High Performance Thin Layer Chromatography method for estimation of Rosuvastatin Calcium and Teneligliptin Hydrobromide Hydrate from its tablet dosage form

Nansi Hadvani*¹, Dr. Dinesh Dangar², Dr. Amitkumar Vyas³

1.      Pharmaceutical Quality Assurance Department, Dr. Subhash University, Dr. Subhash Road, Joshipura, Junagadh, Gujarat 362001

2.      School of Pharmacy, Dr. Subhash University, Dr. Subhash Road, Joshipura, Junagadh, Gujarat 362001           

3.      Pharmaceutical Quality Assurance Department, B.K. Mody Govt. Pharmacy College, Polytechnic Campus, Bhavnagar Rd, Near Aji Dam Road, GIDC, Rajkot, Gujarat 360003

*Correspondence: nansihadvani@gmail.com; Tel.: +91-6351833860

DOI: https://doi.org/10.71431/IJRPAS.2025.4704 

INTRODUCTION

Rosuvastatin Calcium, chemically [3R,5S,6E3R,5S,6E3R,5S,6E-7‑(4‑fluorophenyl)‑3,5‑dihydroxy‑6‑[(4‑phenyl)(1H‑pyrrol‑1‑yl)methyl]hept‑6‑enoic acid] calcium salt, is a potent HMG‑CoA reductase inhibitor widely used for lowering low-density lipoprotein (LDL) cholesterol and reducing cardiovascular risk. [1] Teneligliptin Hydrobromide Hydrate, or 2S,4S2S,4S2S,4S-4-4−(3‑methyl−1−phenyl−1H−pyrazol−5−yl)piperazin−1−yl4-(3‑methyl-1-phenyl-1H-pyrazol-5-yl) piperazin-1-yl4−(3‑methyl−1−phenyl−1H−pyrazol−5−yl)piperazin−1−ylpyrrolidin‑2‑yl(1,3 thiazolidin‑3‑yl)methanone hydrobromide hydrate, is a dipeptidyl peptidase‑4 (DPP‑4) inhibitor prescribed for glycemic control in type 2 diabetes mellitus. [2]

This fixed‑dose combination of Rosuvastatin and Teneligliptin has been introduced to manage dyslipidemia co-existing with type 2 diabetes, offering synergistic benefits in controlling lipid profiles and blood glucose simultaneously. Since its regulatory approval around 2021, this combination has gained attention for its dual therapeutic potential in reducing macrovascular complications.

High-Performance Thin-Layer Chromatography (HPTLC) is an advanced form of thin-layer chromatography, using finer particles on silica gel plates and capable of higher resolution, throughput, and quantitative accuracy with minimal solvent consumption. The technique enables parallel analysis of multiple samples and is highly amenable to stability-indicating, forced-degradation studies, making it ideal for pharmaceutical quality control.

While several analytical methods—particularly, UV [3,4,5,6] RP‑HPLC [7] and RP‑UPLC [8] have been reported for individual estimation of both drugs and various other methods like UV and HPLC are developed for simultaneous estimation of Rosuvastatin and Teneligliptin in synthetic mixtures [9,10,11,12]. There is also one HPTLC method which addresses simultaneous quantification of the Rosuvastatin–Teneligliptin combination in tablet form which is a stability indicating HPTLC method [13]. But a novel HPTLC method for simultaneous estimation of Rosuvastatin Calcium and Teneligliptin Hydrobromide Hydrate in a tablet dosage form is developed which improves the separation efficiency and Rf value of both drugs. Method development and validation were performed following ICH Q2(R2) guidelines, to ensure specificity, robustness, accuracy, and precision.

Figure 1. Chemical structure of Rosuvastatin

Figure 2. Chemical structure of Teneligliptin

MATERIALS AND METHODS

Analytically pure Rosuvastatin and Teneligliptin were kindly provided as a gift sample by Sunij Pharmaceuticals, Ahmedabad, Gujarat. High-Performance Thin-Layer Chromatographic Instrument was of Camag. All the solvents used were of analytical grade. TENLIFAST-RV tablets were obtained from local market of Junagadh, Gujarat.

Ratio of drugs

In the tablet of Rosuvastatin calcium and Teneligliptin Hydrobromide Hydrate, 10mg of Rosuvastatin Calcium and 20mg of Teneligliptin Hydrobromide Hydrate is present. Based on the ratio (1: 2), a solution comprising 1000 μg/mL of Rosuvastatin Calcium and 2000 μg/mL of Teneligliptin Hydrobromide Hydrate was prepared in methanol to achieve suitable separation.

Selection of Stationary Phase

A silica gel G 60F254TLC plate precoated on an aluminium support was used to separate rosuvastatin and teneligliptin hydrobromide hydrate. The silica particles were 2 mm in diameter, and the adsorbent layer was 0.2 mm thick. For technique development, the plates were given in a 20 by 20 cm size and reduced to the suitable size (10 x 10).

Selection of Mobile Phase

The mobile phase component and their ratio were selected based on the solubility and polarity and review of literature of both drugs. The solution of drug was prepared in methanol and used for spotting. Methanol gets vaporized soon after application onto the plate under ​nitrogen stream.  After trying a different mobile phase system an ideal system was chosen based on the resolution between compounds. Several trials were performed in order to get optimum resolution. Toluene: Methanol: Ethyl acetate: Glacial acetic acid: TEA (5: 3: 2: 0.1: 0.1 v/v/v/v/v).

Preparation of Solution

Rosuvastatin Calcium + Teneligliptin Hydrobromide Hydrate (10+20mg) were accurately weighed and transferred into 10ml Volumetric flask. 5ml of methanol was added and dissolved them and volume was made up to the mark with methanol (1000 µg/ml + 2000 µg/ml).

