, NMR TLC, UV, LC–ESI–MS/MS, GC–MS, IR, NMR HPLC–DAD, LC–MS, NMR – – – – HPLC–DAD, LC–MS/MS, FTIR, NMR chiral HPLC–DAD, NMR, polarimetry HPLC–DAD, LC–ESI–MS/MS, HR–MS, FTIR, NMR HPLC–DAD,CD, LC–ESI–MS, HR–MS, IR, NMR HPLC–DAD, CD, LC–ESI–MS, HR–MS, IR, NMR – HPLC–DAD, LC–MS/MS, LC–TOF–MS, IR, NMR

[33,34] [35] [36] [37,38] [39] [24] [24] [24] [24] [40] [41] [42] [43] [43] [24] [44]

2010 2010 2010 2010 2011 2011 2011 2011 2011 2011 2011 2011 2012 2012

Capsules – – Tablets Tablets Capsules Capsules Capsules Not specified Capsules Capsules Capsules Capsules Functional coffee Premixed bulk powder Premixed bulk powder Powder Energy drink Bulk powder Capsules Bulk powder

HPLC–DAD, LC–ESI–MS, FTIR, NMR, elemental analysis – – LC–UV–CD, LC–UV–MSn , NIRS, NMR HPLC–DAD, LC– LTQ OrbiTrap XL FTMS/MS, IR, NMR HPLC–DAD, LC–ESI–MS/MS, NMR HPLC–DAD, LC–ESI–MS/MS, NMR LC–DAD–MS, LC–ESI–MS/MS, HRMS, FTIR, NMR UV, LC–MS, IR, NMR HPLC–DAD, LC–ESI–MS/MS, FTIR, NMR HPLC–DAD, LC–ESI–MSn using LC–LTQ orbitrap, NMR HPLC–DAD, LC–ESI–MSn using LC–LTQ orbitrap, NMR LC–TIS–MS, direct infusion MS/MS, FTIR, NMR UV, HPLC–DAD, LC–ESI–MS/MS, LC–TOF–MS, FTIR, NMR

[45] [24] [46] [47] [48] [49] [49] [50] [51] [52] [53] [53] [54] [14]

HPLC–DAD, LC– LTQ OrbiTrap XL FTMS/MS, FTIR–ATR, NMR

[55]

HPLC–DAD, LC– LTQ OrbiTrap XL FTMS/MS, FTIR–ATR, NMR

[55]

UPLC–DAD, direct infusion MS/MS, LC–TOF–MS, FTIR, NMR UV, LC–ESI–MS, FTIR, NMR, elemental analyses HPLC–DAD, LC–q–TOF-MS/MS, FTIR, CD, NMR HPLC–DAD–MS/MS, TOF–MS, FTIR, NMR HPLC–DAD, LC–LTQ OrbiTrap XL FTMS/MS

[56] [13] [57] [58] [59]

7 8 9 10 11 12 13

14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53

Thiosildenafil, thiodenafil, sulfosildenafil, sidenafil thione Thiohomosildenafil, sulfohomosildenafil, homosildenafil thione Benzamidenafil, xanthoanthrafil Imidazosagatriazinone, desulfovardenafil Chloro-pretadalafil Gendenafil Hydroxyvardenafil Norneovardenafil Oxohongdenafil Chlorodenafil Cinnamyldenafil Acetil acid SR-aminotadalafil [(+)-trans-aminotadalafil] Hydroxythiohomosildenafil Cyclopentynafil N-Octyl-nortadalafil Nitrodenafil Interaction product of aminotadalafil and 5-hydroxymethylfurfural Dimethylacetildenafil Hydroxychlordenafil N-desethylvardenafil (−)-Trans-tadalafil Dithio-desmethylcarbodenafil Piperazinonafil, dihydroacetildenafil Isopiperazinonafil Nitroso-prodenafil Mutaprodenafil Acetylvardenafil Hydroxypropylnortadalafil N-butylnortadalafil Depiperazino-thiosildenafil Descarbonsildenafil Propoxyphenyl aildenafil, propoxyphenyl methisosildenafil Propoxyphenyl thioaildenafil, propoxyphenyl thiomethisosildenafil Propoxyphenyl-thiohydroxyhomosildenafil Propoxyphenyl sildenafil Acetaminotadalafil Hydroxythiovardenafil Propoxyphenyl hydroxyhomosildenafil

2012 2012 2012 2013 2013 2013 2013

Candies

– Information not available.

sensitivity compared to TLC and quantitative determination is possible by densitometric analysis of spots [72,73]. Although TLC and HPTLC methods can be used for preliminary identification, these methods are not useful for the identification of analogues where reference standards are not available.

3.2. High performance liquid chromatography (HPLC) methods HPLC is one of the most important and most commonly employed methods in pharmaceutical analysis [74]. HPLC with ultraviolet detection, in particular HPLC-DAD (diode array detector) has been used extensively as a preliminary screening method

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5

Table 2 Chemical formulae, accurate mass, m/z of major ions and UV max of PDE-5 inhibitors and their synthetic analogues. m/z of major ions

UV max

References

231, 282, 289

[47]

218, 286 230, 260, 285

[57] [40]

234, 279

[17,22,25]

216, 250 222, 295

[52] [26]

225, 283

[18,19,22]

291 295 211, 235, 279

[60] [24,87] [24,87] [24]

285

[36]

239

[24]

218, 290 295, 354

[43] [54]

225, 295

[14]

