Development And Validation of The HPTLC Method For the Estimation of Tianeptine Sodium in Bulk and Tablet Formulation and Its Degradation Study

Tianeptine sodium is a serotonin receptor (5 HT2 receptor) blocker. It improves serotonin and adrenaline in the brain. It leads to preventing stress. Hence it works as an antidepressant. Tianeptine sodium is primarily used to treat major depressive disorder (MDD), and it has also shown benefits in treating anxiety, asthma, and irritable bowel syndrome (IBS) due to its influence on the brain-gut axis. Some studies suggest it might help with certain types of pain, particularly those related to inflammation or stress-induced conditions^1,2.

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Fig 1: Chemical structure of Tianeptine sodium

In an extensive literature survey, various methods like Gas chromatography (British pharmacopeia³) and liquid chromatography (British pharmacopeia²) were reported for the estimation of tianeptine sodium. HPLC^4-8, and UV- UV-visible spectroscopic method^9,10 are reported for the estimation of Tianeptine sodium. Ulu et al., describe an optimized HPLC method that uses fluorescence detection to measure Tianeptine concentrations in tablet dose forms⁴. Appala et al., present a method for estimating Trimebutine maleate in tablet dose form using reverse-phase high-performance liquid chromatography (RP-HPLC)⁵. Buridi et al. reported an estimation of tianeptine sodium using UV visible spectroscopic methods^9,10. Tianeptine, sold as the supplement Tianaa™ in the US, is a synthetic drug linked to opioid addiction. This study found tianeptine levels ranging from 3.1 to 10.9 mg per capsule across Tianaa™ White, Red, and green products, with significant variation in both capsule mass and tianeptine content¹⁰. Literature survey ^1-14 reveals that no method reported using HPTLC for Tianeptine sodium in bulk and marketed products HPTLC is the most sophisticated and automated thin-layer chromatography. HPTLC principle is useful for the separation, identification, and quantification of compounds¹². Hence, it is the best choice to separate degradation products in various conditions (acid, base, oxidative, thermal, and photolytic degradation) for Tianeptine sodium. It is also a very useful technique for the degradation kinetic study ^15-20,. Degradation kinetic studies are useful for understanding a drug's stability, determining its shelf life, and ensuring patient safety by identifying the conditions that lead to degradation. They are also useful for the development of stable formulations and compliance with regulatory requirements, ensuring that the drug remains effective throughout its shelf life^15-20. Hence, the work aims to develop and validate stability indicating the HPTLC method for Tianeptine sodium in bulk and tablet dose form.        

2. Experimental

2.1. Instruments and Apparatus, Camag HPTLC system with a Linomat V automatic sample applicator (100μl syringe), Camag TLC Scanner-III, Twin-trough TLC developing chamber (10 ×10 cm) was used for this study. Pre-coated silica gel G₆₀F₂₅₄  HPTLC plates were used for the development and validation. Analytical balance (Shimadzu Corporation, Japan) and Digital ultrasonic cleaner (Mumbai, India) sonicator (APL-digital Ultrasonic Cleaner) were used in the study.

2.2. Materials. Tianeptine Sodium was obtained as a gift sample by Vega Biotech Private Limited, Vadodara, Gujarat. Methanol, acetone, and chloroform were purchased from Loba Chemie Pvt. Ltd. All solvents used for this study, were AR grade. The stable tablet (Serdia Pharmaceuticals Ltd, India.) 12.5 mg was purchased at the local pharmacy, Anand, Gujarat.

2.3. Selection of detection Wavelength. In the present study drug solution of 10 μg/ ml of Tianeptine sodium was prepared in methanol. This solution was scanned from 200 to 400 nm in the UV spectrum (UV 1700-Pharmaspec). The detection wavelength (λ_max) is 220 nm.

2.4. Selection of mobile phase. The standard solution of Tianeptine sodium was 5000 μg/ml which was used to optimize the mobile phase by trials and error method. In optimization, various solvents with different compositions were studied for the selection of the mobile phase. In the study the optimized mobile phase was found to be chloroform: acetone: glacial acetic acid (7:3:0.1 v/v/v).

2.5. Chromatographic Conditions.  Pre-coated silica gel aluminum plate G₆₀ F₂₅₄ with 10 x 10 cm size, layer thickness 0.2 mm was used for the entire study. Prior washing of the HPTLC plate was done with methanol and activated the HPTLC plate with 60°C for 10 minutes to remove the solvent and impurity. For 10 minutes, the HPTLC plate was saturated with the optimal mobile phase chloroform: acetone: glacial acetic acid (7:3:0.1 v/v/v) in the twin-trough chamber. The produced HPTLC plate was scanned using a TLC scanner III to take measurements.

