Analysis of Herbal Dietary Supplements for Sexual Performance Enhancement

Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 135–150

Contents lists available at SciVerse ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

jou rnal homepage: www.elsevier.com/locate/jpba

Analysis of herbal dietary supplements for sexual performance enhancement:

First characterization of propoxyphenyl-thiohydroxyhomosildenaﬁl and

identification of sildenafil, thiosildenafil, phentolamine and tetrahydropalmatine

as adulterants

∗

Stéphane Balayssac, Véronique Gilard, Chantal Zedde, Robert Martino, Myriam Malet-Martino

Groupe de RMN Biomédicale, Laboratoire SPCMIB (UMR CNRS 5068), Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse cedex, France

a r t i c l e i n f o a b s t r a c t

Article history: Nine herbal dietary supplements intended to be beverages for enhancing sexual performance were ana-

Received 8 December 2011

lyzed before their possible launch on the market. Four of them contained a sildenaﬁl analog reported for

Received in revised form 24 January 2012

the ﬁrst time as an adulterant. After isolation and characterization using NMR, MS, IR and UV, this analog

Accepted 26 January 2012

was named propoxyphenyl-thiohydroxyhomosildenaﬁl as the ethoxy chain on the phenyl ring of the

Available online 4 February 2012

already known analog thiohydroxyhomosildenaﬁl was replaced by a propoxy moiety. One formulation

was tainted with thiosildenaﬁl, another unapproved PDE-5 inhibitor. Sildenaﬁl along with the natural

Keywords:

alkaloid tetrahydropalmatine that has no documented effect for enhancing erectile dysfunction were

Dietary supplements

Adulterants identiﬁed in two formulations. Another formulation was adulterated with phentolamine, a drug that is

Propoxyphenyl-thiohydroxyhomosildenaﬁl not approved for boosting male sexual performance when taken orally. The last formulation containing

NMR osthole, a bioactive natural coumarine improving sexual dysfunction, is most probably truly natural.

1. Introduction herbal drugs and dietary supplements [2,3]. Besides PDE-5 inhibitor

analogs, the most common adulterants, many other pharmacologi-

Dietary supplements are products between medicines and con- cally active substances have been used to taint dietary supplements

ventional foods whose consumption is rising steeply. The control and herbal products consumed for enhancing sexual performance

of their quality is therefore of paramount importance in order to [4].

ensure their safety and to protect consumers. There is a grow- In this study, nine dietary supplements intended to be intro-

ing trend in the intentional adulteration of dietary supplements duced into the South European market were submitted for analysis.

with drugs, which represents an alarming emerging risk to pub- Their exact composition was unknown but they were claimed to

lic health. Some recent articles have highlighted this problem and contain only a mixture of various herbs and sugars. They were

demonstrated the complexity to combat it (see for example [1]). presented in unlabeled plastic bags as brown powders (10 g per

Adulterants are frequently detected in dietary supplements or bag) to be dissolved in water for beverages and to be taken once

herbal medicines aimed at increasing sexual function. In addition a day. Four were adulterated with a new sildenaﬁl analog called

to the approved phosphodiesterase-5 (PDE-5) inhibitors, sildenaﬁl propoxyphenyl-thiohydroxyhomosildenaﬁl (PP-THHS), one with

® ® ®

(Viagra ), tadalafil (Cialis ) and vardenafil (Levitra ) in Europe thiosildenafil (THIO), two with sildenafil (SILD) along with tetrahy-

and USA, udenaﬁl (Zydena ) in South Korea and Malaysia, mirode- dropalmatine (THP), one with phentolamine (PHE) and the last

® ®

naﬁl (Mvix ) in South Korea, and lodenaﬁl carbonate (Helleva ) in contained osthole (OST). The chemical structures of these com-

Brazil, it has been reported that “natural” herbal products were also pounds were elucidated using NMR and MS.

adulterated with unapproved analogs in which most often minor

modiﬁcations were brought to the parent structure and for which

no toxicological data are available. To our knowledge, 33 analogs

have been described so far in the literature as illegal additives in 2. Experimental

2.1. Chemicals

∗

Authentic standards of SILD citrate, OST and PHE mesylate

Corresponding author. Tel.: +33 5 61 55 68 90; fax: +33 5 61 55 76 25.

E-mail address: [email protected] (M. Malet-Martino). (methanesulfonate) were purchased from Sigma–Aldrich (St. Louis,

136 S. Balayssac et al. / Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 135–150

MO, USA). THIO was previously puriﬁed from a formulation adul- gradient of 20 to 50% acetonitrile from 0 to 20 min with 0.1% triﬂuo-

terated with this analog [2]. roacetic acid in both aqueous and organic phases. The ﬂow rate was

−1

15 mL min . The eluted fractions were collected and analyzed with

2.2. NMR analysis a Waters Acquity UPLC-DAD system controlled by Waters Empower

2 software. The analysis conditions were: Acquity UPLC BEH C18

2.2.1. Preparation of samples for NMR analysis column (50 mm × 2.1 mm i.d.; 1.7 ␮m particle size); mobile phase:

Around 500 mg of each powder was mixed with 2 mL of (A) demineralized water and (B) acetonitrile (HPLC grade) both

−1

CD3CN:D2O (80:20 v/v) under vortex agitation during 10 min and containing 0.1% (v/v) triﬂuoroacetic acid; ﬂow rate: 0.3 mL min ;

then sonication for 10 min. The suspension was then centrifuged detection wavelength: 270 nm. The gradient condition was as fol-

(10 min, 4000 rpm) and 550 ␮L of the supernatant was analyzed. lows: 0–1 min, isocratic elution with a 90:10 A:B mixture; 1–6 min,

One of the referee of this article underlined that tadalaﬁl which is linear increase to 10:90 A:B ratio; isocratic elution during 1 min; re-

active at low doses (2 mg/day) could have not been extracted in the equilibration for 1 min with a 90:10 A:B mixture before the start of

solvent system used owing to its poor solubility in most organic the next run. The unknown compound eluted at 4.5 min.

solvents and water. To demonstrate that the method was valid for For the puriﬁcation of THP and OST, 500 mg of each formula-

detecting low-dosed tadalaﬁl, 500 mg of powder 9 was spiked with tion 6 and 9 was dissolved in 20 mL of water and the solutions left

◦

2 or 5 mg of pure tadalaﬁl and treated as described above. In both overnight at 4 C. The precipitates were ﬁltered, washed three times

cases, the H NMR signals of tadalaﬁl were easily detected in the with water and evaporated to complete dryness.

CD3CN:D2O extracts, thus showing that the method of extraction

is appropriate even for low quantities of this PDE-5 inhibitor.