233, 276 258, 285, 356 235, 273 291, 230, 292

[45] [5] [48] [38] [15,17,22,32,63]

230, 280

[20,22]

230, 292

[24] [9,17]

222, 284 225, 293, 353

[53] [42]

520.1926 504.2155 312.1586

390, 779 [2M+H]+ , 262, 250, 135 433, 455 [M+Na]+ 357, 329, 300, 285, 268, 256, 242, 166, 131 467, 449, 439, 420, 404, 396, 381, 355, 353, 339, 325, 324, 311, 297, 166, 127, 111, 99, 97 467, 409, 234, 88, 74 467, 449, 439, 420, 404, 396, 381, 355, 353, 339, 325, 324, 311, 297, 166, 127, 111, 99 391, 269, 262, 241, 239, 224, 197, 169 484 551, 377, 283 453, 283 EI+ (70 eV), 388 [M]+ , 360, 311, 291, 254, 183, 136, 68 427, 391, 349, 334, 287, 229, 159, 135 EI+ (70 eV), 554 [M]+ , 488, 354, 297, 283, 215, 166, 117, 91 529, 461, 153 409, 381, 351, 327, 299, 285, 272 463, 418, 377, 360, 311, 299, 283, 255, 151, 87 467, 279, 149, 177 471, 371, 343 355, 327, 285, 298, 313 489, 467, 461, 377, 313, 312, 283, 127, 111, 113, 99, 97, 72 483, 465, 439, 396, 353, 339, 325, 297 505, 487, 461, 423, 377, 311, 312, 283, 284, 225, 166, 129, 112, 99, 97 456 [M+Na], 521, 503, 461, 393, 373, 341, 327, 315, 299, 283, 271, 242 521 505 313, 284, 256, 169, 151

203, 235, 316 216 212, 253

[58] [39] [35]

498.1539

499, 377, 262, 250, 135

-

[44]

482.2642

−

221, 290

[49]

– 250 218, 240, 283, 297, 335 222, 284 – – 212, 298

[61] [87] [51] [53] [46] [46] [24]

241, 301 281 235, 280

[50] [43] [25]

291

[24,87]

215, 241

[24]

– 481

[5] [24]

No.

PDE-5 inhibitors and analogues

Chemical formulae

1

(−)-Trans-tadalafil

C22 H19 N3 O4

389.1376

2 3

Acetaminotadalafil Acetil acid

C23 H20 N4 O5 C18 H20 N4 O4

432.1434 356.1485

4

Acetildenafil

C25 H34 N6 O3

466.2692

5 6

Acetylvardenafil Aildenafil (Methisosildenafil)

C25 H34 N6 O3 C23 H32 N6 O4 S

466.2692 488.2206

7

Aminotadalafil

C21 H18 N4 O4

390.1328

8 9 10 11

Avanafil Benzylsildenafil Carbodenafil Chlorodenafil

C23 H26 N7 O3 Cl C28 H34 N6 O4 S C24 H32 N6 O3 C19 H21 ClN4 O3

483.1786 550.2362 452.2536 388.8485

12

Chloropretadalafil

C22 H19 ClN2 O5

426.0982

13

Cinnamyldenafil

C32 H38 N6 O3

554.3005

14 15

Cyclopentynafil Depiperazino-thiosildenafil

C26 H36 N6 O4 S C17 H20 N4 O4 S2

528.2519 408.0926

16

Descarbonsildenafil

C21 H30 N6 O4 S

462.2049

17 18 19 20 21

Dimethylacetildenafil Dioxo-acetildenafil Dithio-desmethyl-carbodenafil Gendenafil Homosildenafil

C25 H34 O3 N6 C25 H30 N6 O5 C23 H30 N6 OS2 C19 H22 N4 O3 C23 H32 N6 O4 S

466.2771 494.2278 470.6572 354.4039 488.2206

22

Hydroxyacetildenafil

C25 H34 N6 O4

482.2642

23 24

Hydroxychlorodenafil Hydroxyhomosildenafil

C19 H23 ClN4 O3 C23 H32 N6 O5 S

390.1459 504.2155

25 26

Hydroxypropylnortadalafil Hydroxythiohomosildenafil

C24 H23 N3 O5 C23 H32 N6 O4 S2

433.1638 520.6682

27 28 29

C23 H32 N6 O4 S2 C23 H32 N6 O5 S C17 H20 N4 O2 C27 H22 N4 O6

31

Hydroxythiovardenafil Hydroxyvardenafil Imidazosagatriazinone, Desulfovardenafil Interaction product of aminotadalafil and 5-hydroxymethylfurfural Isopiperazinonafil

32 33 34 35 36 37 38

Lodenafil carbonate Mirodenafil Muta-prodenafil N-butylnortadalafil N-desethylvardenafil N-desmethylsildenafil Nitrodenafil