2.6. Preparation of Standard Solution. 10 mg of Tianeptine sodium was accurately weighed and dissolved in a 10 ml methanol (1000 μg/ml). Further dilution with 1 ml of this solution to 10 ml methanol (100 μg/ml).

2.7. Validation of a method: The HPTLC method for Tianeptine sodium was validated as per ICH guideline (Q2) (R1).

2.7.1. Linearity. In methanol, a stock solution of Tianeptine sodium (100 µg/ml) was made. Using the LINOMAT-V automatic sample applicator, Various stock solution volumes of 1, 2, 3, 4, 5, and 6 (100–600 ng per spot) were precisely applied to the TLC plate. After that, the plate was developed using the previously optimized mobile phase and then scanned. Repeat the experiment with five times of TLC plate. Over the concentration range of 100-600 ng/band, the linearity was determined ¹⁵.

2.7.2. Accuracy (% Recovery). The accuracy study was performed by spiking samples of the Tianeptine sodium (100 µg/ml) solution with three different concentrations of the level at 80%, 100%, and 120% and the absorbance of all solutions was taken at 220 nm and percentage recovery of Tianeptine sodium was calculated. The method was repeated three times¹⁵.

2.7.3. Method Precision (Repeatability). By repeatedly spotting the standard solution of Tianeptine sodium (300 ng/band) and scanning the same place (n = 6) of Tianeptine sodium without moving the plate, the precision was assessed. Relative standard deviation (% RSD) is used to describe the outcomes ¹⁵.

2.7.4. Intermediate Precision (Reproducibility). The corresponding responses for three different concentrations of Tianeptine sodium (100, 300, and 500 ng/spot) were calculated three times on the same day and on three other days to assess the Intraday and Interday precisions. The data is shown as a relative standard deviation (% RSD) ¹⁵.

2.7.5. Limit of Detection and Limit of Quantitation. Using the formulas provided below, the LOD and LOQ were derived from the standard deviation (SD) of the intercepts and slope of the calibration curves of Tianeptine sodium ¹⁵.

                    LOD = 3 σ/S

                    LOQ = 10 σ/S

Where σ is the standard deviation (SD) of intercepts and S is the slope of the calibration curve.

2.7.6. Robustness of the Method.

To assess robustness, purposeful variations in technique variables were made, including mobile phase composition (±0.5 ml for each component), and saturation duration (±2 min) for the 300 ng/band Tianeptine sodium. The %RSD was computed^21-23.

2.8. Assay.  The weight of the twenty tablets (Stablon) was measured, the average weight was calculated, and then they were crushed into a fine powder. The powder, equivalent to 10 mg of Tianeptine sodium, was placed into a 10 ml volumetric flask along with 5 ml of methanol. After sonication for 15 minutes, the solution was diluted with methanol up to the mark to achieve a concentration of 1000 µg/ml. The resulting solution was filtered using filter paper, and from this 1 ml volume was taken and transferred into a 10 ml volumetric flask and diluted to mark with methanol to obtain a working standard solution Tianeptine 100 µg/ml. 3 µl of this solution was put on the HPTLC plate at 300 ng per spot. Three times repeat the experiment, with peak areas recorded at 220 nm and sample concentrations calculated ^21-23.

2.8. Forced Degradation study for Tianeptine sodium in API and tablet dose form.  To evaluate the stability-indicating method, the standard drug and tablet sample was subjected to acid, base, oxidative, thermal, and photolytic degradation.

2.8.1 acid-induced degradation study for API. Accurately weighed 10 mg of Tianeptine sodium, transferred it into a 10 ml volumetric flask, and makeup with methanol. Transfer 1 ml of aliquot from the stock solution into a 10 ml volumetric flask. Add 2 ml of 0.1N hydrochloric acid to the flask and allow it to sit for 60 minutes. The neutralized solution was directly applied to the HPTLC plate followed by development and scanning under optimized chromatographic conditions ^15-17. Acid-induced degradation study for Tablet sample. Equivalently weighed 10 mg of tablet powder, transferred it into a 10 ml volumetric flask, and makeup with methanol. Sonicated for about 15 minutes with occasional shaking. Transfer 1 ml of aliquot from the stock solution into a 10 ml volumetric flask. Add 2 ml of 0.1N hydrochloric acid to the flask and allow it to sit for 60 minutes. Apply the neutralized solution directly to an HPTLC plate, then proceed with development and scanning under optimized chromatographic conditions^15-17.

