1. Rapid HPTLC method for the detection of adulteration in dietary supplements with synthetic drugs
Tiên DO

2. 11/27/2017
State of art
Since the opening of Internet sales it is possible to find a multitude of dietary supplements in different forms such as capsules, tablets, pills.

3. State of art Adulteration
11/27/2017
State of art
Weight loss products
DS are uses for various plant-related properties
DS follow the trend of the natural
Sexual enhancement
Adulteration
Sport-performance enhancement
Do the supplements you’re taking really contain what you think it is?
These dietary supplements are often used for various plant-related properties. Some of these products are used for specific effects.
Inside those dietary supplements there is 3 classes of products which are particularly known.
There are first of all erectile products, sports performance boosters, and slimming products.
These products follow the trend of the natural and are therefore potentially subject to adulteration.
Do the supplements you’re taking really contain what you think it is?

4. 11/27/2017
State of art
Undeclared adulterants present a serious risk for the consumers
In 2016, the United States Pharmacopeia Convention (USP) issued the General Chapter <2251>
Screening for undeclared drugs and Drugs Analogs
Numbers of analytical strategies for PDE5-Is screening including HPTLC
Expanded-scope screening procedures are needed
Weight loss and sport performance enhancement products will follow

5. Focused on weight loss products
11/27/2017
State of art
FDA has identified emerging trend where products contain hidden active ingredients
Focused on weight loss products
Sibutramine (anorexiant)
Phenolphtalein: laxative
Caffeine
Fluoxetine: antidepressant
Phosphodiesterase type 5 Inhibitors (PDE5-Is)
Expanded-scope screening procedures for undeclared pharmaceutical constituents are needed
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Carvalho et al. (2011) Forensic Science International 204, 6-12

6. Setting-up of the project
USP provided commercial products (12) to a number of laboratories
Screening methods are developed with different techniques:
Liquid chromatography with UV and MS detection
Nuclear Magnetic Resonance (NMR)
Direct Sample Analysis (DSA)
Direct Analysis Real Time (DART)
HPTLC
Cross-confirming the findings

7. Detection and documentation
Method development
Sample preparation
Development
Evaluation
Detection and documentation
Information and goals:
Diversity of marketed finished dosage forms
Universal sample preparation procedure
A single unit dose, e.g., a thoroughly pulverized tablet or capsule, contents of a single softgel or a sachet, was ultrasonically extracted with 10 mL of methanol for 30 minutes, and centrifuged. The supernatants were used as test solutions
Do et al. (2015) Journal of AOAC International 98,

8. Detection and documentation
Method development
Sample preparation
Development
Evaluation
Detection and documentation
Information and goals:
Detection of nine adulterants
Simple and reproducible chromatography
Literature
Sibutramine
PDE5-Is
Toluene–methanol (9 :1) (v/v)
MTBE–methanol–ammonia 28% (20:2 1, v/v/v)
Mathon et al. (2014) Food additives & contaminants. Part A, Chemistry, analysis, control, exposure & risk assessment 31, 15-20
Do et al. (2015) Journal of AOAC International 98,

9. Method development Sibutramine method General Chapter <203> USP
Toluene–methanol
(9:1, v/v)
Extensive analyte coelution at low RF values
1: fluoxetine;
2: sildenafil citrate;
3: vardenafil HCl;
4: caffeine;
5: theophylline;
6: tadalafil;
7: acetaminophen;
8: phenolphthalein;
9: sibutramine

10. Method development PDE5-Is method General Chapter <203> USP
MTBE–methanol–ammonia 28% (20:2:1, v/v/v)
Coelution
1: fluoxetine;
2: sildenafil citrate;
3: vardenafil HCl;
4: caffeine;
5: theophylline;
6: tadalafil;
7: acetaminophen;
8: phenolphthalein;
9: sibutramine

11. MTBE–methanol–ammonia 28% (20:2:1, v/v/v)
Method development
Toluene–methanol
(9:1, v/v)
MTBE–methanol–ammonia 28% (20:2:1, v/v/v)
Different selectivity (Snyder classification)
Toluene: VII
MTBE: I
Water: VIII
1: fluoxetine;
2: sildenafil citrate;
3: vardenafil HCl;
4: caffeine;
5: theophylline;
6: tadalafil;
7: acetaminophen;
8: phenolphthalein;
9: sibutramine

12. Method development New method General Chapter <203> USP
Ammonia: critical for separation reproducibility
---> left out
MTBE–toluene–methanol
(9:1:1, v/v/v)
Overall separation better
1: fluoxetine;
2: sildenafil citrate;
3: vardenafil HCl;
4: caffeine;
5: theophylline;
6: tadalafil;
7: acetaminophen;
8: phenolphthalein;
9: sibutramine

13. MTBE–toluene–methanol (9:1:1, v/v/v)
Method development
MTBE–toluene–methanol (9:1:1, v/v/v)
Optimization
Selectivity
MTBE–toluene–methanol
(9:1:1, v/v/v)
(7:3:1, v/v/v)
(6:4:1, v/v/v)
(5:5:1, v/v/v)
(8:2:1, v/v/v)
Optimization on the solvent ratio:

14. MTBE–toluene–methanol
Results
Final method
MTBE–toluene–methanol
(8:2:1, v/v/v)
1: fluoxetine;
2: sildenafil citrate;
3: vardenafil HCl;
4: caffeine;
5: theophylline;
6: tadalafil;
7: acetaminophen;
8: phenolphthalein;
9: sibutramine

15. Results

16. Results
Test on the 12 samples from the USP, 9 were adulterated with: phenolphtalein, sibutramine, sildenafil, theophylline, acetaminophen, and fluoxetine.
HPTLC screening results compared favorably with those obtained by LC-UV, LC-MS, NMR, DSA and DART

17. Conclusion
The proposed method will facilitate rapid and reliable screening for the various adulterants in DS.
In finished products, each test solution was 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.
HPTLC can be the technique of choice for a “general screening” method for multiple adulterants
Method submitted to the USP

18. Thank you for your attention