C47 H62 N12 O11 S2 C26 H37 N5 O5 S C27 H35 N9 O5 S2 C25 H25 N3 O4 C21 H28 N6 O4 S C21 H38 N6 O4 S C17 H19 N5 O4

39 40 41

Nitroso-prodenafil N-octylnortadalafil Noracetildenafil

C27 H35 N9 O5 S2 C29 H33 N3 O4 C24 H32 N6 O3

629.2203 487.2471 452.2536

42

Norneosildenafil, piperidino sildenafil

C22 H29 N5 O4 S

459.1940

43

Norneovardenafil

C18 H20 N4 O4

356.1485

44 45

Nortadalafil Oxohongdenafil

C21 H17 N3 O4 C25 H32 N6 O4

375.1219 480.2485

30

C25 H34 N6 O4

Accurate mass

1034.4102 531.6698, 629.2203 431.1845 460.1893 470.2675 357.3647

481 [M−H] , 453, 422, 379, 336, 325, 311, 309 – 532 630 432 – – EI+ (70 eV), 358 [M]+ , 307, 289, 261, 217, 176, 154, 136, 107, 89, 77 652 (M+Na), 630 (M+H)+ , 142 488, 366, 227 453, 435, 425, 406, 396, 380, 367, 355, 353, 339, 325, 324, 313, 297 EI+ (70 eV), 459 [M]+ , 431, 387, 312, 283, 282, 254, 166, 136, 84, 55 EI+ (70 eV). 357[M]+ , 329, 307, 289, 176, 154, 136, 107, 99, 77, 39 – EI+ 481[M]+ , 451, 396, 354, 339, 312, 297, 289

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6 Table 2 (Continued) No.

PDE-5 inhibitors and analogues

Chemical formulae

Accurate mass

m/z of major ions

UV max

References

46

Piperazinonafil, dihydroacetildenafil

C25 H34 N6 O4

482.2642

221, 290

[49]

47 48

Piperidino acetildenafil piperidinovardenafil, piperidenafil,pesudovardenafil

C24 H31 N5 O3 C22 H29 N5 O4 S

437.2427 459.1940

276 230

[22,23] [21,22]

49

Propoxyphenyl aildenafil, propoxyphenyl methisosildenafil Prproxyphenyl hydroxyhomosildenafil

C24 H34 N6 O4 S

502.2362

481 [M−H]− , 453, 435, 348, 336, 321, 311, 309, 282, 267 438, 410, 353, 341, 325, 288 460, 432, 403, 391, 377, 349, 329, 312, 301, 299, 284, 270, 256, 169, 151 503, 252

215, 225, 295

[55]

C24 H34 N6 O4 S2

518.2311

225, 295, 354

[59]

Propoxyphenyl sildenafil Propoxyphenyl thioaildenafil, propoxyphenyl thiomethisosildenafil Propoxyphenyl thiohydroxyhomosildenafil Sildenafil

C23 H32 N6 O4 S C24 H34 N6 O3 S2

488.2205 518.2133

215, 292 227, 295, 355

[13] [55]

C24 H34 N6 O4 S2

534.2083

227, 295, 353

[56]

C22 H30 N6 O4 S

474.2049

230, 292

[17,21,22,32,62]

SR-aminotadalafil, (+)-trans-aminotadalafil Tadalafil

C21 H18 N4 O4

390.1328

225, 283

[41]

C22 H19 N3 O4

389.1376

230, 283

[22,32,62]

C23 H32 N6 O3 S2

504.1977

390, 302, 268, 262, 250, 240, 197, 169, 135 505

227, 248, 296, 355

[27,28,87]

C23 H32 N6 O3 S2

504.1977

505, 477, 421,393, 357, 355, 343, 327, 315, 299, 271, 113, 99

225, 293, 353

[31,32]

C24 H25 N6 OS C22 H30 N6 O3 S2

448.2045 490.1821

211, 268, 363 225, 295, 355

[29,30] [31,32]

C25 H36 N6 O4 S C23 H32 N6 O4 S

516.2519 488.2206

– 230

[87] [22,32,62]

C19 H23 N3 O6

389.1587

449, 363, 246, 225, 204, 121 491, 407, 393, 343, 341, 327, 315, 312, 299, 283, 271, 163, 99 517 489, 461, 420, 377, 376, 375, 346, 339, 329, 312, 299, 283, 169, 151,123, 99 390, 252, 223, 151

275, 377, 391

[33,34]

50 51 52 53 54

55 56 57

61 62

Thioaildenafil, thio- methisosildenafil, sulfodimethyl sildenafil Thiohomosildenafil, sulfohomosildenafil, homosildenafil thione Thioquinapiperifil, KF31327 Thiosildenafil, thiodenafil, sulfosildenafil, sidenafil thione Udenafil Vardenafil

63

Xanthoanthrafil, benzamidenafil

58

59 60

519, 501, 325, 299, 283, 129, 112, 99 489.2, 325, 283, 299 519, 260 535, 517, 359, 341, 315, 299, 271, 129, 112, 99 475, 447, 418, 391, 377, 374, 346, 329, 311, 297, 283, 255, 163, 160, 100 –

– Information not available.

for adulteration with synthetic PDE-5 inhibitors (Table 1). A library of UV spectra of known PDE-5 inhibitors analogues can be created with the use of reference standards [75,76]. The UV spectra, retention time and relative retention time matching with those of

reference PDE-5 inhibitors can be used to identify the adulterant [75,76]. A library match score of 1000 represents a perfect match [75,76]. In addition, it can also provide some structural information on related compounds. For example, hydroxyactildenafil, an

Scheme 1. Chemical structures of sildenafil and its analogues (a) with pyrazolo pyrimidine-7-one, ethoxyphenyl and sulfonamide moieties and (b) with pyrazolo pyrimidine7-one, propoxyphenyl and sulfonamide moieties.

