Experimental

Chemicals

Sibutramine hydrochloride monohydrate (purity 98.4 %, CAS No. 125494-59-9) was used as the analytical standard. Graphite powder (Sigma-Aldrich, USA), sulfuric acid (H₂SO₄, 98 %), phosphoric acid (H₃PO₄, 85 %), hydrochloric acid (HCl, 37 %), hydrogen peroxide (H₂O₂, 30 %), potassium chloride (KCl), and other analytical-grade reagents were obtained from Merck (Germany). Potassium permanganate (KMnO₄) was purchased from Scharlau (Spain). Nafion solution (5 wt.%) was supplied by Aldrich. All solutions were prepared using deionized water.

Britton-Robinson (BR) buffer (0.25 M) was prepared from boric acid, phosphoric acid, and acetic acid, and adjusted to the desired pH (2 to 12) using 1.0 M NaOH. Commercial herbal weight-loss products were collected from pharmacies and e-commerce platforms in Vietnam.

Apparatus

Electrochemical measurements were performed using a PCPA HH5 electrochemical workstation in a conventional three-electrode system consisting of a modified glassy carbon electrode (GCE) as the working electrode, a platinum wire as the counter electrode, and an Ag/AgCl (3 M KCl) electrode as the reference. Material characterization was carried out using X-ray diffraction (Bruker D8 Advance), Fourier-transform infrared (FTIR) spectroscopy (PerkinElmer), scanning electron microscopy (SEM, Hitachi S-4800) coupled with energy-dispersive X-ray mapping, Raman spectroscopy (Horiba Xplora Plus, 785 nm), and high-resolution transmission electron microscopy (JEOL JEM-2100F). For validation, an HPLC with a diode-array detection (HPLC-DAD) system (Shimadzu LC-20AD with a PDA detector) was used for comparison. The HPLC-DAD method was validated in accordance with the AOAC guidelines [15] and demonstrated satisfactory analytical performance. The chromatographic conditions were as follows: an InertSustain™ C18 column (4.6×250 mm, 5 μm) was used for separation. The mobile phase consisted of acetonitrile (ACN, solvent A) and KH₂PO₄ buffer solution (pH 3.2, solvent B) under the following gradient elution program: 0 to 1 min, 20 % A; 1 to 3 min, 40 % A; 3 to 11 min, 50 % A; 11 to 12 min, 50 % A; 12 to 13 min, 20 % A and 13 to 28 min, 20 % A. The flow rate was maintained at 0.8 mL min-1, the injection volume was 20 μL, and the column temperature was set at 40 °C. Detection was carried out at a wavelength of 225 nm.

The synthesis of electrochemically reduced graphene oxide, AgNPs and AgNPs-ErGO for material characterization

Synthesis of graphene oxide and electrochemically reduced graphene oxide

First, graphene oxide (GO) was synthesized from graphite using a modified Hummers method [16]. Subsequently, electrochemically reduced graphene oxide (ErGO) was synthesized from GO via a one-step electrochemical reduction method [17] according to the following procedure:

A 0.1 M phosphate buffer solution (PBS, pH 7) was prepared and GO powder was dispersed into the PBS solution at a concentration of 1 mg mL-1. The mixture was ultrasonicated for 2 h to obtain a homogeneous GO-PBS suspension. Stainless steel mesh (Inox 304) was used as the working electrode. Prior to use, the mesh was cleaned by ultrasonication in 2 M HNO₃ and 96 % ethanol to remove impurities and the native oxide layer and then dried. The electrochemical synthesis was carried out in a conventional three-electrode system consisting of the stainless steel mesh as the working electrode, a platinum wire as the counter electrode and an Ag/AgCl|3 M KCl electrode as the reference electrode. The stainless steel mesh was immersed in the GO-PBS suspension. The electrochemical reduction and deposition of ErGO were performed by cyclic voltammetry (CV) in the potential range from 0 to -2.0 V at a scan rate of 0.100 V s-1 for 10 consecutive cycles.

After electrochemical deposition, the ErGO-coated stainless steel mesh was removed and immersed in distilled water. The porous ErGO film was completely detached from the mesh surface by ultrasonication for approximately 15 min. To completely remove residual salt ions from the buffer solution, the dispersed ErGO suspension was centrifuged at 6000 rpm, the supernatant was discarded, and the solid was washed three times with distilled water. Finally, the purified ErGO product was freeze-dried to obtain a lightweight porous powder, which was then ground into a fine powder for further characterization.

Synthesis of silver nanoparticles

The synthesis of AgNPs was carried out electrochemically using a three-electrode system as follows: Stainless steel mesh (Inox 304) was used as the working electrode and was cleaned by ultrasonication in 2 M HNO₃ and 96 % ethanol, followed by drying. A solution containing 50 mL of 0.1 M KNO₃ as the supporting electrolyte and 0.05 M AgNO₃ as the silver precursor was prepared in the electrochemical cell.

The electrochemical synthesis was performed using a three-electrode configuration comprising the stainless steel mesh working electrode, platinum wire counter electrode, and Ag/AgCl|3 M KCl reference electrode. The reduction and deposition process was carried out by cyclic voltammetry over the potential range from 0 to -0.6 V at a scan rate of 0.100 V s-1 for 10 consecutive cycles.

After the reduction process, the Inox 304 mesh electrode was removed from the electrochemical cell and rinsed thoroughly with distilled water to eliminate residual AgNO₃. The electrode was then immersed in distilled water and ultrasonicated for approximately 15 min to detach the deposited AgNPs layer from the mesh surface. During this process, the solution gradually turned yellowish-brown, while the mesh regained its metallic appearance.

