1226 Do et al.: Journal of AOAC International Vol. 98, No. 5, 2015

DIETARY SUPPLEMENTS

Received December 10, 2014. Accepted by AP March 13, 2015.1 Corresponding author’s e-mail: eike.reich@camag.comDOI: 10.5740/jaoacint.14-285

An HPTLC method is proposed to permit effective screening for the presence of three phosphodiesterase type 5 inhibitors (PDE5-Is; sildenafil, vardenafil, and tadalafil) and eight of their analogs (hydroxyacetildenafil, homosildenafil, thiohomosildenafil, acetildenafil, acetaminotadalafil, propoxyphenyl hydroxyhomosildenafil, hydroxyhomosildenafil, and hydroxythiohomosildenafil) in finished products, including tablets, capsules, chocolate, instant coffee, syrup, and chewing gum. For all the finished products, the same simple sample preparation may be applied: ultrasound-assisted extraction in 10 mL methanol for 30 min followed by centrifugation. The Rf values of individual HPTLC bands afford preliminary identification of potential PDE5-Is. Scanning densitometry capabilities enable comparison of the unknown UV spectra with those of known standard compounds and allow further structural insight. Mass spectrometric analysis of the material derived from individual zones supplies an additional degree of confidence. Significantly, the proposed screening technique allows focus on the already known PDE5 Is and provides a platform for isolation and chemical categorization of the newly-synthesized analogs. Furthermore, the scope could be expanded to other therapeutic categories (e.g., analgesics, antidiabetics, and anorexiants) that are occasionally coadulterated along with the PDE5-Is. The method was successfully applied to screening of 45 commercial lifestyle products. Of those, 31 products tested positive for at least one illegal component (sildenafil, tadalafil, propoxyphenyl hydroxyhomosildenafil, or dimethylsildenafil).

The sale of “Natural Aphrodisiacs” seems to be a prospering business and not just on the Internet. Frequently these products are adulterated with synthetic

phosphodiesterase type 5 inhibitors (PDE5-Is), including the active principles of Viagra® (sildenafil), Levitra® (vardenafil), and Cialis® (tadalafil). Not only are these regular pharmaceuticals

found in lifestyle products, but also their unapproved analogs such as hydroxyacetildenafil, homosildenafil, thiohomosildenafil, acetildenafil, acetaminotadalafil, propoxyphenyl hydroxyhomosildenafil, hydroxyhomosildenafil, and hydroxythiohomosildenafil (1). Because of the propensity of causing serious health risks, PDE5-Is have become a significant focus for the U.S. Food and Drug Administration (FDA; 2) and other authorities (e.g., police and custom authorities in Poland and the German Health Care Inspectorate; 1). A recent research letter concerning “Presence of Banned Drugs in Dietary Supplements Following FDA Recalls” describes the severity of the situation: 274 product recalls were initiated in the United States between January 1, 2009 and December 31, 2012 because of the undisclosed active pharmaceutical ingredients (3).

Several methods using HPLC coupled with a diode array detector and/or a mass spectrometer have been developed for detection of PDE5-Is in finished products (4–6). Using conventional TLC, Moriyasu et al. (7) developed a method for identification of sildenafil in health food. One year later, Mikami et al. (8) proposed another mobile phase for identification of sildenafil in soft drinks. In 2003, Hsu et al. (9) reported the use of a TLC/MS interface for identification of sildenafil by MS with an electrospray ionization source (ESI). The first use of HPTLC plates for determination of sildenafil in pharmaceutical products and aphrodisiac herbal preparations was published in 2005 by Abourashed et al. (10). Since that time, methods for quantification of sildenafil (11) and for multidetermination of PDE5-Is were developed (12–14). The power of HPTLC was demonstrated in determination of eight PDE5-Is, but to the best of our knowledge there is no published HPTLC method for detection of the three target PDE5-Is and eight of their analogs in (15). In response to an increasing demand for simple and reliable routine analytical methods for screening of lifestyle products for the presence of PDE5-Is, we have developed a comprehensive, yet straightforward, HPTLC approach including several levels of confirmation. The method was successfully applied to screening of 45 commercial products suspected of containing PDE5-Is.