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7

Scheme 2. Chemical structures of sildenafil analogues with pyrazolo pyrimidine-7-one and ethoxyphenyl moieties but without sulfonamide moiety.

analogue of acetildenafil, was suspected to be an adulterant by HPLC-DAD analysis of a pre-mixed bulk powder [20]. The UV spectrum of the unknown compound (hydroxyacetildenafil) matches that of acetildenafil but gives a different retention time [20]. Such observation can help to preliminarily detect the presence of structurally similar analogues such as hydroxyacetildenafil as adulterants. The most widely used stationary phase is octadecyl silane (C18 ), while commonly used mobile phase involved combination of acetonitrile and acidic aqueous phase, e.g. 0.1% formic acid, 0.011 M ammonium formate, 0.1% sodium-1-hexanesulfonic acid [9]. The advent of ultra high-pressure liquid chromatography (UHPLC) has significantly reduced the time required for analysis [74]. The UHPLC

uses core–shell packing of 1.7 mm internal diameter with 1.8 ␮m particle size to substantially improve the resolution, efficiency and speed of separation [74]. For example, Sacré et al. reported an UHPLC–UV method which was able to separate three licensed PDE5 inhibitors and five of their analogues in less than 4.5 min. [77]. However, the UHPLC is a more costly instrument compared to HPLC [77]. Although, HPLC–UV/DAD based methods can be used effectively in the screening of known adulterants, it has limited scope in the identification of new analogues with exotic structural modification. Nevertheless, it can be used as a preliminary screening tool to get a fair idea about the structural resemblance of potential unknown analogue with known PDE-5 inhibitors [5,9].

Scheme 3. Chemical structures of (a) sildenafil analogues with pyrazolo pyrimidine-7-thione, ethoxyphenyl/propoxyphenyl moieties with or without sulfonamide moieties and (b) other miscellaneous analogues.

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Scheme 4. Chemical structures of tadalafil and its analogues.

3.3. Mass spectrometry (MS) based hyphenated methods MS is currently the method of choice for structural elucidation because of its selectivity for different masses in a complex mixture of compounds [78–80]. Various types of commercially available mass spectrometers include single quadrupole, triple quadrupole, linear trap, time of flight (TOF) and Orbitrap (Fourier transform mass spectrometry, FTMS). MS alone has been used in the structural elucidation of new analogues of PDE-5 inhibitors such as piperidenafil, aildenafil and nor-acetildenafil. Direct infusion and collision-induced dissociation (CID) MS experiments provided useful information in structural elucidation of piperidenafil and nor-acetildenafil [21,25]. The advances in technology used in mass spectrometry such as the modern ionization techniques, emergence of high resolution mass spectrometry, tandem or multi-stage mass spectrometry (MS/MS or MSn ) have resulted in considerable

improvement in the accuracy and resolution of mass spectrometers [78–80]. The multi-stage or tandem mass studies where two or more MS experiments are combined often generate rich information regarding origin of the observed fragment ions of unknown structure. The comparisons between the characteristic fragmentation patterns of unknown analogues with those of known PDE-5 inhibitors provide good confidence on the predicted structure of the unknown analogue of an approved PDE-5 inhibitor. The evolution of time-of-flight (TOF) technology in high resolution mass spectrometers has enabled mass spectrometers to provide accurate mass up to 4–6 decimal places. This helps in deducing elemental composition and hence the molecular formula of a compound [18,31,42,78–80]. In other such developments, Fourier transform ion cyclotron resonance MS (FT-ICR-MS), was applied in the identification of sildenafil, hydroxyacetildenafil and piperidinoacetildenafil despite the analytes having similar product ions

Scheme 5. Chemical structures of vardenafil and its analogues (a) with imidazosagatriazinone and sulfonamide moieties, and (b) with imidazosagatriazine-thione and sulfonamide moieties and (c) other related compounds.

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[22]. The high resolving power and mass accuracy of FT–ICR–MS provides unambiguous assignments of elemental composition. In addition, mass spectrometric techniques such as the atmospheric solid analysis probe (ASAP) mass spectrometry and desorption corona beam ionization (DCBI) mass spectrometry offer an advantage of direct analysis of samples without a need for sample preparation [81–85]. These techniques were used for the rapid detection of PDE-5 inhibitors as adulterants. In ASAP, the sample is vaporized by exposing it to the hot nitrogen gas from an ASAP probe and then analyzed by using an atmospheric ionization source present in LC–MS instruments [81,82]. In DCBI, the sample is desorbed using a visible heated helium beam in the atmospheric pressure region of the ion source and it is ionized by the energetic particles embedded in the gas stream [83,84]. However, these techniques have limited scope in quantitative and trace analyte determinations [81–85]. Furthermore, combination of MS with different chromatographic methods has provided some of the most powerful techniques available for pharmaceutical analysis. Hyphenated techniques such as liquid chromatography–mass spectrometry (LC–MS), gas chromatography–mass spectrometry (GC–MS), capillary electrophoresis–mass spectrometry (CE–MS) etc. are preferred choices for the structural elucidation of unknown compounds [78–80]. The following sections give an overview of the application of these MS based hyphenated techniques in detecting and identifying adulterants. 3.3.1. LC–MS LC–MS based methods are the most popular among all the hyphenated techniques for characterization of adulterants in pharmaceutical products. The ability of LC–MS based methods to render near unequivocal structural information on their own makes them indispensible in the structural elucidation of unknown analogues [5,9,78]. LC–MS gives higher sensitivity and is capable of easily and selectively separate target molecules in a complex matrix without an extensive sample preparation procedure [5,9,78]. LC–MS also offers an advantage of combining an online DAD detector which empowers the acquisition of both UV and MS data in a single run [50]. Modern atmospheric ionization techniques, such as electrospray ionization (ESI) where solvent elimination and ionization steps are combined in the source and take place at atmospheric pressure allows easy and direct transfer of most of the HPLC methods to LC–MS [9]. LC–MS and its variants, including LC–MS/MS, LC–MS-TOF, LC–LTQ Orbitrap XL FTMS, LC–FT–ICR–MS are predominantly the principal techniques used in characterization of unknown analogues. In addition, various MS experiments such as selected ion monitoring (SIM), product ion scanning (PIS), constant neutral loss scanning (CNLS), precursor ion scanning (PREC), selected reaction monitoring (SRM) or multiple reactions monitoring (MRM) can be performed in LC–MS for the detection and identification of adulterants in multi-component herbal dietary supplements. For example, MS2 fragmentation patterns were used in identification of unlicensed analogues such as homosildenafil, acetildenafil, hydroxyhomosildenafil and licensed PDE-5 inhibitors, sildenafil and vardenafil in capsules and premixed bulk powder [76]. Similarly, two new unlicensed analogues of tadalafil, 2hydroxypropylnortadalafil and butylnortadalafil were identified by MS3 experiments [53]. The potential use of HPLC-DCBI-MS/MS has also been explored for rapid determination of sildenafil, tadalafil and vardenafil in adulterated products [85]. UHPLC in combination with TOF/MS has also been applied for the rapid detection of PDE-5 inhibitors and their analogues as adulterants [86]. Furthermore, the combination of different scanning modes can be used to maximize the structural information obtained from LC–MS such as the information dependent acquisition (IDA) [87]. IDA is an artificial intelligence system where two or more scanning