2.4. MS, MS/MS and high resolution MS analyses

For the quantitative analysis, around 40 mg of each powder

was mixed with 10 mL of methanol under magnetic stirring dur-

The methanolic extracts of powders and the puriﬁed com-

ing 15 min, then sonicated for 10 min. After centrifugation (5 min,

pounds dissolved in methanol were directly infused in an Applied

4000 rpm), an aliquot of 1 mL was evaporated to dryness and the

Biosystems API 365 triple-quadrupole mass spectrometer (Applied

residue dissolved in 1 mL of methanol-d4. The experiments were

Biosystems Inc., Foster City, CA, USA), equipped with a TIS inter-

done in triplicate. The quantiﬁcation was performed on the H

face and controlled by the Analyst software (version 1.4). The mass

NMR signals of the aromatic protons H15 and H18 (doublets) for

spectrometer was operated in positive ionization mode. Nitrogen

SILD, THIO and PP-THHS, H1 and H4 (singlets) for THP, H9/H13 and

served both as auxiliary and collision gas and oxygen served as

H10/H12 (respectively XX and AA parts of an AA XX system) for

nebulizer gas. The operating conditions for TIS interface were as

PHEN and H4 and H5 (doublets) for OST (see Fig. 1 for numbering).

follows: (1) in MS mode: scan range m/z 100–1000, step size 0.1.

The solid residue from the ﬁrst extraction was re-extracted with

Q1 TIS MS spectra were recorded in proﬁle mode, IS 4700 V, DP

the same protocol. The mixture obtained was centrifuged (5 min,

65 V, FP 330 V for the new analog and IS 5000 V, DP 25 V, FP 200 V

4000 rpm) and the supernatant evaporated to dryness. The residue

for PHE and OST; and (2) in MS–MS mode: precursor ions at m/z

was dissolved in 1 mL of methanol-d4 for H NMR analysis. The

535 for the unknown analog, 282 for PHE and 245 for OST; scan

proton signals used for quantiﬁcation could not be detected.

range m/z 30–600 for the unknown analog and 50–300 for PHE and

OST, step size 0.1; MS–MS spectra were recorded in proﬁle mode,

2.2.2. NMR recording conditions

IS 5000 V, DP 65 V, FP 330 V, CE 50 V, CAD 3 for the new analog, and

The NMR experiments were performed on a Bruker Avance

IS 5000 V, DP 25 V, FP 200 V, CE 25 V, CAD 3 for PHE and OST.

500 spectrometer (Bruker BioSpin AG, Fällanden, Switzerland)

The accurate masses of the new analog, PHE and OST were

1 13

equipped with a 5 mm dual H– C TCI cryoprobe. Sodium 2,2,3,3-

determined on a Waters GCT Premier time-of-ﬂight (TOF) mass

tetradeutero-3-trimethylsilylpropionate (TSP; Sigma–Aldrich) was

spectrometer equipped with a Desorption Chemical Ionization

used as an internal reference for chemical shift (ı) measurement

(DCI) probe employing methane as the reagent gas and controlled

and quantiﬁcation. A solution of TSP (10 ␮L) was added before the

by the MassLynx 4.1 software. The TOF-MS was operated between

NMR analysis at a ﬁnal concentration of 0.2 mM in all the sam-

m/z 150 and 850 in positive ionization mode. The accurate mass

ples analyzed. The recording and processing conditions for H NMR,

measurements of THP and its product ions were acquired using a

1 1

quantitative H NMR and 2D DOSY H NMR spectra have already

Waters XEVO G2 QTOF mass spectrometer. The instrument param-

been described [5,6].

eters were as follows: positive ionization mode; for MS analysis:

The identiﬁcation of SILD and THIO was achieved by spiking

cone voltage 30 V, scan range m/z 100–1200; for MS/MS analy-

the formulation extracts with authentic standards and comparing

sis: coil energy 35 V, cone voltage 40 V, scan range m/z 100–440.

the peak heights in the H NMR spectra recorded before and after

The samples were dissolved in methanol and analyzed after direct

addition. The structural elucidation of PP-THHS, THP and OST was infusion.

performed on isolated puriﬁed compounds, and that of PHEN on

1 13

crude extract, using 1D ( H and C) and 2D (gCOSY, gHSQC and

2.5. IR spectroscopy

gHMBC) experiments. To conﬁrm the structures determined, stan-

dard OST and PHE were also added to solutions of puriﬁed OST or

The FT-IR spectrum of the new SILD analog was recorded on a

to crude extracts containing PHE.

Nicolet 6700 total reﬂectance device (Thermo Nicolet Corporation,

Madison, WI, USA) using a diamond crystal at an incidence angle of

2.3. Isolation of the new SILD analog, THP and OST

◦ −1

45 over the spectral range 4000–500 cm .

For the puriﬁcation of the new SILD analog, 3 × 1 g of powder

was extracted with 3 10 mL of methanol. The liquid phases were 3. Results and discussion

pooled, evaporated to dryness and then redissolved in 4 mL of a

mixture of acetonitrile:water (80:20). The puriﬁcation was per- 3.1. Overview of the H NMR spectra of the nine formulations

formed on a XBridge Prep C18 column (150 mm × 19 mm; 5 ␮m analyzed

particle size; Waters Corporation, Milford, MA, USA), using a Waters

Delta Prep 4000 system equipped with a 486 tunable absorbance The H NMR spectra of CD3CN:D2O (80:20 v/v) extracts of for-

detector set at 270 nm. The elution was carried out using a linear mulations 1–4 on one hand and 6–7 on the other hand were

S. Balayssac et al. / Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 135–150 137

O S A 10 B 10 7 7 6 8 N1 6 8 N1 O HN O HN 25 24 N 2 25 24 N 2 22 15 5 22 15 5 S 23 S

26 23 16 14 26 16 14

9 9

29 N N N 3 29 N N N 3

O 4 11 O 4 11

19 19

27 28 17 12 27 28 17 12 18 O 18 O

13 13

20 21 20 21

sildenaﬁl (SILD) thiosildenaﬁl (THIO)

S 10

C 7 6 1 8 N O HN 25 24 22 15 N 2 5 29’ 29 26 23 S 16 14

9 HOCH2CH2 N N N 3 O 4 11 19

17 27 28 12 18 O

13 20 21

21’

propoxy phenyl-thiohydroxyhomosildenaﬁl (PP-THHS)

9’

D O E 5 1 6 8 9 O NH 7 8a 4 5 N 10 10’ 2 N 4a 6 11 3 4 13a 12a

13b

13 12 16 H 9 O 3’ 3 8 N 17 1 10 7 O 2 15 11 20 18 13 O 14

12 19

2’

tetrahydropalmane (THP) phentolamin e (PHE)

F 5 4 10 6 3

11 2

9 7 O O 8 O 1 12 13 14

16 15

osth ole (OST)

Fig. 1. Chemical structures of the actives identified in the dietary supplements analyzed: (A) sildenafil (SILD), (B) thiosildenafil (THIO), (C) propoxyphenyl-thiohomosildenafil

(PP-THHS), (D) tetrahydropalmatine (THP), (E) phentolamine (PHE), (F) osthole (OST).