The obtained suspension was centrifuged at 6000 rpm, the supernatant was discarded, and the precipitate was washed three times with distilled water. The purified AgNPs were finally freeze-dried and used for further characterization.

Synthesis of AgNPs-GO and AgNPs-ErGO

The AgNPs-GO composite was prepared by mechanically mixing AgNPs and GO at a mass ratio of 1:5. The AgNPs-ErGO composite was synthesized electrochemically using a three-electrode system according to the following procedure: Stainless steel mesh (Inox 304) was cleaned as described above and used as the working electrode. A precursor suspension was prepared directly in the electrochemical cell by mixing 1.8 mL of 0.05 M AgNO₃, 48.2 mL of GO suspension (1.0 mg mL-1) and 0.51 g of KNO₃ as the supporting electrolyte. These quantities were selected to obtain an AgNPs:GO mass ratio of approximately 1:5, assuming the complete electrochemical reduction of Ag⁺ ions to metallic Ag nanoparticles. The electrochemical synthesis was then carried out using a conventional three-electrode system consisting of a stainless steel mesh working electrode, a platinum wire counter electrode, and an Ag/AgCl reference electrode. The reduction and deposition process was performed by cyclic voltammetry over the potential range from 0 to -2.0 V at a scan rate of 0.100 V s-1 for 10 consecutive cycles. During this process, both the oxygen-containing functional groups on GO and Ag⁺ ions were simultaneously reduced, leading to the formation of an AgNPs-ErGO nanocomposite film on the stainless steel mesh surface. The immersed portion of the stainless steel mesh gradually turned dark grey, confirming the successful deposition of the AgNPs-ErGO film.

After electrochemical reduction, the modified mesh electrode was removed and rinsed with distilled water. The electrode was dried at 60 °C for 2 h to remove residual water and promote stronger anchoring of AgNPs within the folded ErGO network. Subsequently, the mesh was immersed in distilled water and ultrasonicated for approximately 15 min to detach the AgNPs-ErGO film from the mesh surface.

The obtained suspension was centrifuged at 6000 rpm, washed three times with distilled water, and finally freeze-dried to obtain purified AgNPs-ErGO powder for material characterization.

Preparation of modified electrodes for electrochemical measurements

Bare GCE: The surface of the glassy carbon electrode (GCE) was polished using 0.05 μm Al₂O₃ powder until a mirror-like surface was obtained. The polished GCE was then immersed in 2 M HNO₃ solution for 15 min. Subsequently, the electrode was rinsed twice with 96 % ethanol and distilled water, with each wash repeated 3 times. Finally, the electrode was dried naturally at room temperature.

GO/GCE electrode: GO suspension was prepared by dispersing 0.1 g of GO in 100 mL of distilled water followed by ultrasonication for 2 h to obtain a homogeneous GO suspension (1.0 mg mL-1). Then, 400 μL of 1% nafion solution (used as a binder) was added to 10 mL of the GO suspension. Afterward, V μL of the obtained mixture was drop-cast onto the cleaned GCE surface and dried under an infrared lamp until complete solvent evaporation, yielding the GO/GCE electrode.

ErGO/GCE electrode: 10 mL of the synthesized ErGO suspension was mixed with 400 μL of 1 % nafion solution. Subsequently, V μL of the mixture was drop-cast onto the cleaned GCE surface and dried under an infrared lamp.

AgNPs/GCE electrode: 10 mL of AgNPs suspension (1 mg mL-1) was mixed with 400 μL of 1 % nafion solution. Then, V μL of the resulting suspension was drop-cast onto the cleaned GCE surface and dried under an infrared lamp.

AgNPs-GO/GCE electrode: AgNPs were mechanically mixed with GO at an appropriate mass ratio. Subsequently, 0.1 g of the obtained composite material was dispersed into 100 mL of distilled water to form an AgNPs-GO suspension (1 mg mL-1). Then, 400 μL of 1 % nafion solution was added to 10 mL of the AgNPs-GO suspension. Finally, V μL of the resulting mixture was drop-cast onto the cleaned GCE surface and dried under an infrared lamp.

AgNPs-ErGO/GCE electrode: 10 mL of the AgNPs-ErGO composite suspension was mixed with 400 μL of 1% Nafion solution. Subsequently, V μL of the mixture was drop-cast onto the cleaned GCE surface and dried under an infrared lamp to obtain the AgNPs-ErGO/GCE modified electrode.

Placebo preparation

The placebo sample used in this study was Slimtosen slimming capsules (0.5790 g capsule-1), containing herbal ingredients including Folium Nelumbinis, Alisma plantago, Gynostemma pentaphyllum, Garcinia cochinchinensis, Cassia tora L., chitosan, L-carnitine fumarate, and other excipients. Ten capsules were randomly selected, and their entire powder contents were thoroughly homogenized in an agate mortar for 2 h.

The placebo sample preparation procedure was performed as follows: 0.5 g of the homogenized powder was accurately weighed into a 50 mL Falcon tube. Subsequently, 25 mL of n-hexane acidified with HCl was added, and the mixture was shaken for 5 min to remove nonpolar interfering substances. After discarding the supernatant, the remaining residue was collected and dried at room temperature. Then, 25 mL of absolute methanol was added to the dried residue, followed by vortex mixing for 5 min and ultrasonication for 10 min to facilitate analyte extraction. The resulting clear solution was filtered through a 0.45 μm membrane filter prior to analysis. Three placebo samples were independently prepared following the same procedure.