9

modes can be used in sequential order to obtain MS and MS/MS data simultaneously [87]. The IDA first uses a survey scan, such as an MRM or precursor ion, to generate “information” based on predefined ion selection criteria and a second scan, such as a product ion scan, is then started using information obtained in the survey scan [87]. In addition to identification of known compounds, IDA can also predict the structure of unknown analogues [87]. Such IDA method combining a MRM scan and a precursor ion scan with triple quadruple linear ion trap (TQ-LIT) was developed for 22 PDE5 inhibitors [87]. Characteristic marker fragment ions generated by MS/MS experiments are very useful in identifying unknown analogues. Sildenafil analogues can be identified by characteristic fragment ions with m/z 377, 311 and 283. Analogues of acetildenafil (m/z 449, 396, 353), thiosildenafil (m/z 299), tadalafil (m/z 268, 262, 250) and vardenafil (m/z 461, 377, 299) can also be similarly identified by their characteristic fragment ions [5,9]. However, it is important to note that the use of different collision gases (He, Ar, N2 ) may give rise to different marker ions [5,37]. 3.3.2. GC–MS GC–MS was the first hyphenated technique introduced in 1960s. Volatility and thermal stability are the two main criteria for any compound to be successfully analyzed by GC–MS. Only sporadic reports can be found in literature for its use in the analysis of PDE5 inhibitors despite its low cost compared to LC–MS [88–91]. This could be attributed to the thermal instability of PDE-5 inhibitors and their analogues and difficulty in derivatising them using standard silylation reagents [88]. Heretofore, the use of GC–MS is only limited to three approved PDE-5 inhibitors, sildenafil, vardenafil and tadalafil [88–91]. Nevertheless, in situations where LC–MS analysis alone is not able to define the structure of an analogue or its fragment, GC–MS analysis after derivatization and/or chemical reactions such as hydrolysis can be used to characterize the structure. For example, acid hydrolysis followed by GC–MS analysis was employed to elucidate structures of piperidinafil [21], noracetildenafil [25], aildenafil [26], thioaildenafil [28] and desulfovardenafil [35]. The cleavage of the sulfonamide bond yields sulfonic acid and amine. GC–MS analysis of the hydrolysis products provides useful information for structural elucidation. Hence GC–MS can be employed as a complementary tool along with LC–MS for identification of new analogues. 3.4. Vibrational spectroscopy Vibrational spectroscopic methods such as infrared (IR), near infrared (NIR) or Raman spectroscopy or their combinations have been used for screening for known analogues [5] and in identification of counterfeit pharmaceutical products [92–94]. Vibrational spectroscopy is based on the vibrations or rotations of organic molecules at specific frequencies, corresponding to distinct vibrational energy levels. The changes in the vibrational energy levels of a molecule are induced by the absorption of IR radiation in IR spectroscopy and by the scattering of light in the Raman spectroscopy. IR and Raman spectroscopy are complementary to each other and are often used in the identification of various functional groups in an unknown structure [92–94]. However, Raman spectroscopy is preferred over IR for aqueous samples such as biological samples because water is a weak Raman scatterer. Compared with IR, NIR has a higher precision and an easier sample preparation. These techniques when used in conjunction with chemometrics, generates chromatographic fingerprints which help to differentiate between authentic and fake or counterfeit samples [94]. Such approach using several explorative (e.g. principal component analysis (PCA), discriminant partial least squares (D-PLS), projection pursuit (PP) and hierarchical clustering) as well as classification methods (e.g. classification and regression trees (CART), soft