Experimental

Materials and Chemicals

All reference substances (sildenafil, vardenafil, tadalafil, hydroxyacetildenafil, homosildenafil, thiohomosildenafil, acetildenafil, acetaminotadalafil, propoxyphenyl hydroxyhomosildenafil, hydroxyhomosildenafil, and

Simultaneous Detection of Three Phosphodiesterase Type 5 Inhibitors and Eight of Their Analogs in Lifestyle Products and Screening for Adulterants by High-Performance Thin-Layer ChromatographyTiên T.K. Do, Grigorios Theocharis, and Eike Reich1

CAMAG Laboratory, Sonnenmattstrasse 11, 4132 Muttenz, Switzerland

Do et al.: Journal of AOAC International Vol. 98, No. 5, 2015 1227

hydroxythiohomosildenafil) were from TLC PharmaChem (Ontario, Canada). Tert-butyl methyl ether (TBME) was of LC grade and purchased from Acros Organics (Morris Plains, NJ), and methanol and water were LC grade (Fisher Scientific, Loughborough, UK). Acetonitrile (Carlo Erba, Val de Reuil, France) and formic acid (Fluka, St. Louis, MO) were of LC grade. Ammonia solution (35%) was obtained from Fisher Scientific and KSCN (>98%) was obtained from Merck ( Darmstadt, Germany)

Instrumentation

(a) HPTLC system.—HPTLC analyses were performed using a VisionCATS 1.4 SP 2 software controlled HPTLC system (CAMAG, Muttenz, Switzerland) consisting of

an automatic TLC sampler (ATS 4), automatic developing chamber (ADC2) with humidity control, visualizer, and a TLC/MS Interface equipped with an oval elution head (2 × 4 mm).

(b) Documentation.—Densitometric peak detection on the plates was performed at 232 nm (deuterium lamp) using a CAMAG TLC Scanner 4 in the reflection/absorption mode. The slit dimension was 5 × 0.30 mm. Plates were subsequently scanned to acquire the in-situ UV spectra (190 to 550 nm) of the individual zones.

(c) Mass spectrometer.—HPTLC/ESI-MS spectra were directly recorded using the TLC-MS Interface coupled to the ESI interface of a single-quadrupole mass spectrometer (Expression CMS; Advion, Ithaca, NY). Data processing and

Figure 1. HPTLC profiles of SST and reference substances under 254 nm UV light (A), UV 366 nm UV light (B), and 366 nm UV light after 12 h (C). 1: SST sildenafil, vardenafil, tadalafil (with increasing Rf); 2: mixture of 3 PDE5-Is and eight of their analogs; 3: hydroxyacetildenafil; 4: hydroxyhomosildenafil’ 5: propoxyphenyl hydroxyhomosildenafil; 6: acetildenafil; 7: hydroxythiohomosildenafil; 8: sildenafil; 9: acetaminotadalafil; 10: vardenafil; 11: homosildenafil; 12: tadalafil; and 13: thiohomosildenafil.

1228 Do et al.: Journal of AOAC International Vol. 98, No. 5, 2015

evaluation of mass spectra were performed with Advion Mass Express 2.0 and Advion Data Express 2.0.50.9.

Preparation of Solutions

(a) Elution solvent for HPTLC/ESI-MS.—The respective zone was directly eluted with water–acetonitrile (80 + 20, v/v) containing 0.1% formic acid.

(b) Mobile phase (MP) for HPTLC.—TBME–methanol–ammonia 28% (20 + 2 + 1, v/v/v).

Preparation of Sample Extracts

(a) Standard preparation.—Each reference substance (2 mg) was dissolved in 10 mL methanol.