138 S. Balayssac et al. / Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 135–150

identical, whereas those of all the other formulations displayed dif- of the aromatic protons of this compound called compound 1 (1)

ferent ﬁngerprints. As an illustration, we present in Figs. 2 and 3 the resembled the coupling pattern of a 1,2,4-substituted phenyl ring

characteristic H NMR spectra of formulations 4, 5, 6, 8 and 9. All system and suggested that its chemical structure could be related

the spectra revealed the presence of sugar(s) (S) with anomeric sig- to that of a PDE-5 inhibitor derived from SILD or vardenaﬁl.

≈ ≈

nal(s) at 5.3 ppm and highly overlapped peaks between 3.4 and The LC-DAD analysis of extracts of these formulations showed

≈

3.8 ppm, and fatty acids (FA) with characteristic signals at 0.9 (ter- the presence of a major peak at RT 4.5 min whose UV spectrum

≈ ≈

minal CH3 protons), 1.3 ppm ( (CH2)n protons), 1.5 ppm (CH2 with absorption bands at max 227, 295 and 353 nm (Fig. 4) was

␤ ≈ ␣

protons to the carboxyl group) and 2.2 ppm (CH2 protons to similar to that of THIO (max 224, 295 and 354 nm), and different

the carboxyl group). S (Fig. 3) and FA resonances were respectively from that of SILD (max 211, 224 and 291 nm). All the pyrazolopy-

lined up in the 2D DOSY H NMR spectra. Ethanol (EtOH; triplet at rimidinethiones (THIO and analogs) and PDE-5 inhibitors with

1.13 ppm) was detected in formulations 2, 6, 7 and 9, and citrate imidazoquinazolinethione-type chemical structure present a band

ı ı

(AB system with A 2.68 ppm and B at 2.62 ppm, J = 15.4 Hz) in for- at 350–370 nm, which was ascribed to the conjugated heterocyclic

mulations 6 and 7. Mesylate (singlet at 2.61 ppm), a non-toxic acid thiones ([7–10] for some examples), whereas the UV absorp-

often used to form salts of active pharmaceutical ingredients con- tion bands of pyrazolopyrimidinones (SILD and analogs) as well

taining basic centers, was observed in formulation 8. Other major as PDE-5 inhibitors with imidazotriazenone core (vardenaﬁl and

resonances were present in all formulations. They could correspond analogs) and piperazinedione ring (tadalaﬁl and analogs) appear

≤

to PDE-5 inhibitors in formulations 1–7. at 300 nm ([7,8,11] for some examples). It can be thus inferred

that 1 was a THIO analog.

1 13

H and C NMR data of THIO and isolated 1 are summarized

1 13

3.2. Characterization of the new SILD analog in formulations 1–4 in Table 1. The 1D H and C NMR spectra of isolated 1 are

presented in Fig. 5, while the H NMR spectrum of THIO in a

2D DOSY H NMR spectra of formulations 1–4 presented a pat- commercial formulation was reported in a previous paper [2].

1 13

tern of identical lined up resonances which strongly looked like The strong resemblance of H and C NMR spectra of the two

that of a PDE-5 inhibitor (data not shown). Moreover, the proﬁle compounds also indicated that 1 is a THIO derivative. Indeed,

Fig. 2. H NMR spectra of CD3OD:D2O (80:20) extracts of (A) formulation 4, (B) formulation 5, (C) formulation 6 and (D) formulation 9.

PP-THHS: propoxyphenyl-thiohydroxyhomosildenafil, THIO: thiosildenafil, SILD: sildenafil, THP: tetrahydropalmatine, OST: osthole, ᭹: imperatorine; S: sugars, FA: fatty

acids, EtOH: ethanol, TSP: sodium 2,2,3,3-tetradeutero-3-trimethylsilylpropionate (internal reference).

S. Balayssac et al. / Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 135–150 139

Fig. 2. (Continued).

13 1

the C signal at 175.7 ppm and the H singlet at 4.46 ppm are between this signal and those at 4.26 ppm (t) and 1.10 ppm

respectively characteristic of the N C S and N CH3 moieties of (t) suggested the presence of a propoxy moiety instead of an

a THIO pyrazolopyrimidine ring as the corresponding resonances ethoxy group. The correlations observed in HSQC and HMBC

in a SILD pyrazolopyrimidine ring would appear in the range experiments supported this assignment and established that

148–154 ppm (for the N C O group) and 4.15–4.3 ppm [11–13]. the substitution occurred at the C19 position of the phenyl ring.

{

Moreover, the NMR characteristics of protons (ı and J) and pro- 1 was thus identiﬁed as 5- 5-[4-(2-hydroxyethyl)piperazine-

ı }

tonated carbons ( ) of pyrazolopyrimidine (and its N-methyl and 1-sulfonyl]-2-propoxyphenyl -1-methyl-3-propyl-1,6-

C-propyl lateral chains) and phenyl rings are very similar for 1 dihydropyrazolo[4,3-d]pyrimidine-7-thione hereinafter referred

and THIO (within 0.03 ppm for H and with a systematic mean to as propoxyphenyl-thiohydroxyhomosildenaﬁl (PP-THHS)

deshielding of 1.25 ppm (range 0.8–1.55 ppm) for C). However, (Fig. 1C) since the THIO analog with an ethoxyphenyl group is

1 13

the H (s, 2.38 ppm) and C (44.9 ppm) methyl resonances of named as thiohydroxyhomosildenaﬁl (THHS).

the N-methylpiperazine (NMP) entity of THIO are missing in the To conﬁrm the structure of 1, a MS study was conducted. The

spectra of 1. On the other hand, two triplets at 2.49 and 3.57 ppm, full scan mass spectrum exhibited the presence of a very major

with a H H COSY correlation between them, each integrating pseudo-molecular ion [M + H] at m/z 535. The accurate mass of

1 13

for two protons, are present in the H spectrum of 1. Their C this ion was found at m/z 535.2162 in agreement with the cal-

resonances were assigned at 61.9 and 60.9 ppm, respectively culated mass 535.2161 of C24H35N6O4S2 within 0.2 ppm, which

from HSQC correlations. These data indicated the presence of a corresponds to the atomic composition of PP-THHS C24H34N6O4S2.

hydroxyethyl moiety. HMBC correlations between the H triplet The parent ion was selected to be fragmented in the MS/MS sys-

at 2.49 ppm and the C resonance at 54.8 ppm (C-25/27) of the tem. It produced two prominent ions at m/z 299 and 99 and other

piperazine ring as well as those between the protons 25/27 at representative fragment ions at m/z 517, 359, 341, 315, 271, 129,

2.58 ppm and the C signal at 61.9 ppm demonstrated that the 112, 84 and 58 (Fig. 6). A summary of proposed structures for these

NMP moiety of THIO was replaced by an N-hydroxyethylpiperazine ions is depicted in Fig. 7. The product ion at m/z 517 (loss of H2O

(NHEP) in 1. Moreover, a H signal at 1.93 ppm (tq) which is not from the hydroxyethyl group) was very probably formed by an ini-

present in THIO was observed in 1. The H–H COSY correlations tial migration of the hydroxylethyl group to the sulfur atom of the

140 S. Balayssac et al. / Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 135–150

B PHE PHE HODMes PHE CHD2 CN PHE PHE S S FA TSP FA

4000 A CHD CN 3000 HOD 2 Mes 2000 PHE

1000 )

-1 S .s 2 µ 500 D ( m

Formulation 8

100

7 6 5 4 3 2 1 0 δ (ppm)

1 1 1

Fig. 3. (A) 2D DOSY H NMR spectrum of a CD3CN:D2O (80:20) extract of formulation 8. (B) 1D H NMR spectrum of phentolamine (PHE) extracted from the 2D DOSY H

2 −1

NMR spectrum at D = 1356 ␮m s .