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independent modelling by class analogy (SIMCA) and support vector machines (SVM)), had been applied to PDE-5 inhibitors [94]. These techniques are often fast and require less samples and minimal sample preparation. Hence, they are preferred over chromatography for the identification of counterfeit products [94]. For the identification of known analogues, a substantial library of reference materials (analogues and excipients) is required. Inhouse FTIR library with attenuated total reflectance (ATR) sampling technique has been used in characterization of propoxyphenyl thioaildenafil [55]. In ATR, sample extraction is not usually required and herbal dietary supplement or food can be analyzed after removing water to reduce the interference from water. In addition, these techniques could also provide some important information about salt forms, polymorphs and excipients [5]. 3.5. Nuclear magnetic resonance (NMR) spectroscopy NMR spectroscopy is one of the most powerful and indispensible tool to unambiguously elucidate the structure of known and novel compounds. Compared to other analytical techniques such as LC–MS and GC–MS, NMR has the advantage of simpler sample preparation and high reproducibility [95–97]. However, NMR is less sensitive than LC–MS and GC–MS and often requires a larger quantity of sample [95–97]. Nevertheless, the technological advances in the field of magnetic resonance have dramatically improved the sensitivity by developing new NMR experiments and data processing tools [95–97]. 1 H NMR, 13 C NMR, 1D (e.g. DEPT, NOE), 2D homonuclear (e.g. COSY, NOESY) and heteronuclear 2D (e.g. HMQC, HMBC) experiments are the most frequently used NMR experiments for structural elucidation. Technological advances have also enabled NMR to overcome its requirement for pure and isolated compounds. For example, the diffusion ordered spectroscopy (DOSY) allows the measurement of translation selfdiffusion of molecules in solution [95–97]. Depending on mono and multi-exponential decays, NMR spectra of components of mixture can be separated from each other based on their diffusion coefficients [95–97]. Higher molecular weight compounds have lower diffusion coefficient. 2D DOSY 1 H NMR spectra offer distinct fingerprints for pharmaceutical preparations [95–97]. These fingerprints include active ingredients as well as excipients which makes DOSY NMR a global method allowing the analysis of the pharmaceutical preparation as a whole. It hence gives a “global signature” of a product [95–97]. Such method was developed to differentiate genuine and fake formulations of tadalafil [95]. Further extension of this method in the form of 2D DOSY and 3D DOSY-COSY 1 H NMR was also used to distinguish authentic and fake formulations of sildenafil [96,97]. In addition to the structural elucidation, NMR can also be used for the quantification of adulterants. 3.6. X-ray diffraction (XRD) spectrometry XRD has been explored to detect fake and counterfeit products of licensed PDE-5 inhibitors [98,99]. The advances in X-ray optics in X-ray sources and radiation detection such as multilayer mirrors and position sensitive counters have made XRD much easier and faster to use [98]. Maurin et al. described its use in differentiating fake Viagra tablets from originals [98]. XRD was found to be very fast and reliable method for detecting counterfeits and imitation [98]. Novel X-ray fluorescence (XRF) allied to chemometrics method was developed for the inorganic fingerprinting of 41 commercial samples containing sildenafil citrate or tadalafil and 56 of their counterfeit products [99]. XRF technique is based on the presence of metals. XRF was found to be an excellent analytical methodology for identifying counterfeits by semi-quantitative determination of active ingredient (e.g. sildenafil citrate that has sulphur in its structure) and excipients such as calcium phosphate,

titanium oxide and iron oxide, detecting phosphorus, calcium, titanium and iron respectively [99]. 3.7. Other methods In addition to the analytical techniques discussed, techniques such as the ion mobility spectroscopy (IMS), immunoassay based methods have also been reported for characterization PDE-5 inhibitors and their analogues. IMS is a high throughput analytical technique used to separate and identify chemical substances based upon their mobilities in a carrier buffer gas [100,101]. The ion mobilities can be used for qualitative purposes. The ion mobility depends on the size, shape, and charge of a molecular ion. IMS is popularly used at the ports of entry for security purposes to detect any explosives and drugs of abuse [102]. In pharmaceutical sciences, IMS is used for quality assurance, process monitoring, cleaning verification, direct analysis of formulated products etc. [101–103]. Due to its high speed, sensitivity, selectivity and low cost, it can be used as a screening tool to detect adulteration. Such IMS method has been reported to correctly identify PDE-5 inhibitors as adulterants and adulterated products [101,102]. Although, no adulterant was misindentified, it could not differentiate structurally similar analogues, namely, homosildenafil and methisosildenafil [101]. Nevertheless, such a method has a great potential to become a rapid pass/fail screening tool for monitoring the adulteration with PDE-5 inhibitors [100–102]. Furthermore, immunoassay methods have also been investigated in the quest for developing rapid screening tools to detect PDE-5 inhibitors as adulterants [104,105]. Multi-component immunoassays have become the centre of attraction for simultaneous determination of small molecules [104]. Such assays are based on indirect ELISA using monoclonal antibodies specific to the common structure of certain classes of small molecules. An immunoassay was developed for the screening of vardenafil and two of its analogues, piperidenafil and desulfovardenafil by preparing group specific monoclonal antibody [104]. The method was found to be rapid, sensitive and specific to vardenafil and related analogues with minimal cross reactivity with sildenafil and tadalafil [104]. Similarly, an enzyme-linked immunosorbent assay was developed for rapid determination of sildenafil in functional foods [105]. Such methods could be developed as cost-effective tools for preliminary screening for adulterants. 4. Challenges Various challenges in detecting and identifying PDE-5 inhibitors and their analogues as adulterants are listed in List 2. The preponderance of adulteration with PDE-5 inhibitors and their unlicensed analogues is of great concern for drug regulatory authorities. The ever increasing demand for effective natural sexual performance enhancement health supplements makes it difficult to curb the supply chain. The enchanting label claims, low price, ready availability through discreet alternatives such as the internet and apparent efficacy (often due to adulteration) are the main reasons for the high demand of such products despite reports of severe and sometimes fatal cases of adverse events [6]. In many cases, the manufacturers indicated on product labels do not exist [6]. This presents additional challenge for drug-enforcement agencies in taking punitive actions against perpetrators of such adulteration. The dishonest manufacturers are now embarking into functional food such as coffee to expand their profitable business [14,106]. For example, a coffee sample submitted to the HSA, Singapore, was found to be adulterated with a sildenafil analogue, descarbonsildenafil