(b) Sample preparation.—Commercial lifestyle products were either provided by U.S. Pharmacopeial Convention (Rockville, MD), or obtained from local markets and pharmacies in different countries. To detect the presence of PDE5-Is and their analogs in lifestyle products, each unit (e.g., powdered pill, content of a capsule, piece of chewing gum, or piece of chocolate) was crushed using a mortar and pestle, then ultrasonically extracted with 10 mL methanol for 30 min, and centrifuged. The supernatants were used as test solutions and were diluted 10-fold with methanol if the chromatographic bands appeared overloaded and in-situ UV spectra were distorted.

(c) System suitability test (SST).—On track 1 of each plate, a mixture of sildenafil, vardenafil, and tadalafil was applied as an SST; after chromatography the Rf values of each of the three reference substances must be located within a window of 0.03 units.

Figure 2. UV spectra of acetildenafil, hydroxythiohomosildenafil, and sildenafil.

Figure 3. UV spectra of acetaminotadalafil and vardenafil.

Do et al.: Journal of AOAC International Vol. 98, No. 5, 2015 1229

Analysis

(a) HPTLC.—HPTLC analyses were performed on Merck silica gel 60 F254 Premium Purity HPTLC glass plates (20 × 10 cm, 0.2 mm). Aliquots (3 µL) of reference and test solutions (15 tracks/plate) were applied as 8 mm bands (50 nL/s delivery speed, track distance 11.4 mm, 8 mm distance from the lower plate edge, and 20 mm from the left plate edge). Prior to development, the plates were conditioned to a relative humidity of 47% with saturated KSCN solution in an ADC2 chamber lined with filter paper and saturated for 20 min; the development distance was 70 mm from the lower edge of the plate. The developed plates were imaged under 254 and 366 nm

UV light. After 12 h, an additional image was taken under 366 nm UV light.

(b) MS.—The MS system was operated in the full scan mode (total ion current) between m/z 100 and 1000 using a detection gain of 2100 in the positive ESI mode [(+)-ESI]. The capillary voltage was set at 3.0 kV and cone voltage was 50 V. The temperature of the ESI probe was 105°C, and the desolvation temperature 150°C. Nitrogen was used as the nebulizer gas and drying gas. The desolvation gas flow was set at 300 L/h and the cone gas flow at 80 L/h. Mass spectra of the analytes were corrected by subtracting the plate background at a migration distance comparable to that of the analyte zone.

Results and Discussion

At the onset of our investigation of an HPTLC separation of three available PDE5-Is and eight of their analogs, we compared two MPs: MP1 and MP2. MP1, proposed by Cai et al. (14), consisted of the lower layer of chloroform–ethyl acetate–methanol–water (40 + 40 + 15 + 11, v/v/v/v). In our evaluation it provided insufficient resolution, with the four pairs of coeluting substances: sildenafil and hydroxyhomosildenafil, acetildenafil and hydroxyacetildenafil, thiohomosildenafil, and acetaminotadalafil, and propoxyphenyl hydroxyhomosildenafil and vardenafil. Additional shortcomings of MP1 were the presence of chloroform, an undesirable toxic solvent, and the fact that the composition of a two-phase mixture of solvents was highly sensitive to temperature changes. The latter factor and missing details about the chamber setup may have been responsible for our inability to reproduce the published Rf values and the elution order of standards. MP2, TBME–methanol–ammonia 28% (20 + 2 + 1, v/v/v), was adopted from the diploma thesis of Caprez (12) and provided significantly better resolution of the target substances. Only two sets of coeluting substances were found: acetildenafil (track 6), hydroxythiohomosildenafil (track 7), and sildenafil (track 8) as well as acetaminotadalafil (track 9) and vardenafil (track 10) had similar Rf values and were not well separated (Figure 1). However, the UV spectra (Figures 2 and 3) were distinctly different. It is generally uncommon for adulterated products to contain closely related chemicals (2); therefore, MP2 was selected for further work, even though the concentration of ammonia was identified as a critical factor for good reproducibility of the separation. Consequently, throughout this work, the MP was prepared using

Figure 4. HPTLC profile of “Triple Miracle Zen Platinum” capsule. 1: SST (sildenafil, vardenafil, tadalafil); 2: test solution (1 unit/10 mL); 3: test solution diluted 10-fold.