S: sugars, Mes: mesylate, FA: fatty acids, TSP: sodium 2,2,3,3-tetradeutero-3-trimethylsilylpropionate (internal reference).

thiocarbonyl function, followed by elimination of water (Fig. 7(a)), of the corresponding PP-THHS ion [16]. The second fragmentation

as recently demonstrated for THHS [14]. The heterolytic cleav- is identical to that reported in the literature for the ethyl group

age of the S N sulfonamide bond generated peaks corresponding of an ethoxy substituent leading to the same ion at m/z 299 for

to NHEP ion at m/z 129 and fragment ions at m/z 112 (loss of THIO, thiohomosildenaﬁl and THHS and to an ion at m/z 283 for

hydroxyl radical) and 99 (loss of methanal) which respectively SILD, homosildenaﬁl and hydroxyhomosildenaﬁl with a mass shift

give other ions at m/z 84 (loss of methyleneamino radical) and of 16 Da as the C S group is replaced by a C O [8,10,14]. The ion at

58 (loss of neutral azirine entity). The structures of all these ions m/z 271 was observed not only in the MS/MS spectrum of PP-THHS

(Fig. 7(b)) were proposed by Ahn et al. [15]. The product ions at m/z but also in those of THIO, thiohomosildenaﬁl and THHS. It could

341 and 299 arise respectively from successive losses of a neutral be generated from the ion at m/z 299 by loss of a neutral ethene

molecule of 194 Da corresponding to the sulﬁnic acid NHEP–SO2H (C2H4) moiety from the propyl chain of the pyrazolopyrimidine ring

(C6H14N2O3S) (Fig. 7(c)) and neutral propene (C3H6) entity from [8,10,14] (Fig. 7(e)). Indeed, this fragmentation pathway from the

the propoxy substituent of the phenyl ring (Fig. 7(d)). The ﬁrst propyl chain of vardenaﬁl, an isomer of SILD, was reported by Gratz

fragmentation was previously described for SILD and THIO with et al. [17]. Moreover, SILD, homosildenaﬁl and hydroxyhomosilde-

formation of analogous ions at m/z 311 and 327 which have an naﬁl also exhibit a similar fragmentation as an ion at m/z 255 most

expected mass shift of 30 and 14 Da respectively compared to that probably formed by the loss of a neutral C2H4 molecule from the

Fig. 4. UV spectrum of the new sildenaﬁl analog, propoxyphenyl-thiohydroxyhomosildenaﬁl, obtained using the chromatographic conditions indicated in the Section 2.

S. Balayssac et al. / Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 135–150 141 J 4 H-18 H-10 H-17 H-15 H-20

H-17,

J 3 H-12 H-15 H-10 H-11 H-13 H-11 H-18 H-17 H-18 H-15 H-15, H-21 H-20 H-29 H-25/27

H-13 H-17 H-21

J HMBC 2 H-11 H-12 H-11, H-12 H-15 H-15, H-17 H-18 H-21 H-20, H-21 H-25/27 H-24/28 H-29 H-29

(ppm)

C 13 60.9 41.6 29.9 24.8 16.0 74.4 24.9 12.9 48.6 54.8 61.9 130.1 ı 148.4 152.0 175.7 135.6 137.2 125.8 133.0 134.6 116.5 163.3

J 4 H-17 H-15

H-13 H-21

J H,H-COSY 3 H-12 H-11, H-12 H-18 H-17 H-21 H-20, H-21 H-25/27 H-24/28 H-29 H-29

(Hz))

signal.

8.9) 6.3)

signal

(7.4)

(2.5, (7.4,

(HMBC)

(2.5) (8.9)

(7.4) (7.4) (6.3) (7.4) (4.8) (5.9) (5.9)

80/20. Multiplicity s t sext t d dd d t tq t broad t t t

O 2 )

carbon sextuplet.

(

or CN/D

3 sext: (ppm)

H 1 in

) 0.99 PP-THHS ␦ 4.46 2.90 1.80 8.40 7.94 7.40 4.26 1.93 1.10 3.06 2.58 2.49 3.57 1

(H,H-COSY)

quadruplet;

(ppm)

(PP-THHS,

C hydrogen

13 40.3 28.4 23.3 14.6 67.7 15.4 45.6 54.3 44.9 triplet ␦ 147.3 148.5 173.6 133.7 135.0 122.2 131.6 128.8 133.8 115.7 162.0

tq: either

(Hz))

J ( indicates

b quadruplet;

8.9) q:

signal

(7.4)

(2.5,

(2.5) (8.9) (7.0) (7.4) (7.4) (7.0) (4.3)

numbering) triplet; Multiplicity s t sext t d dd d q t broad t s

the

for

doublet; (ppm)

c and propoxyphenyl-thiohydroxyhomosildenaﬁl H of

1 B 0.98 ␦ 4.45 2.91 1.81 8.41 7.91 7.38 4.36 1.54 3.13 2.72 2.38 1

and

Fig.

doublet

2 (THIO) (see

dd: CH

signals.

2 piperazine piperazine or

number

3 2 2 3 2

S doublet;

OH CH CH CH CH CH aromatic aromatic aromatic aromatic aromatic C aromatic aromatic aromatic aromatic aromatic 2 2 3 3 3 2 d:

thiosildenaﬁl broadened (Pos)

Group Cq Cq Cq Cq Cq N Cq CH Cq CH CH Cq O CH CH N N N CH of

singlet; 1

data a

3 5 9 7 8 Position s: Strongly 10 20 21 21 25/27 11 CH 13 CH 1214 CH 16 17 18 24/28 29 29 15 19 c a THIO Pos b Table NMR

142 S. Balayssac et al. / Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 135–150

C9 C8 C21 C12

CD3CN

C25 C24

C27 C28

C29 ’

C17 C15 C20 C29 C11

C14

C7 C19 C5 C3 C16

C18 C10 C13 C21’ 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 δ (ppm)

1 13

Fig. 5. H (A) and C (B) NMR spectra of the new sildenafil analog, propoxyphenyl-thiohydroxyhomosildenafil, after isolation and purification, recorded in CD3CN:D2O (80:20).

S. Balayssac et al. / Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 135–150 143

Fig. 6. MS–MS spectrum of pure propoxyphenyl-thiohydroxyhomosildenaﬁl (product ion spectrum of the molecular peak at m/z 535).

S+ S

H+ N O HN O O HN O N

(a) S (b)

S + N N N NNH HN N N HO HO

O O m/z 517 m/z 535 m/z129

S (c) .

