Standard of sibutramine hydrochloride monohydrate was purchased from Sigma-Aldrich, Steinheim, Germany. The toluene, ethyl acetate, and methanol were of analytical grade purchased from Sigma-Aldrich, Steinheim, Germany. Bismuth subnitrate, potassium iodide, glacial acetic acid were purchased from Sigma-Aldrich, Steinheim, Germany. Forty DSs were purchased in pharmacies, herbal shops, and via the Internet.

The stock solution of sibutramine was prepared by diluting with methanol in concentration of 1 mg/mL. Better dilution was accomplished using ultrasonic bath (BANDELIN, Berlin, Germany).

Dragendorff’s reagent was prepared by dissolving 0.85 g of bismuth subnitrate in a mixture of 40 mL of water and 10 mL of glacial acetic acid (Solution A) and dissolving 8 mg of potassium iodide in 20 mL of water (Solution B). Five milliliters of both solutions were diluted with 20 mL of glacial acetic acid and 100 mL of water.^[21]

The samples of DSs were prepared by diluting 250 mg of each sample with 10 mL methanol. After that they were homogenized by vortex. The next step involved the use of an ultrasonic bath for 30 minutes and then the samples were filtered through 0.45 μl PTFE Syringe filters (GVS North America Sanford, USA).

The method was developed using a CAMAG HPTLC system (CAMAG, Muttenz, Switzerland). The configuration of the HPTLC system consisted of Limomat 5; Automatic Developing Chamber 2, and TLC Visualizer 2. The system was controlled by VisionCATS version 3. An ultrasonic bath (BANDELIN, Berlin, Germany) and а dipping chamber (Biostep-Desaga, Burkhardtsdorf Germany) were also used.

The analyses were carried out using silica gel 60 F254 aluminum HPTLC plates, 10×20 cm, 200 µm layer thickness (E. Merck KGaA, Darmstadt, Germany). The mobile phase comprised toluene, ethyl acetate, and methanol at a ratio of 7:2:1 v/v/v. The volume of the mobile phase was 10 mL. Application type: band. Front: 70 mm. Time for development: 25 minutes. Drying: 5 minutes. After development, derivatization was performed with Dragendorff’s reagent in a dipping chamber for 20 seconds. The plate was observed with the CAMAG TLC visualizer at 254 nm.

To achieve good linearity, accuracy, precision, robustness, limit of detection, and limit of quantification different chromatographic conditions were tested, including a reversed-type chromatography, using RP-18 modified silica gel-coated aluminum plates, with F254 fluorescent indicator.

However, the best results were achieved using silica gel 60 F254 aluminum HPTLC plates, 10×20 cm, 200 µm layer, and mobile phase – toluene : ethyl acetate : methanol at a ratio of 7:2:1 v/v/v.

Compared to the classical TLC, this technique requires a smaller volume of the mobile phase (10 mL). The standard solutions and the samples were applied as bands. All steps of the progress development were carefully monitored using specialized software. The time for the development was 25 minutes. After reaching a front of 70 mm, the plates were dried for 5 minutes. In the HPTLC techniques, the drying process is independent of human interaction, it is much safer for the researcher who performs the analysis, and it is performed automatically in a specific place of the development chamber. The next step involved derivatization with Dragendorff’s reagent in a dipping chamber for 20 seconds. The plate was observed with the CAMAG TLC visualizer after 5 minutes at 254 nm.

The optimized HPTLC method was validated for linearity, accuracy, precision, limit of detection and quantification, and robustness according to the guidelines of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH).^[22]

According to the ICH guidelines, the linearity must be evaluated using a minimum of five concentrations. The calibration curve was built using concentrations of sibutramine standard solutions in range from 0.250 to 1.250 μg/band.

A stock solution of sibutramine 100 μg/ml was prepared in methanol. Different volumes of the stock solution were applied by automatic sampler on HPTLC plate to obtain concentrations of 0.250 μg/band, 0.500 μg/band, 0.750 μg/band, 1.000 μg/band, and 1.250 μg/band.

After the development and derivatization, the plate was observed with the CAMAG TLC visualizer at 254 nm (Fig. 1).

The peak areas of the standard solutions were measured with CAMAG HPTLC system. Isometric profile of sibutramine in different concentrations is represented in Fig. 2. The linear regression line was y=0.0055933+0.000995. R²=0.9977. The concentration range 0.250–1.250 μg/band showed a good linearity.

Figure 1.

HPTLC chromatogram of sibutramine after derivatization with Dragendorff’s reagent under 254 nm. Concentrations 250 ng/band, 500 ng/band, 750 ng/band, 1000 ng/band, and 1250 ng/band.

Figure 2.

Isometric profile of sibutramine in different concentration levels.

Accuracy was assessed as a percentage of recovery. Recovery was evaluated using 3 known concentrations and 6 replicates for each concentration level. For accuracy tests from each examined substance were used 3 different quality control (QC) levels: lower QC (LQC; 0.500 μg/band), middle QC (MQC; 0.750 μg/band), and high QC (HQC; 1.000 μg/band) with six replicates. The results from the accuracy evaluation are presented in Table 1.

The interday and intraday precision of the method were determined by evaluating the coefficient of variation for the 3 known level concentrations, each concentration with 6 replicates (Table 2).

The detection limit and quantification limit were expressed by the standard deviation of the slope (s) and the slope of the calibration curve (S). We used the following formulas: LOD=3.3 s/S and LO-Q=10 s/S. The limit of detection was 0.0765 μg/band, and the limit of quantification was 0.2318 μg/band.