Figure 5. UV spectra of two questionable zones in comparison to spectra of reference substances.

1230 Do et al.: Journal of AOAC International Vol. 98, No. 5, 2015

to verify the specificity of the proposed method, plant extracts were analyzed with and without spiking them with PDE5-Is reference substances. The proposed MP does not facilitate migration of the polar plant components far from the application zone and thus sufficiently resolves all PDE5-Is from excipients and other legitimate components (Figure 6).

Twenty-four commercial products provided by the U.S Pharmacopeial Convention and suspected to contain analogs of PDE5-Is were tested together with 21 additional commercial products purchased in local markets. Sildenafil, tadalafil, propoxyphenyl hydroxyhomosildenafil, and dimethylsildenafil (aildenafil) were found in 31 of those products without being declared on the product label (Table 1).

Figure 7 illustrates another valuable feature of the proposed method: a potential for identification of novel adulterants by MS. For one of the investigated samples, three zones (Rf = 0.25, 0.31, and 0.50) were obtained that did not match the Rf values of the available reference substances. Comparison of their UV spectra with those of the reference substances did not provide a match either. However, zones 1 and 3 displayed UV features characteristic of thione analogs, while the spectrum of zone 2 was sildenafil-like. For identification of those zones, MS spectra were obtained from the separated zone on the plate using a TLC/MS interface (Figure 7). ESI-MS of unknown compound 1 gave [M+H]+ at m/z 500, which did not help with identification. However, unknown zone 2 revealed [M+H]+ at m/z 489, suggesting a molecular formula of C23H32N6O4S (Figure 8). This formula could correspond to sildenafil analogs such as dimethylsildenafil (aildenafil), propoxyphenyl sildenafil, or isobutyl sildenafil. Figure 9 shows the ESI-MS spectrum of compound 3. The peak at m/z 505 corresponds to the [M+H]+ ion and suggests a compound with a molecular mass of 504. This mass could match thione analogs of sildenafil (C23H32N6O3S2): sulfoaildenafil, propoxyphenyl thiosildenafil, or thiovardenafil. It has been noted that dimethylsildenafil and sulfoaildenafil have been found as adulterants previously and have been listed by the FDA (2).

This method allows a preliminary detection or identification of adulterants by using the similarities of the Rf values and UV profiles to those of reference standards. Confirmation of the identity can be achieved with the mass spectrum.

freshly diluted 28% aqueous ammonia following titrimetric assessment of the 35% ammonia. This way, the Rf values of each of the three reference substances (SST) were located within a range of 0.03 units.

To estimate the individual detection limits for all available substances, dilutions of standard stock solutions were made to achieve absolute amounts of 30 ng/zone. At this level, all substances could be clearly detected by scanning densitometry. Visual detection is achievable down to 60 ng/zone under 254 and 366 nm UV light. The performance of the proposed method appears satisfactory, considering that the lifestyle products are commonly adulterated to contain dosages similar to, or exceeding, those of the approved drugs. A single dose of Viagra (25 to 100 mg) would generate from 7.5 to 30 µg/zone. From a practical standpoint, false-negative results due to insufficient method sensitivity appear unlikely.

Due to the considerable segment of the population affected by erectile dysfunction and the concomitant demand for lifestyle products that enhance sexual performance, a substantial diversity of finished dosage forms is available. Therefore, universal sample preparation procedure becomes highly desirable in adulterant profiling. Most published methods rely on the extraction with an organic solvent (1); we have successfully used methanol. One unit dose of each product (45 commercial products including capsules or tablets, chocolate, instant coffee, chewing gum, and topical cream) was finely powdered, mixed with 10 mL methanol, sonicated for 30 min, and then centrifuged.