2.8.2 Base-induced degradation study for API. Accurately weighed 10 mg of Tianeptine sodium, transferred it into a 10 ml volumetric flask, and made it with methanol. Transfer 1 ml of aliquot from the stock solution into a 10 ml volumetric flask. Add 2 ml of 0.1N sodium hydroxide to the flask and allow it to sit for 60 minutes. Apply the neutralized solution directly to an HPTLC plate, then proceed with development and scanning under optimized chromatographic conditions ^15-17. Base-induced degradation study for Tablet sample. Equivalently weighed 10 mg of tablet powder, transferred it into a 10 ml volumetric flask, and makeup with methanol. Sonicated for about 15 minutes with occasional shaking. Transfer 1 ml of aliquot from the stock solution into a 10 ml volumetric flask. Add 2 ml of 0.1N sodium hydroxide acid to the flask and allow it to sit for 60 minutes. Apply the neutralized solution directly to an HPTLC plate, then proceed with development and scanning under optimized chromatographic conditions ^15-17.

2.8.3 Hydrogen peroxide-induced (oxidation) degradation study for API. Accurately weighed 10 mg of Tianeptine sodium, transferred it into a 10 ml volumetric flask, and made it with methanol. Transfer 1 ml of aliquot from the stock solution into a 10 ml volumetric flask. Add 2 ml of 3% w/v H₂O₂ to the flask and allow it to sit for 60 minutes made up to the mark. The solution obtained was applied directly onto the HPTLC plate, and the chromatogram was then documented using optimized chromatographic conditions ^15-17. Hydrogen peroxide-induced (oxidation) degradation study for Tablet sample. Equivalently weighed 10 mg of tablet powder, transferred it into a 10 ml volumetric flask, and made up with methanol. Sonicated for about 15 minutes with occasional shaking. Transfer 1 ml of aliquot from the stock solution into a 10 ml volumetric flask. Add 2 ml of 3% w/v H₂O₂ to the flask and allow it to sit for 60 minutes made up to the mark. The solution obtained was applied directly onto the HPTLC plate, and the chromatogram was then documented using optimized chromatographic conditions.

2.8.4 Photolytic degradation study for API.  Accurately weighed 10 mg Tianeptine sodium was irradiated with sunlight for 60 min. A solution was prepared by dissolving a drug sample in the diluent. From this stock solution of 1000 µg/ml, a working standard solution of 100 µg/ml was prepared. The resulting solution was applied on a TLC plate and chromatograms were run. Photolytic degradation study for Tablet sample. Equivalently weighed 10 mg of tablet powder of Tianeptine sodium was irradiated with sunlight for 60 min. A solution was prepared by dissolving a drug sample in the diluent. Sonicated for about 15 minutes with occasional shaking. From this stock solution of 1000 µg/ml, a working standard solution of 100 µg/ml was prepared. The resulting solution was applied on a TLC plate and chromatograms were run ^15-17.

2.8.5 Thermal degradation for API. Accurately weighed 10 mg Tianeptine sodium was placed in a hot air oven at 75?C for 60 min. The solution was prepared by dissolving the drug sample in the diluent. From this stock solution of 1000 µg/ml, a working standard solution of 100 µg/ml was prepared. The resulting solution was applied on a TLC plate and chromatograms were run. Thermal degradation for Tablet sample. Equivalently weighed 10 mg of tablet powder Tianeptine sodium was placed in a hot air oven at 75?C for 60 min. The solution was prepared by dissolving the drug sample in the diluent. Sonicated for about 15 minutes with occasional shaking. From this stock solution of 1000 µg/ml, a working standard solution of 100 µg/ml was prepared. The resulting solution was applied on a TLC plate and chromatograms were run ^15-17.

2.8.6 Degradation kinetics In acidic medium

The stock solution of Tianeptine sodium was prepared up to 1000 µg/ml. 1 ml of tianeptine sodium was refluxed at different temperatures (40°C, 50°C, 60°C, 70°C, and 80°C). After refluxing, 2 ml of 0.1 N HCl was added. The neutralized solution of Tianeptine sodium was applied on a chromatographic plate under optimized chromatographic conditions. Each study was repeated three times at different temperatures with different internal. The concentration of the drug was calculated. Various parameters like shelf life, degradation rate constant, half-life, and activation energy were determined by using degradation kinetics and the Arrhenius plot. From the Arrhenius plot, ????_1/2 and ????₉₀ were determined at 25 °C for acid degradation ^15-17.

In a basic medium

The stock solution of Tianeptine sodium was prepared up to 1000 µg/ml. 1 ml of Tianeptine sodium was refluxed at different temperatures (40°C, 50°C, 60°C, 70°C, and 80°C). After refluxing, 2 ml of 0.1 N NaOH was added. The neutralized solution of Tianeptine sodium was applied on a chromatographic plate under optimized chromatographic conditions. Each study was repeated three times at different temperatures with different internal. The concentration of the drug was calculated. Various parameters like shelf life, degradation rate constant, half-life, and activation energy were determined by using degradation kinetics and the Arrhenius plot. From the Arrhenius plot, ????_1/2 and ????₉₀ were determined at 25 °C for basic degradation ^16,17.