The study by Phattanawasin et al. was the first validated TLC method for quantification of sibutramine in DSs. The analysis was carried out using silica gel 60 F254 TLC plates, where the mobile phase comprised toluene-n-hexane-diethylamine, and for visualizing the spots Dragendorff’s reagent was used. The calculated LOD and LOQ were 190 ng/spot and 634 ng/spot respectively.^[10]

Later on, Hayun et al. developed a TLC-densitometry method using TLC silica gel 60 F254 aluminum plate, toluene-diethylamine (10:0.3) as mobile phase, and densitometric scanning at 227 nm. Lower amounts of LOD/LOQ were reported, 217.5 ng and 724.9 ng/spot, respectively.^[23]

With the advent of new improved techniques based on the principles of thin-layer chromatography, HPTLC instruments were developed. HPTLC represents a significant advancement in the TLC methodology, owing to the incorporation of several enhancements: automatic and improved sample application, automatic development and drying, automatic monitoring of the analytical procedure, increased resolution, etc. All these essential improvements lead to more precise results and better safety for the researchers.^{[18, 19]}

Ariburnu et al. developed and reported much sensitivity HPTLC technique compared to previous TLC methods, where the LOD and the LOQ were 77.34 and 257.79 ng respectively. HPTLC was performed with glass plates silica gel 60 F254, n-hexane-acetone-ammonia as a mobile phase, where the densitometric observation was set at 225 nm.^[24]

The discussed studies reported not only novel methods for sibutramine detection but also, positive samples adulterated with sibutramine in various concentrations, reaching up to 35 mg per capsule.^{[10, 23, 25-27]} A comparison between previous HPTLC/TLC techniques for the detection of sibutramine in DSs is presented in Table 3.

The method established and validated in this study demonstrated excellent linearity, accuracy, and precision. The accuracy of sibutramine was between 99.911% and 103.278%. The limit of detection was 0.0765 μg/band, and the limit of quantification was 0.2318 μg/band which shows good sensitivity of the proposed method. The HPTLC assay for the determination and quantification of sibutramine could be used for the analysis of DSs.

In contrast to prior approaches used for analysis, derivatization was performed in a dipping chamber. Dipping allows for a more controlled and even application of the sample onto the HPTLC plate. Dipping provides better resolution of the separated spots on the HPTLC plate compared to spraying. The controlled application of the sample by dipping ensures that the spots remain well-defined and do not merge, leading to more accurate identification and quantification of the analytes.

The research carried out by Hachem et al. investigated 164 dietary supplements, of which 43 samples had sibutramine adulteration, with concentrations varying from 0.1 mg to 22 mg.^[28] In 2016, a study conducted by Adela Krivohlavek and her team revealed that sibutramine was present in 20% of the 123 analyzed supplements, with the highest recorded level reaching 26.41 mg/g.^[29] A comparable investigation was conducted by Zeng et al., who analyzed 447 weight loss dietary supplements, among which 55 out of 119 contaminated samples were found to contain sibutramine.^[30] In 2014, Kim et al. discovered that 29 out of 188 DSs contained sibutramine, with quantities ranging from 0.03 mg/g to 132.40 mg/g.^[11]

The results indicated that a large number of DSs contain undeclared sibutramine.

For determining sibutramine in DSs, the validated HPTLC method was used. The samples and standard solutions were spotted on the same plate and analyzed. Bands of dietary supplements were identified by comparing the Rf value, after dipping in Dragendorff’s reagent, with those from standard of sibutramine. Forty DSs were tested and the presence of sibutramine in 5 dietary supplements was confirmed. The chromatogram and isometric profile of sibutramine standards and 5 dietary supplements are presented in Figs 3, 4, respectively.

Three DSs showed a higher amount of sibutramine. The results from HPTLC were confirmed through GC-MS analysis by a previously validated method.^[12]

Figure 3.

Chromatogram of sibutramine standard in concentrations from 0.250 μg/band to 1.250 μg/band and 5 dietary supplements.

Figure 4.

Isometric profile of sibutramine standards and 5 dietary supplements. 1. Sibutramine 0.250 μg/band. 2. Sibutramine 0.500 μg/band. 3. Sibutramine 0.750 μg/band. 4. Sibutramine 1.000 μg/band. 5. Sibutramine 1250 μg/band. 8–12 DSs.

Despite the high level of consumption of DSs, the lack of regulations is currently a critical issue.^[31] The majority of DS users believe these products are safe and represent a healthy lifestyle. In general, consumers are unaware of the risks associated with the intake of DSs.^[32] Unintentional intake of sibutramine from DSs can affect levels of norepinephrine and serotonin in the body, resulting in an increased heart rate and tachycardia. These effects raise the risk of cardiovascular events such as heart attacks, strokes, and arrhythmias.^{[3, 5, 7, 9]} Moreover, sibutramine intake can cause sleep disorders, anxiety, dizziness, and restlessness.‌^[33] People with cardiovascular disease, including those with a history of heart attack, stroke, arrhythmias, congestive heart failure, or uncontrolled hypertension, are at a higher risk of adverse cardiovascular events when using sibutramine. Sibutramine intake can potentially increase the likelihood of cardiovascular events, as mentioned above.^[3]

Furthermore, DSs are essential components of professional athletes’ diets and may offer advantages like quick recovery from strenuous exercise regimens, improved exercise performance, and dietary enrichment.^[34] However, unintentional doping can occur after intake of DSs, if an athlete unknowingly consumes substances that are prohibited by the World Anti-Doping Agency (WADA). Sibutramine falls into this category because it is classified as a banned substance by WADA.^[35] The reason for its prohibition is primarily due to appetite suppression and weight loss.

The five contaminated DSs, from our study, could expose consumers not only to serious health side effects but also expose many professional athletes to a significant risk of unintentional doping.