For lifestyle products screening, the SST was applied to each plate to qualify the data after chromatography. Each test solution was then visually evaluated for the presence of zones matching in Rf value those of the previously analyzed reference substances. UV spectra of suspect zones were recorded and compared with those of the reference substances. As shown in Figure 4, one of the samples was found to contain two zones matching the Rf values of (1) sildenafil and (2) tadalafil. The UV spectra of the zones did not match well with the spectra of the reference substances, but it was suspected that this was caused by the high concentration of the target zones. Spectra obtained after chromatography of a 1:10 diluted solution of the sample gave a better match and confirmed the presence of the suspected drugs (Figure 5).

Some plants, such as Eurycoma longifolia, Tribulus terrestris, and Pausinystalia johimbe, have an established traditional medical use in the treatment of erectile dysfunction. Extracts of those plants are available as commercial products. In order

Figure 6. HPTLC profiles under 254 nm UV light (A), and 366 nm UV light (B). 1: mixture of PDE5-Is (sildenafil, propoxyphenyl hydroxyhomosildenafil, homosildenafil, and hydroxyhomosildenafil with increasing Rf); 2: P. johimbe; 3: T. terrestris; 4: E. longifolia; 5: P. johimbe + mixture of PDE5-Is; 6: T. terrestris + mixture of PDE5-Is; 7: E. longifolia + mixture of PDE5-Is.

Do et al.: Journal of AOAC International Vol. 98, No. 5, 2015 1231

Table 1. Analysis of 45 commercial products suspected to contain PDE5-Is

Sample description Source and date of purchase Manufacturer, Lot, Expiry PDE5-Is detected

Aphrodisiaque (paste) Istanbul Grand Bazaar– December 2009a

None None

Bali mojo (capsule)b www.balimojo.com, January 2014a

Production: 10/01/2014, Expiry: 09/01/2017

Tadalafil + extra zone unknown

Stree overlord (black pill and yellow gelcap)b

Provided by Dr. John Edwards, June 2014a

None Yellow gelcap: none, black pill: sildenafil

Control (capsule)b Provided by Dr. John Edwards, June 2014a

None Sulfoaildenafil and sildenafil analog

Triple miracle zen platinum (capsule)b

Maryland gas station, August 2014a

Power Life Distributors, Lot OAWF 1259, Expiry: 12/31/2016

Sildenafil, Tadalafil

O.M.G. (capsule)b www.rakuten.com, July 2014a S.M.I., Lot A758964, Expiry: 02/2015

Sildenafil

Niterider plus Maryland Gas Station, August 2014a

Maximus Niterider Int. Group Inc., Lot MXGM7513, Expiry: 06/01/2015

None

S.W.A.G (round black pill and white capsule)b

Maryland Gas Station, August 2014a

221 Enterprises LLC, batch No. SWAG130, Expiry: 02/20/2015

Black pill: none, white capsule: sildenafil

Ying Da Wang (yellow tablet) http://www.2daydiet-shopper.com, June 2014a

Date: 9/2/2013, Expiry: 3 years Sildenafil

Santi bovine (blue/white capsule) http://www.2daydiet-shopper.com, June 2014a

Production: 6/5/2014, Expiry: 5/5/2016

Sildenafil

ManKing (capsule)b http://www.2daydiet-shopper.com, June 2014a

None Sildenafil

African superman (oval black tablet) http://www.2daydiet-shopper.com, June 2014a

Production: 1/4/2014, Expiry: 5 years

Sildenafil

Indian stud horse (white capsule) http://www.2daydiet-shopper.com, June 2014a

Production: 1/11/2014, Expiry: 3 years

Unknown tadalafil analog

Stiff Delay Cream (white cream) http://www.2daydiet-shopper.com, June 2014a

Lot: HK14EQ5, Production: 05/2014, Expiry: 3 years

None

Magna Rx (octagonal white tablet) http://www.2daydiet-shopper.com, June 2014a

None Sildenafil and tadalafil

Africa black ant (white capsule)b http://www.2daydiet-shopper.com, June 2014a

None Sildenafil, + extra zone unknown

Super power pills (octagonal red tablet)b http://www.2daydiet-shopper.com, June 2014a