3. RESULTS

3.1. Optimization of Chromatographic condition:

Various solvents with different compositions and ratios were tried for the HPTLC plate for Tianeptine sodium. The optimized mobile phase was chloroform: acetone: glacial acetic acid (7:3:0.1, v/v/v). The optimized saturation time with a twin-trough developing chamber was 10 minutes with the same mobile phase. (Figure 1)

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Fig 2: (a) Densitogram of Tianeptine sodium standard 100 ng/band using Chloroform: Acetone: Glacial acetic acid (7:3:0.1 v/v/v) as mobile phase (b) View of all tracks of Tianeptine sodium

3.2.1. Calibration curve

 The calibration curve for Tianeptine sodium showed a good linear relationship over the range of 100-600 ng per spot (r² = 0.999, slope = 4.609, intercept = 148.5).  (Table 1 & Figure 2)

Table 1: Linearity study of Tianeptine sodium at 220nm

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Fig 3: Calibration curve of Tianeptine sodium at 220 nm

3.2.2. Precision

Repeatability for Tianeptine sodium was found to be 0.91% for six replicates with the same concentration. The % RSD values obtained for intraday and interday variation were 0.72-1.51 % and 1.42-1.64 %, respectively.

3.2.3. Robustness

The % RSD value of the robustness study was found to be 0.54 for mobile phase composition and 0.30 for saturation time. The result indicated that the optimized method was robust.

3.2.4. LOD and LOQ

The LOD and LOQ were found to be 16.75 and 50.77 ng per band for Tianeptine sodium.

3.2.5. Specificity

The specificity of Tianeptine sodium was determined by analysis of the standard and sample of the drug. It indicates the no interference of degradation product or impurity (Figure 3).

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Fig 4: Overlain spectra of standard solution and dose form solution

3.2.6. Accuracy

% Recovery of Tianeptine sodium was found to be 100.4 % which is within the limit (98%-102%) as per ICH guidelines. So, the developed method was found to be accurate.

3.3. Analysis of marketed formulations.

The amount of Tianeptine sodium (12.1 mg) obtained in tablets concerning the label claim (12.5 mg). The drug content of Tianeptine sodium in tablets was found to be 99.15 % ± 10.10 %.

There is a summary of validation parameters in Table 2.

Table 2: Summary of validation parameters of the proposed HPTLC method

3.4. Forced degradation study for Tianeptine sodium in API and table dose form.

The percentage degradation for Tianeptine sodium was found to be 25.46 % in API and 23.42 % in tablet at R_f value 0.62 in the acid-induced degradation study. In basic conditions, the percentage degradation was found to be 12.20 % in API and 13.87 % in tablet at R_f value 0.61. For their oxidative study one degradation peak was found at 0.61 R_f value (11.64 % in API and 12.25% in tablet). The photolytic degradation was also found at the R_f value of 0.62. The Tianeptine sodium was also susceptible to thermal conditions. The % degradation peak was found 13.67% in API and 14.86% in tablet dose form. The result also indicated that the degradation mixture may be found to be the same. Hence it was obtained at the same R_f value (Figure 4).

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Fig 5 : Densitogram of API of Tianeptine sodium and its degradation products in the (a) acidic, (b) basic, (c) oxidative, (d) photolytic, and (e) thermal degradation study

3.5 Degradation kinetics

In the data (Table 3,4) and graph (Figure 5) of degradation kinetics, it was observed that the concentration of the drug decreased with time. This means when the time increases, the concentration of the drug also decreases, the effect of temperatures 40°, 50°, 60°, 70° and 80°C in the degradation kinetics was shown in Fig 5 in acidic conditions and basic conditions. The K_obs the apparent first-order degradation rate constants, t_90, and half-lives (t_1/2) were calculated from the graph of degradation. In the context of degradation studies, pseudo-first-order kinetics (Figure 5) is a situation where the reaction rate appears to follow first-order kinetics concerning one reactant. The data obtained from the first-order kinetics treatment were subsequently analyzed using the Arrhenius equation.

Log K = Log A – Ea/2.303 RT

Where K is rate constant, A is frequency factor, E_a is energy of activation (Cal mol-¹), R is gas constant (1.987 cal/deg . mol) and T is absolute temperature (°K), In the Arrhenius plot, a linear relationship was observed between the values of (2+ log Kobs) and (1/T × 103) within the temperature range of 40-80°C.

Table 3. Summary Of Degradation Products of Tianeptine Sodium Under Different Stress Conditions.

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Fig 6:  Pseudo first-order plots for the degradation of Tianeptine sodium with (a) 0.1 N HCl and (b) 0.1 N NaOH at various temperatures using the HPTLC method.

Table 4. The degradation rate constant (Kobs), half-life(t_1/2), and t₉₀ for Tianeptine sodium in the presence of 0.1 N HCl and 0.1 N NaOH.