Selection of Analytical Wavelength

A standard solution of ROSUVA (10 μg/ml) and TENELI (20 μg/ml) in methanol was scanned between 200 and 400 nm, and 246 nm was chosen as the analytical wavelength based on the observation of overlain spectra. Where both of the components had a sufficient UV response.

RESULT AND DISCUSSION

The proposed HPTLC method was systematically validated according to the ICH protocols. The methods performance was evaluated based on system suitability, linearity, precision, accuracy, sensitivity (LOD & LOQ), and robustness.

System Suitability Parameter

Three times a solution of Rosuvastatin Calcium + Teneligliptin Hydrobromide Hydrate (1+2 μg/band) was spotted to determine the system appropriateness parameter of peak purity and retardation factor. RSD determined the system suitability parameter for the specified concentration.

Figure 3. Optimized Densitogram of ROSUVA & TENELI at 246 nm

Table 1. System Suitability Parameters

Linearity and Range

The solution containing ROSUVA+ TENELI (1000+2000 μg/mL) has been applied in concentration 1-5 μL for determination of Linearity and Range. Responses were linear for ROSUVA in range of 1-5 μg/band, and TENELI in range of 2-10 μg/band.

Table 2. Linearity data of Rosuvastatin

Table 3. Linearity data of Teneligliptin

Figure 4. Calibration curve of Rosuvastatin

Figure 4. Overlain 3D Spectra of Rosuvastatin and Teneligliptin

Figure 5. Calibration curve of Teneligliptin

Repeatability

Using optimal chromatographic conditions, standard mixes containing Rosuvastatin Calcium (1-5 μg/Band) and Teneligliptin Hydrobromide Hydrate (2-10 μg/Band) were spotted on TLC plate. The peak area of each standard mixture was measured five times and RSD checked the consistency of each concentration. According to ICH criteria, the C.V. value should be less than 2, and the observed C.V. is less than 2 for all concentrations of Rosuvastatin and Teneligliptin.

Table 4. Repeatability studies for Rosuvastatin and Teneligliptin

Intraday and Inter-day Precision

Intraday and interday precision were used to determine method precision. For intraday precision, a mixture representing the whole range (ROSUVA+TENELI = 1+2, 3+6, and 5+10 μg/band) was examined on the same day at different time intervals. On separate days, a mixture representing the overall range (ROSUVA+TENELI = 1+2, 3+6, and 5+10 μg/band) was evaluated for inter-day precision.

Table 5. Intraday and Interday Precision study for Rosuvastatin and Teneligliptin

Limit of Detection (LOD) and Limit of Quantification (LOQ)

LOD and LOQ were calculated using a statistical approach using repeatability data. According to ICH, the approach based on the standard deviation of the response and mean of slope was used for determining the Limit of detection (LOD) and limit of quantitation (LOQ). The detection limits for TEN and ROS were found to be 0.12 µg/band and 0.037 µg/band, respectively, while quantitation limits were found to be 0.38 µg/band and 0.11 µg/band, respectively. The above data shows that a microgram quantity of both the drugs can be accurately and precisely determined. The values of LOD and LOQ of TEN and ROS respectively indicate the sensitivity of proposed method.

Accuracy

The accuracy of the test was determined by spiking the placebo with the standard. ROSUVA and TENELI (3+6 μg/spot) were the concentrations to aim for. Hydroxypropyl methylcellulose (40 mg) + microcrystalline cellulose (124 mg) + Talc (4 mg) + Magnesium stearate are the ingredients in the placebo (2mg). 10 mg ROSUVA and 20 mg TENELI diluted in 10 mL methanol (master stock solution). (1000 μg/mL+2000 μg/mL).

Table 6. Accuracy study for Rosuvastatin and Teneligliptin

Robustness

To determine the method's robustness, the following parameters were modified one by one, and the effect was seen using standard preparation.

1) Development distance (about + 5mm), with an optimal development distance of

80mm.

2) Detection wavelength (+ 2nm), using 246nm as the optimal wavelength.

3) Optimized ratio of mobile phase composition (+ 0.2ml)

4) Saturation time (about + 5 minutes); the optimal saturation duration was 20 minutes.

The percent C.V. levels were discovered to be under 2% of the acceptable standard.

At concentrations of 3 μg/band (ROSUVA) and 6 μg/band (TENELI), robustness was tested.

Assay

20 tablets were crushed and powder equivalent to 10 mg ROSUVA and 20mg TENELI was dissolved in 10ml methanol and a stock solution of 1000 μg/ml and 2000 μg/ml of ROSUVA and TENELI was prepared respectively. Then withdraw 1 ml from master stock solution in 10ml volumetric flask; make up the volume with methanol, which will contain 100 μg/ml of ROSUVA and 200 μg/ml of TENELI. Using optimal chromatographic conditions, 3 μL spot from stock solution was taken three times on TLC plate.

Table 7. Assay study for Rosuvastatin and Teneligliptin

CONCLUSION

A robust, dependable and accurate HPTLC method was successfully formulated and confirmed for the combined analysis of Rosuvastatin and Teneligliptin in both active drug substance (API) form and combined tablet dosage formulation. The method demonstrated excellent validation parameters, in compliance with ICH protocols. The retardation factor for both analytes were well-resolved, unaffected by excipients or breakdown compounds, confirming the methods specificity. The proposed method is therefore appropriate for regular quality testing & stability examination of Rosuvastatin and Teneligliptin within medicinal preparations.

CONFLICT OF INTEREST

Authors declare no conflict of interest.

ACKNOWLEDGEMENT

I would like to express my sincere gratitude to Dr. Dinesh Dangar and Dr. Amitkumar Vyas for their valuable guidance, encouragement, and support throughout the work.

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