None Sildenafil

Stree overlord strong version (black pill and yellow gel cap)b

http://www.2daydiet-shopper.com, June 2014a

Production: 1/08/2014, Expiry: 3 years

Yellow gel cap: none, black pill: sildenafil

Maximum powerful triangular (black tablet) http://www.2daydiet-shopper.com, June 2014a

Production: 4/15/2013, Expiry: 3 years

Sildenafil

Wolf (oval red tablet) http://www.2daydiet-shopper.com, June 2014a

None Sildenafil

Arize (capsules)a www.CalCompNutrition.com, August 2014a

Lot: 3108, Expiry: 01/10/2016

Sulfoaildenafil

MaxMan IV (white capsule and black round pill)

http://www.2daydiet-shopper.com, June 2014a

Lot: A0080012180, Expiry: 06/10/2015

None

Super lover chewing gum (green chiclets) http://www.2daydiet-shopper.com, June 2014a

Expiry: 04/2016 Sildenafil

Vegetal Vigra (white capsule) http://www.2daydiet-shopper.com, June 2014a

Production: 07/03/2013, Expiry: 07/03/2016

Sildenafil

Caviarqtonical Purchased locally None Sildenafil

MOOOMR chocolate for men Purchased locally Production: 10/2011, Expiry: 10/2014

Sildenafil

Hercules Purchased locally None Sildenafil

MaxMan Purchased locally None Black part: none, orange part: tadalafil

Tongkat ali power plus tablet Purchased locally None Sildenafil

MaxMan capsules (Vigrx) Purchased locally None Black part: none, white part: sildenafil

Men’s VIP increased (tablet) Purchased locally None Sildenafil

Potaskuschka (tablet) Purchased locally None None

1232 Do et al.: Journal of AOAC International Vol. 98, No. 5, 2015

Conclusions

HPTLC can be used for rapid screening of commercial products for adulteration with three known PDE5-Is and eight of their analogs. The method affords selective and sensitive analysis of PDE5-Is and analogs and has been found applicable to a variety of finished products without interference from matrix and excipients. The adulterants may be detected by their Rf values and UV spectra compared to those of the reference substances. Availability of a TLC-MS interface permits added

certainty in confirmation of known adulterants, and reinforces analysis even in the absence of reference substances.

Acknowledgments

We wish to thank Anton Bzhelyansky (U.S. Pharmacopeial Convention) for providing standards and samples. We appreciate his valuable comments and discussions of the subject matter. We also thank Mahmoud Elghamrawi and John Edwards

Figure 7. Strategy for identification of unknown PDE5-Is and their analogs in a finished product.

Sample description Source and date of purchase Manufacturer, Lot, Expiry PDE5-Is detected

ViaMAX power sexy coffee (only for female)

Purchased locally None None

ProLatis 2.0 Purchased locally None None

ViaMAX power sexy coffee (only for male) Purchased locally None None

TiQTalk Purchased locally Production: none, Expiry: 09/2014

Propoxyphenyl hydroxyhomosildenafil

MaxMan instant coffee Purchased locally None Tadalafil

Human energy 107 Purchased locally Production: none, Expiry: 04/2013

Tadalafil

Reflex-it Purchased locally Production: none, Expiry: 31/07/2013

Tadalafil

Tiger king Purchased locally None Sildenafil

Tongkat ali extract (nu-prep Lelaki) Purchased locally Production: 11/2013, Expiry: 11/2016

None

Tongkat ali extract (Tonex) Purchased locally Production: 28/06/2013, Expiry: 27/06/2016

None

Tongkat ali plus Purchased locally Production: 03/02/2013, Expiry: 02/02/2016

None

Speman Purchased locally Production: 08/2013, Expiry: 07/2016

None

SimiPower capsules Purchased locally Production: none, Expiry: 03/2016

None

a Sample provided by USP.b FDA Public Notification available at http://www.fda.gov/Drugs/ResourcesForYou/Consumers/BuyingUsingMedicineSafely/MedicationHealthFraud/

ucm234539.htm.