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[14]. The United States Food and Drug Administration (USFDA) also reported adulteration of instant coffee sample, marketed as a dietary supplement for sexual performance enhancement with hydroxythiohomosildenafil [106]. In addition, adulterated products to increase the libido of women have also been reported [6,107]. Flibanserin was detected as an adulterant in a health supplement used for female sexual performance enhancement [6,107]. Flibanserin is a serotonin-1A (5-HT (1A)) receptor agonist and the serotonin-2A (5-HT (2A)) receptor antagonist. It was developed as a novel non-hormonal treatment for hypoactive sexual desire disorder. However, its New Drug Application (NDA) was rejected by the USFDA advisory committee for reproductive health because of insufficient evidence of its efficacy [107,108]. Herbal dietary supplements and health supplements are not drugs and hence are not tightly regulated in most countries [5]. The unscrupulous manufacturers are intelligibly investing time and resources to find novel means to elude the standard testing protocols for quality checking. An example is the adulteration of gelatin capsule shells with tadalafil [109,110]. The presence of tadalafil throughout the gelatin matrix was determined by microscopy and Raman spectroscopy [110]. The adulterants might be concealed in unusual and complex matrices. Therefore, besides routine screening protocols, proactive and criminological investigative approach may be useful for the analysis of suspected products. Although significant advances have been made in the analytical technologies and methods for screening for adulteration with PDE-5 inhibitors and their analogues, the ideal screening method is still not available. The ideal screening method should be one that is rapid, specific, sensitive, accurate and have lower cost in simultaneous determination of various PDE-5 inhibitors and their analogues with minimal sample preparation. Different analytical techniques have their own advantages and limitations. Hence, one must be mindful of the need to use a combination of techniques (at least two) to detect and elucidate the structure of potential adulterant. Successful detection is also dependent on sample preparation and instrument parameters [5,9]. For example, tadalafil is poorly soluble in water and most of the organic solvent, except for chloroform in which it is sparingly soluble [67]. Therefore, selection of appropriate extraction solvent is essential. Currently reported methods could not simultaneously determine more than 22 PDE-5 inhibitors [87]. In addition, the lack of reference standards for most of the unapproved analogues remains an unwieldy challenge. Development of an international or national database of currently known and other possible analogues of PDE-5 inhibitors containing their chemical structures and data from various analytical techniques is helpful in the absence of reference standards. Stricter regulatory control and pharmacovigilance of products such as herbal dietary supplements and food supplements could help in combating the adulteration of such products with unknown analogues. However, less than expected reports of adverse events associated with the use of such products denigrate the cause for taking urgent regulatory actions [5,11,111,112]. The under-reporting of adverse events due to adulterated products could be attributed to the unwillingness of patients to disclose the use of such illegal products and hence the healthcare professionals could not discern the signals of harm [11,111,112]. Therefore, reporting of adverse events associated with the use of herbal products and health supplements should be encouraged even if the causality is not confirmed because any signs of clustering will allow rapid regulatory actions to be taken [11]. From another perspective, millions of people may be consuming illicit ED drugs but reports of their harm are usually scanty except during outbreaks. In a previous report, only 3.8% (627 cases) of total adverse events reported (16,696) during the period 1998–2009 were associated with the use of complementary medicine products and health supplements [11]. Of these, only 1.7%