(f) -OH -CH 2O

N - C H N O S HN 6 14 2 3 N S +

. N+ NH + N N N N HN N

O O m/z 341 + N m/z112 m/z99

- C H (d)

3 6 . - C H N

-CH2N 2 3 S m/z 359

N HN

+ N (g) - C3H8 N N+ + N

OH S m/z 84 m/z58 m/z 299

N - C H (e) HN 2 4 S N O N + N HN NH O + N m/z 315

m/z 271

Fig. 7. Summary of proposed fragmentation pathways for the new analog propoxyphenyl-thiohydroxyhomosildenaﬁl.

144 S. Balayssac et al. / Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 135–150 1166

0.10 1492 1350 1256 0.08 946 0.06

absorbance 0.04 3267

0.02

4000 3500 3000 2500 2000 1500 1000

wav enumber (cm-1)

Fig. 8. Infrared spectrum of the new sildenaﬁl analog, propoxyphenyl-thiohydroxyhomosildenaﬁl.

prominent ion at m/z 283 is observed [8]. The structure of the 3.4. Characterization of SILD and THP in formulations 6 and 7

ion at m/z 359 was tentatively proposed to be the same than that

reported for SILD ion at m/z 329 [17]. Indeed, it exhibits a mass In addition to the lined up resonances of S (translational diffu-

2 −1 2 −1

shift of 30 Da as expected due to replacements of the C O and sion coefﬁcient (D) = 865 ␮m s ) and FA (D = 1118 ␮m s ), the

ethoxy groups of SILD with C S and propoxy groups, respectively 2D DOSY H NMR spectra displayed those of ethanol and two other

(Fig. 7(f)). The ion at m/z 315 could be generated from the ion at major compounds with close D values. The pattern of resonances

2 −1

␮

m/z 359 by the cleavage of the O C bond of the propoxy moiety of the ﬁrst one (D = 947 m s ) was consistent with a PDE-5

followed by the formation of a cationic 1,4-benzoquinone moi- inhibitor of the SILD class. It was deﬁnitely identiﬁed as the citrate

ety resulting in the loss of the neutral propane (C3H8) molecule salt of SILD (Fig. 1A) by comparison of the H NMR spectra of the

(Fig. 7(g)). The structure of this ion is similar to that reported crude formulation extracts before and after addition of authentic

for the vardenaﬁl fragment ion at m/z 299 with a mass shift of SILD citrate. The H NMR characteristics of the second main com-

2 −1

16 Da as the carbonyl group is replaced by a thiocarbonyl group pound (D = 1069 ␮m s ), called compound 2 (2), did not match

[17]. with a usual PDE-5 inhibitor structure. 2 was isolated with a purity

The IR spectrum of isolated 1 is reported in Fig. 8. It displayed an of 80% with residual presence of less than 15% of SILD.

−1 1 13

absorption band at 3267 cm , characteristic of the N H stretching The H and C NMR spectra of 2 indicated the presence

vibration of an amine moiety. The three bands at 1492, 1256 and of two aromatic rings with eight quaternary carbons (Cq), four

−1

946 cm suggested the presence of a thioamide (N C S) group. methoxy groups and ﬁve other aliphatic carbon signals. The 1D

Indeed, in such a group, the C S stretching vibration is strongly cou- and 2D COSY H NMR spectra exhibited resonances of CH2 CH2

pled to that of the C N part leading to three signals in the regions and CH CH2 spin systems as well as a pair of doublets each inte-

−1 −1 −1

1570–1395 cm , 1420–1260 cm and 1140–940 cm that are grating for one proton at 3.62 and 4.22 ppm with a large JH H

partly due to the C S vibration [18]. The strong absorption bands (16.1 Hz) characteristic of geminal protons. The corresponding C

−1

at 1350 and 1166 cm correspond to the asymmetric and symmet- signal of these two protons at 55.6 ppm as determined from the

ric stretching vibrations of SO2 in a sulfonamide group that occur HSQC experiment indicated that this isolated methylene group was

−1 −1

respectively at 1360–1315 cm and 1180–1140 cm in the solid probably a N CH2 moiety. The aromatic region displayed two sin-

phase [18]. These characteristic group frequencies are very similar glets each integrating for one proton consistent with two para

−1

to those observed in THIO ( NH at 3271 cm , C S at 1498, 1253 related hydrogens in one of the aromatic rings and an AB pat-

−1 −1

and 939 cm and SO2 at 1352 and 1171 cm , conﬁrming that 1 tern integrating for two protons with J = 9.1 Hz which suggested

is a THIO analog. the presence of two ortho hydrogens in the second aromatic ring.

To our knowledge, PP-THHS already patented by Kim et al. [19] Unambiguous assignments of Cq were achieved considering that

in 2004 was never detected as an adulterant in a dietary supple- the cross-peaks in the HMBC experiment were more intense for

3 2 4

ment. JC H correlations than for J or JC H across the aromatic sys-

tems. The H H, direct C H and long-range C H bond correlations

observed in the COSY, HSQC and HMBC experiments supported

3.3. Characterization of THIO in formulation 5

1 13

the assignment of all H and C signals summarized in Table 2

1 and allowed identifying 2 as THP (5,8,13,13a-tetrahydro-2,3,9,10-

In the 2D DOSY H NMR spectrum of formulation 5, the res-

tetramethoxy-6H-dibenzo[a,g]quinolizine) (Fig. 1D). The H and

onances pattern of the major compound was very similar to

C NMR assignments were slightly different from those previously

that observed in formulations 1–4. This compound was identi-

reported data [20].

ﬁed as THIO (Fig. 1B) by spiking with an authentic standard and

1 High-resolution MS (HR-MS) and MS/MS studies of 2 corrob-

comparing the H NMR spectra recorded before and after its

addition. orated its identiﬁcation as THP. Indeed, the accurate mass of

S. Balayssac et al. / Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 135–150 145 system

unsaturated

the

p-eq to

J quinolizidine

4 H-4 H-1 H-1 H-8 H-4 adjacent

p-eq trans-fused

H-8

p-eq , carbons

the p-eq p-ax

p-ax H-13a on

H-13 ,

, H-8

H-9 H-8 , H-13

, , p-eq , p-ax eq because p-ax

p-eq p-ax protons H-8

, H-13a H-6 H-13 H-10 H-8

, ,

H-8 p-eq H-2 H-3 H-5

the

p-ax p-ax ax ax C-13a

equatorial.

J on

H-4 H-6 H-12 3 H-4, H-1, H-5 H-1 H-8 H-12, H11, H-12, H-13 H-11, H-6 H-4, the

than

proton

p-eq cyclohexene,

p-eq the

p-eq p-eq spectrum.