Table 1. (continued )

Do et al.: Journal of AOAC International Vol. 98, No. 5, 2015 1233

(Process NMR Associates, LLC, Danbury, CT) for contributing additional samples.

References

(1) Singh, S., Prasad, B., Savaliya, A.A., Shah, P.R., Gohil, M.V., & Kaur, A. (2009) TRAC–Trends Anal. Chem. 28, 13–28. http://dx.doi.org/10.1016/j.trac.2008.09.004

(2) Nickum, E.A., & Flurer, C.L. (2014) J. Chromatogr. Sci. 24, 24(3) Cohen, P.A., Maller, G., DeSouza, R., & Neal-Kababick, J.

(2014) J. Am. Med. Assoc. 312, 1691–1693. http://dx.doi.org/10.1001/jama.2014.10308

(4) Gratz, S.R., Flurer, C.L., & Wolnik, K.A. (2004) J. Pharm. Biom. Anal. 36, 525–533. http://dx.doi.org/10.1016/j.jpba.2004.07.004

(5) Gratz, S.R., Gamble, B.M., & Flurer, R.A. (2006) Rapid Commun. Mass Spectrom. 20, 2317–2327. http://dx.doi.org/10.1002/rcm.2594

(6) Zou, P., Hou, P., Oh, S.S.-Y., Chong, Y.M., Bloodworth, B.C., Low, M.-Y., & Koh, H.-L. (2008) J. Pharm. Biomed. Anal. 47, 279–284. http://dx.doi.org/10.1016/j.jpba.2008.01.016

(7) Moriyasu, T., Shigeoka, S., Kishimoto, K., Ishikawa, F., Nakajima, J., Kamimura, H., & Yasuda, I. (2001) Yakugaku Zasshi 121, 765–769. http://dx.doi.org/10.1248/yakushi.121.765

(8) Mikami, E., Ohno, T., & Matsumoto, H. (2002) Forensic Sci. Int. 130, 140–146. http://dx.doi.org/10.1016/S0379-0738(02)00368-7

(9) Hsu, F.-L., Chen, C.-H., Yuan, C.-H., & Shiea, J. (2003) Anal. Chem. 75, 2493–2498. http://dx.doi.org/10.1021/ac0203746

(10) Abourashed, E., Abdel-Kader, M., & Habib, A.-A. (2005) J. Planar Chromatogr.–Mod. TLC 18, 372–376

(11) Reddy, T.S., Reddy, A.S., & Devi, P.S. (2006) J. Planar Chromatogr.–Mod. TLC 19, 427–431

(12) Caprez, S. (2005) Development of Methods for Analysis of Synthetic Adulterants in Herbal Medicines by HPTLC, Diploma Thesis, University of Basel, Basel, Switzerland, p. 80

(13) Lai, K.-C., Liu, Y.-C., Liao, Y.-C., Lin, Y.-L., Tsai, L.-Y., Lin, J.-H., & Lo, C.-F. (2010) J. Food Drug Anal. 18, 269–278

(14) Cai, Y., Cai, T.-G., Shi, Y., Cheng, X.-L., Ma, L.-Y., Ma, S.-C., Lin, R.-C., & Feng, W. (2010) J. Liq. Chromatogr. Relat. Technol. 33, 1287–1306. http://dx.doi.org/10.1080/10826076.2010.488979

(15) Patel, D.N., Li, L., Kee, C.-L., Ge, X., Low, M.-Y., & Koh, H.-L. (2014) J. Pharm. Biomed. Anal. 87, 176–190. http://dx.doi.org/10.1016/j.jpba.2013.04.037

Figure 8. MS spectrum of unknown zone (2).

Figure 9. MS spectrum of unknown zone (3).