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(291 cases) was associated with the use of illegal sexual performance enhancement products [11]. The Traditional Herbal Medicinal Products Directive (THMPD) enforced by the European Union (EU) in 2011 has been claimed to be the most stern regulatory control measure in the world [113,114]. Under the THMPD, it is illegal to sell unlicensed herbal products without prior registration, e.g. Marketing Authorization or a Traditional Herbal Registration [113]. This directive also enforces uniform criteria across the member states to classify whether a product is a food supplement or a medicine [114]. The products are not allowed to make label claims without providing evidence of its use for specified condition for at least 30 years, including minimum of 15 years in Europe [113,114]. Although, such evidence does not require clinical trial, it must provide acceptable source of documented longstanding use [113,114]. In addition, herbal products must be of acceptable quality, contain correct ingredients, free from contamination and indicate the correct shelf life. THMPD does not apply to the traditional medicine practitioners who are still allowed to dispense their remedies. Directives such as THMPD could help in regulating health products sold over the counter in health shops, pharmacies and in supermarket but they might not have the same effectiveness over the products sold illegally outside the official distribution system via e.g. the internet, street peddlers and makeshift stalls. Moreover, the implementation and harmonisation of THMPD in EU is complex because herbs and their preparations are also used as food supplements, dietary food or as functional food [115,116]. The classification of the herbal product as a medicinal product or food supplement may be ambiguous and largely depends on the interpretation and application of regulation by competent national authority or the manufacturer, rather than its intrinsic medicinal properties [115–117]. Since the food supplements are not strictly regulated, the perpetrators of adulterated products can circumvent official registration procedure required under THMPD by presenting and marketing their products as food supplements [115,116]. This has resulted in non-uniform regulations of food supplements within member states of EU. This allows legal marketing of a large number of food supplements, but unfortunately some were subsequently found to be adulterated. Unlike herbal medicinal products, food supplements do not require premarketing registration and hence they may be adulterated with PDE-5 inhibitors and their analogues. In Table 1, at least six of the analogues were detected in food products [13–17,39,44]. Therefore, there is a need to have greater quality control of food supplements. In addition, a balanced and harmonised regulatory framework is also required to eliminate any ambiguity between food and herbal medicinal products [115,116]. Collaborative efforts such as increased surveillance and raids by regulatory authorities, police, customs, postal and courier services (to track suspected mails) and immigration check point authorities are required to curb the supply chain of illegal products. For example, enhanced surveillance involving seven major raids over a two month-period in red-light district of Singapore led to a haul of 175 illegal sexual performance enhancement products by HSA, of which 123 were found to be adulterated with sildenafil [6]. However, such intensive surveillance may not be feasible all the time. Moreover, the stigma associated with erectile dysfunction and cheaper cost of adulterated products might encourage unscrupulous suppliers and unwitting consumers to find loopholes in regulatory and enforcement activities. Therefore, greater public awareness of risk associated with the consumption of such adulterated products is important to complement regulatory and enforcement actions. An effective public education programme targeted at high risk regions and people at-risk could help to curtail the high demand of adulterated products. Individual regulatory authorities may not have the access to some of the more advanced analytical instruments. However, with greater information sharing

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and readily accessible information in the internet, the race against time to detect adulterants before they can cause harm can be won.

5. Conclusion Extensive and steady adulteration of herbal dietary supplements and health products with new designer analogues of prescription PDE-5 inhibitors has become a major area of concern for regulatory authorities. There are now more than 50 unapproved analogues of PDE-5 inhibitors reported as adulterants and the number is still growing. The information presented in this paper could be tapped to rapidly identify such analogues. The persistent demand of such products encourages shoddy suppliers to take risk and invest resources to find new analogues and new means to avoid the detection of adulteration. Nevertheless, continuous efforts to screen health products for adulterants via developing novel methods as well as structural elucidation of previously unknown analogues remain important. Collaboration between regulators, healthcare professionals, academics and the relevant industry, as well as education of healthcare professionals and the general public will go a long way in sussing out adulterated products, identifying adulterant and hence safe guarding the interest of the general public. List 1. List of various analytical techniques used to detect and identify unknown adulterants. Analytical techniques Atmospheric solid analysis probe mass spectrometry (ASAP-MS) Collision-induced dissociation-mass spectrometry (CID-MS) Direct infusion-mass spectrometry (MS) Direct infusion-tandem mass spectrometry (MS/MS) Direct infusion-MSn (multistage mass spectrometry) Fourier ion transform infrared (FTIR) spectroscopy Fourier ion transform infrared attenuated total reflectance (FTIR-ATR) spectroscopy Gas chromatography-mass spectrometry (GC–MS) High performance liquid chromatography-desorption corona beam ionization-tandem mass spectrometry (HPLC-DCBI-MS/MS) High performance liquid chromatography with UV or diode array detector (HPLC–UV/DAD) High performance thin layer chromatography (HPTLC) High resolution mass spectrometry (HRMS) Immunoassay Ion mobility spectrometry (IMS) Liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry (LC–FT-ICR-MS) Liquid chromatography-linear ion orbitrap XL Fourier transform mass spectrometry (LC–LTQ Orbitrap XL FTMS) Liquid chromatography-mass spectrometry (LC–MS) Liquid chromatography-multi-stage mass spectrometry (LC–MSn ) Liquid chromatography-tandem mass spectrometry (LC–MS/MS) Liquid chromatography-quadrupole-time-of-flight-tandem mass spectrometry (LC–q-TOF-MS/MS) Liquid chromatography-turbo ion spray mass spectrometry (LC–TIS-MS) Liquid chromatography-time of flight-mass spectrometry (LC–TOF/MS) Liquid chromatograph-hybrid triple quadrupole linear ion trap mass spectrometer with information dependent acquisition Near infrared (NIR) spectroscopy Nuclear magnetic resonance (NMR) spectroscopy Optical polarimetry Raman spectroscopy Thin layer chromatography (TLC) Ultra-high performance liquid chromatography with diode array detector (UHPLC-DAD) Ultra-high performance liquid chromatography-time of flight-mass spectrometry (UHPLC–TOF/MS) Ultraviolet-visible (UV) spectroscopy X-ray diffraction (XRD) spectrometry X-ray fluorescence (XRF) spectrometry

List 2. List of challenges to detect and identify adulteration with PDE-5 inhibitors and their analogues and to curb adulteration.

Challenges Increasing demand for natural sexual performance enhancement products Readily available starting material for the synthesis of sildenafil analogues Structural diversity and unlimited possibilities of analogues Absence of reference standards Non-uniform naming of analogues Novel routes of adulteration e.g. capsule shell Difficulty in classifying product as a herbal medicinal product or as a food supplement Adulteration of functional food, e.g. coffee Detection dependent on sample preparation and instrumental parameters Ideal screening method (low cost, rapid, sensitive, specific, accurate and requires minimal sample preparation) is still not available Lack of suitable or advanced analytical instruments in some laboratories Under reporting of adverse events Need for public education and increase awareness

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