H-13

H-13 , H5 shielded

, H8 ,

except

p-ax similar p-ax HSQC

p-ax p-eq

p-ax more

J is the H-5 HMBC 2 H-5 H8 H-12 H-11 H13 H-13 H-13a

orientation,

proton

conformation

axial

cross-peaks

the

(ppm) adopt

(p-ax/p-eq) the

C to

of 13

ı 111.7 150.3 150.2 114.4 129.0 30.4 53.6 55.6 129.8 147.4 153.1 114.4 127.0 129.9 61.8 131.3 58.1 58.3 62.6 58.3 37.5 group,

core

structure p-eq

signal.

p-ax methylene ax ﬁne

H-13

, H-5

, quinolizidine H-6

the

, each p-ax

(HMBC)

eq eq p-eq p-eq for

J from

3 H-6 H-6 H-5 H-5 H-13a H-13a H-13 carbon

that,

non-planar

or pseudo-axial/pseudo-equatorial

p-eq p-ax the

p-eq p-ax eq ax p-eq p-ax to

determined

J H,H-COSY 2 H-5 H-5 H-6 H-6 H-8 H-8 H-13 H-13 considering

were

(H,H-COSY) J

carbons

orientation

and (Hz)) the

(9.1) (9.1)

J ı

assigned

(

b (B) (A)

hydrogen force

8.8) 12.4) 3.3) (ax/eq)

were

either system system respective (11.9, (15.8, (16.1,

(16.9) (9.3) (16.1) (16.1) (10.1)

skeleton

Multiplicity s s d m dd d d d AB AB dd dd d s s s s protons

Their

indicates 80/20.

d d

axial/equatorial

e d O 2 3.07 4.22 3.46 2.74 3.62 2.73 p-ax/p-eq multiplet.

(ppm)

5 e 5 5

3.27 2.71 usual or

CN/D m: 1 p-ax p-eq ax eq p-ax p-eq p-ax p-eq overlapped.

3 ı 6.87 6.76 H H H 6.94 6.95 H 3.69 3.80 3.81 3.79 3.84 H H H H the

numbering) CD are

ax/eq

from the ) p-ax

2 doublet; The

tetrahydroprotoberberine for

H-13 D tilted )

1 the

ax (THP;

in are and

(H 2 2 3 3 3 3 Fig.

ax doublet

CH CH CH CH CH CH CH aromatic aromatic aromatic aromatic aromatic aromatic aromatic aromatic aromatic aromatic aromatic aromatic substituent. 2 2

(see

H-6

dd:

C-13a)

Group CH Cq Cq CH Cq CH N N Cq Cq CH CH Cq CH N Cq O O O O signals. moieties

p-ax and

oriented

number H-5

doublet;

C-13

benzene

tetrahydropalmatine broadened (Pos)

axially C-8,

two of

signals

the (C-5,

singlet; 2

data a

Position s: As The Strongly c c c c a e Pos 1 3 4 6 8 8a 9Cq 10 11 12a 13 2 13a 3 9 10 12 2 4a 13b 5 b d Table NMR bonds requires

146 S. Balayssac et al. / Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 135–150

OCH3 OCH3 6 8 9 OCH OCH H 3 H 8a 3

N 5 N+

+ 7 10

B C D -CH 4 4a 11 4 13a 12a 13b 13 12 A 3 1

O H CO m/z 340 3 2 m/z 356 Retro Diels-Aldercleavage OCH3 OCH3

H + NH + OCH3

OCH3

H3CO m/z 192 m/z 165 OCH3

-CH4 -CH3 -CH 3 H NH + NH + + OCH3

O H3CO

m/z 176 m/z 177 m/z 150

OCH3 O

Fig. 9. Tentatively proposed structures of representative product ions of protonated tetrahydropalmatine (THP).

the protonated molecular ion was measured at m/z 356.1859 3.5. Characterization of PHE in formulation 8

in agreement with the calculated mass 356.1862 of C21H26NO4

within an error of −0.8 ppm, which corresponds to the elemen- The H NMR spectrum of formulation 8 showed unknown reso-

tal composition of THP, C21H25NO4. Moreover, the HR MS/MS nances along with those of S, FA, and mesylate (Fig. 3). The fact that

spectrum revealed representative product ions at various m/z all the major unknown resonances were lined up in the 2D DOSY H

2 −1

within 1.2 ppm relative to calculated masses, except for ion at NMR spectrum at D = 1356 ␮m s supports the presence of a sole

m/z 192 for which the error reached 3.1 ppm (Table 3). The major product, called compound 3 (3) (Fig. 3A). The corresponding

elemental formulae of these protonated ions correspond to char- H slice DOSY spectrum enables the attribution of the resonances

acteristic fragments due to a retro Diels–Alder cleavage of ring of 3 even if two signals of FA are detected due to the closeness of

2 −1

C of a tetrahydroberberine skeleton into two pieces that can be their D values (1492 ␮m s for FA) (Fig. 3B).

1 13

protonated (see for example [21]) (Fig. 9). Moreover, these two The H and C NMR characteristics of 3 were determined on

product ions and the parent ion can release a neutral methyl the crude extract of formulation 8 and are listed in Table 4. The

radical and/or a neutral methane molecule from a methoxy aliphatic region of the H NMR spectrum exhibited three aliphatic

ı group. singlet signals at 2.32, 3.87 and 4.71 ppm integrating respectively

Table 3

Accurate masses of protonated THP (2) and of its major product ions.

Mass found Mass calculated Error (ppm) Elemental formula

356.1859 356.1862 −0.8 C21H26NO4

340.1550 340.1549 0.3 C20H22NO4

192.1031 192.1025 3.1 C11H14NO2

177.0791 177.0790 0.6 C10H11NO2

176.0714 176.0712 1.1 C10H10NO2

165.0918 165.0916 1.2 C10H13O2

150.0681 150.0681 0 C9H10O2

S. Balayssac et al. / Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 135–150 147

for 3, 4 and 2 protons with corresponding HSQC direct carbon

J correlations at 21.0, 45.8 and 50.2 ppm. The aromatic region of

4 H-18 H-20 13

the C spectrum displayed six protonated carbon signals corre-

sponding to eight carbon atoms from comparison of their relative

intensities, and ﬁve Cq signals indicating the presence of two aro- 1

(C10)

matic rings. The H spectrum showed resonances of eight aromatic (C9)

protons. The AA XX system at ı 7.03 and 7.21 ppm had long- H-12

H-13

range H,H COSY correlations with the singlet signal of the methyl H-6

group (2.32 ppm) providing ﬁrm evidence of a para-methyl substi- H-6 H-20H-20 H-18 H-19 (C12),

(C13),

tuted aromatic ring. The multiplicity, coupling constants and H,H

3 H-4/5 H-10/12, H-10 H-9/13 H-10/12 H-19, H-18, H-19 H-16, H-16, COSY correlations suggested that the second aromatic ring was

1,3-disubstituted. The long-range H,H COSY correlations between

the N CH2 singlets at 3.87 and 4.71 ppm and their HMBC cor-

(C4) relations with the Cq at 171.6 ppm which does not belong to

the two aromatic rings suggested the presence of an imidazoline H-5

moiety with a CH2 N substituent located at C2. Moreover, the H-20 H-18

(C5),

other HMBC correlations between the N CH2 protons at 4.71 ppm

and Cq at 145.5 (part of one aromatic ring) and 150.0 ppm (part

HMBC 2 H-6 H-4 H-9/13 H-10/12H-9/13 H-14 H-16, H-9 H-16, H-19

of the second aromatic ring) implied that the N atom was con-

nected to these two moieties. The meta substituent on the second

aromatic ring could be assigned to an hydroxyl group based on (ppm) 5

the high value of the connected Cq (159.0 ppm). 3 was thus

13 identiﬁed as PHE (3-[(4,5-dihydro-1H-imidazol-2-ylmethyl)(4- 135.3

␦ 171.6 45.8 50.2 145.5 131.3 21.0 150.0 159.0 109.9 131.4 111.1

methylphenyl)amino]phenol) (Fig. 1E), whose structure was

conﬁrmed by comparison of the H NMR spectra before and after

addition of authentic PHE mesylate.

MS, MS/MS and HR-MS were carried out in positive ionization J

5 H-6 H-4/5 H-9/13 39.7

mode on the crude extract of formulation 8. The full scan mass spec-

trum showed the presence of a pseudo-molecular ion [M + H] at

m/z 282. Based on the accurate mass determination, 3 has a chemi-

H-20 106.6

cal formula of C H N O for the [M + H] ion (measured mass m/z

17 20 3

282.1648, calculated mass m/z 282.1606, error 14.9 ppm) which J signal. 4 H-14 H-10/12 H-16 H-16

corresponds to the atomic composition of PHE C17H19N3O. The

MS/MS fragmentation of the parent ion at m/z 282 produced a char-

(HMBC) acteristic, dominant base peak at m/z 212 which is achieved with

H-20 the loss of neutral imidazoline molecule.

carbon

J H,H-COSY 3 H-9/13 H-18, H-19

3.6. Characterization of OST in formulation 9

) H-10/12 H-14 124.4 ) The H NMR spectrum of formulation 9 exhibited resonances of

S, FA, EtOH and unknown compounds among which the major one (Hz)) (XX (AA

(H,H-COSY) J

0.8)0.8) H-19 (

was isolated for identiﬁcation. b 1 13

2.2, 2.3,

The H and C NMR data of the puriﬁed compound, called system system

compound 4 (4), are summarized in Table 5. In the aromatic (8.8, (8.1, hydrogen

XX XX 1

region of the H spectrum, two H,H COSY correlated pairs of (2.2)(8.1) H-18,

Multiplicity s s AA s t ddd ≈

doublets with a large coupling constant ( 9 Hz) were observed either

indicating the presence of two ortho protons located on two

inverted.

different aromatic rings. The methyl proton singlet at 3.94 ppm 80/20.

be O

indicates with a HSQC direct carbon correlation at 59.0 ppm was consistent 2

(ppm)

H with a methoxy group on an aromatic ring. H,H COSY correla- 1 could

CN/D

␦ 3.87 4.71 7.21 2.32 7.10 6.34 s

3 tions between the proton at 5.21 ppm and methylene (3.51 ppm) doublet.

and methyl (1.68 and 1.84 ppm) signals indicated the presence CH-20 in

numbering)

of a (CH2)HC C(CH3)2 moiety. The C spectrum exhibited ﬁf- and the

teen carbon signals: ﬁve aromatic and ethylenic CH, one CH and

2 doublet

for

of three CH3 in accordance with proton observations, and six Cq,

E mesylate

1 ) CH-18

one of them being ethylenic. Therefore, the two aromatic sys- 3

(imidazoline) (phenyl)

Fig.

tems contained nine carbon atoms, four CH and ﬁve Cq. Based

N N doublet

(PHE;

aromaticaromatic aromatic 7.03aromaticaromatic aromatic AA 6.31 6.45 t ddd

on the Cq chemical shift values, one corresponded to an O C O aromatic aromatic aromatic aromatic aromatic 2 2 3 (see

ddd: Group Cq CH CH CH CH Cq CH CH CH 2.61 carbon suggesting the presence of a coumarine entity, and a sec-

ond bore a methoxy group. HSQC and HMBC spectroscopic data assignments

number

provided ﬁrm evidence that 4 was OST (7-methoxy-8-(3 -methyl- triplet;

2 -butenyl)-2H-1-benzopyrane-2-one) (Fig. 1F), whose structure phentolamine (Pos)

and

was authenticated by spiking with standard OST and comparing H 1

(mesylate) the H spectra before and after addition. Moreover, an unequivocal singlet; 4

data a

c c 1 13

Position s: The

assignment of all H and C resonances of OST was accomplished c a Pos 2 8Cq 20 4/5 14 15 Cq 1618 CH 10/12 9/13 CH 11 Cq 6 17 19 CH b Table

NMR

by a careful inspection of the correlations observed in the H,H COSY

148 S. Balayssac et al. / Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 135–150

Table 6

Contents of adulterants identiﬁed in the formulations analyzed. H-5

J Formulation Actives identiﬁed Content 4 H-4, H-6

1 PP-THHS 80 ± 3

2 PP-THHS 65 ± 5

3 PP-THHS 103 ± 5

4 PP-THHS 81 ± 5

5 thiosildenaﬁl 89 ± 2 H-12

H12 c

6 sildenaﬁl 117 ± 6

H-16 H-16 H-15

tetrahydropalmatine 96 5 H-11, H-5, H-6

7 sildenaﬁl 66 ± 2

J ±

3 H-4 H-5 H-4 H-5, H-6 H-4, H-3, H-15, H-12 H-13, H-13, tetrahydropalmatine 44 5

8 phentolamine 64 ± 3

9 osthole 41 ± 3

Contents are expressed in mg per 10 g of powder. b

PP-THHS: propoxyphenyl-thiohydroxyhomosildenaﬁl. c

H-16

The formulation contains sildenaﬁl citrate but the amount is that of the free base

H-5

−1

(molecular weight 474.6 g mol ).

J d

HMBC 2 H-3 H-4 H-3 H-6 H-5 H-6 H-12 H-4, H-13 H-12 H-15, The formulation contains phentolamine mesylate but the amount is that of the

−1

free base (molecular weight 281.35 g mol ).

Osthole was not considered as an adulterant in formulation 9 (cf. text).

(ppm)

C 13

and H,C HSQC and HMBC spectra, considering that the C reso- 13

ı 165.0 115.2 148.0 130.1 111.0 163.2 119.9 155.5 116.0 59.0 24.5 124.1 135.7 27.8 20.0

nance of the “Z methyl group” was more shielded (usually 4–6 ppm,

7.8 ppm for OST) than the “E one” due to the ␥ effect.

MS and HR-MS data conﬁrmed the structure determined from

NMR. The full scan mass spectrum of isolated 4 generated pro- +

H-16

tonated monomeric and dimeric peaks at m/z 245 [M + H] and

489 [M2 + H] . The accurate masses of these ions were m/z J

5 H-15, H-12 H-12

245.1082 and 489.2294 giving estimated elemental composi-

tions of C15H17O3 (calculated mass 245.1178, error −39.2 ppm,

−

9.6 mDa) and C30H33O6 (calculated mass 489.2277, error 3.7 ppm,

signal.

1.7 mDa) as the most approximate results. Both compositions cor-

H-16 responded to the atomic composition of OST C H O . The MS/MS

15 16 3

fragmentation of the parent ion at m/z 245 produced promi- (HMBC) J

4 H-15, H-13 H-13

nent ion at m/z 189 generated by the elimination of the neutral

dimethyl-1,1-ethene (H2C C(CH3)2) molecule. This ion was fur-

carbon

ther fragmented to yield another ion at m/z 161 (loss of CO) with or

rearrangement of the 2-oxopyrane ring into a furane one. A subse-

quent loss of CH O occurred with a peak observed at m/z 131. This

H,H-COSY 3 H-4 H-3 H-6 H-5 H-13 H-12 2

fragmentation pattern was characteristic of OST as described by

(H,H-COSY)

Chen et al. [22]. The loss of CH2O from the ion at m/z 189 produced

an ion at m/z 159. (Hz))

hydrogen J

(

3.7. Quantiﬁcation of adulterants in the formulations analyzed b 1.4)

either

(7.2,

The contents of the different adulterants detected are listed

(7.2) (1.1)

(9.5) (9.5) (8.7) (8.7) in Table 6. Even if the amounts of the approved PDE-5 inhibitor hept c c c

Multiplicity d d d d s d t d s

SILD in formulations 6 and 7 (117 and 66 mg per 10 g bag) are indicates

slightly higher than or below the maximum recommended daily

dose (100 mg/day), the consumers are not aware of taking a pre-

scription drug that has severe contraindications. In addition to numbering)

80/20. SILD, these formulations enclosed THP (96 and 44 mg per 10 g bag,

(ppm)

heptuplet. O

the

2 respectively), an alkaloid of the benzylisoquinoline class that is 1 of

ı 6.26 7.88 7.51 7.04 3.94 3.51 5.21 1.68 1.84

for

one of the more bioactive constituent of plants from the botanical F CN/D 1 3

genera Corydalis and Stephania. The molar ratio SILD/THP, deter- triplet

Fig.

mined as the ratio of the mean integrated areas of H15 and H18 in

)

hept: SILD signals in one hand and of H1 and H4 THP signals on the 4

(see

signals.

other hand in H NMR spectra of formulations 6 and 7, is very O) 3

(OST;

close to 1 (range 0.97–0.995), indicating that the same SILD-THP CH aromatic aromatic aromatic aromatic ethylenic (C aromatic aromatic aromatic aromatic ethylenic 2 3 3

number

doublet; mixture was added in the two formulations. Formulation 6 was

Group Cq CH CH CH CH Cq Cq Cq Cq O CH CH Cq CH CH

adulterated by a two-fold SILD-THP dose than formulation 7, as osthole broadened (Pos)

the absolute amount of the two compounds is roughly two-fold

higher in the former one. Moreover in our opinion, added THP did singlet; 5

data a

Position s: Strongly

2 3 5 6 9 4 7 8 not come from a crude extract of plants known to contain great c a 10 Pos 12 15 13 11 14 16

Table NMR quantities of this bioactive such as Stephania and Corydalis species.

S. Balayssac et al. / Journal of Pharmaceutical and Biomedical Analysis 63 (2012) 135–150 149

Indeed, the aromatic region in the H NMR spectra of formulations 4. Conclusion

6 and 7 showed the same pattern of minor intensity resonances

whose ı and J characteristics are consistent with those of alkaloids Among the nine herbal formulations analyzed, eight were adul-

identiﬁed in these plants in addition to THP such as corydaline, terated with a synthetic drug. Four of them contained PP-THHS,

corydalmine, stylopine. . . (see for example [20,23,24]). The inte- an unapproved SILD analog detected for the ﬁrst time as an

grated area of THP aromatic resonances represented more than adulterant. Two were tainted with SILD and THP that has no

85% of that of all alkaloids detected with the reasonable assump- reported effect for enhancing erectile dysfunction, and one with

tion that, on average, these compounds have four aromatic protons THIO, another unapproved PDE-5 inhibitor. Another formulation

as THP. THP represents ≈70% of the alkaloid content extracted was adulterated with PHE that is unapproved for boosting male

from S. bancroftii or yunnanensis [20,24] and less than 25% of the sexual performance as oral formulation. The last formulation con-

tertiary alkaloids extracted from C. yanhusuo [23]. Therefore THP tains the bioactive coumarin derivative, OST, a major compound

found in formulations 6 and 7 probably arises from THP-enriched present in Cnidium monnieri fruits, and is most probably truly

extracts commercially available under the form of powders pre- natural.

pared from natural rhizome Corydalis yanhusuo which can contain

up to 98% of THP. THP is used in traditional Chinese herbal prepara-

Acknowledgements

tions for the treatment of chronic pain and anxious insomnia as it

possesses remarkable analgesic, sedative and hypnotic activities. It

The authors wish to thank Dr. J.C. Garrigues for helpful dis-

also exhibits anticonvulsant, hypotensive, bradycardial, neuropro-

cussions, Dr. C. Routaboul from IR and Raman Department and

tective and antioxydant properties but was not reported as effective

researchers from the Mass Spectrometry Department for technical

for enhancing erectile dysfunction and sexual performance in

assistance.

man [20,21]. However, its possible interactions with SILD are not

known.

The quantities of the two unapproved analogs, THIO and the References

newly identiﬁed compound PP-THHS, in formulations 1–5, ranged

[1] A. Petroczi, G. Taylor, D.P. Naughton, Mission impossible? Regulatory and

between 65 and 103 mg/10 g bag, which could place consumers at

enforcement issues to ensure safety of dietary supplements, Food Chem. Toxi-

risk for potentially serious side-effects. Indeed, THIO and PP-THHS

col. 49 (2011) 393–402.

have an inhibitory activity against the isolated enzyme PDE-5 (IC50) [2] J. Vaysse, V. Gilard, S. Balayssac, C. Zedde, R. Martino, M. Malet-Martino, Iden-

tiﬁcation of a novel sildenaﬁl analogue in an adulterated herbal supplement, J.

of 0.59 and 0.46 nM, respectively, i.e. more than ten-fold higher

Pharm. Biomed. Anal. 59 (2012) 58–66, and references quoted in.

than that of SILD (IC50 = 6.86 nM) [19]. As drug safety is dependent

[3] V.M. Toomey, J.J. Litzau, C.L. Flurer, Isolation and structural characterization of

on both activity and pharmacokinetics, the fact that the pharma- two tadalaﬁl analogs found in dietary supplements, J. Pharm. Biomed. Anal. 59

(2012) 50–57.

cokinetic proﬁles of these two unlicensed compounds are unknown

[4] B.J. Venhuis, M.E. Zwaagstra, J.D.J. van den Berg, A.J.H.P. van Riel, H.W.G. Wage-

could increase the risks for consumers who unwittingly consume

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