INTRODUCTION:

Rosuvastatin calcium (ROSU) is a synthetic lipid-lowering agent that acts on plasma lipids^1,2. Teneligliptin hydrobromide hydrate (TENEG) belongs to the anti-diabetic class of medications. It works by increasing incretin levels such as GLP-1 and GIP, which in turn inhibit the release of glucagon³. The combination of these two drugs is used in the treatment of dyslipidemia associated with type 2 diabetes mellitus⁴.The structure of is given in figure no. 1a and 1b.

Figure 1a: ROSU Figure 1b: TENEG

With respect to regulatory compliance CCD and AQbD contributes a lot for RP-HPLC stability indicating methods or method development or.^5-7Spectrophotometric methods and LC-MS for formulations^8,9. Stability and pharmacological action is critically affects by Presence of impurities.^10-12.

Determination of ROSU and TENEG include Methods like HPLC for ROSU^13,14 and TENEG^15,16. The HPTLC for ROSU^17,18and HPTLC method for TENEG^19, LC-MS method for ROSU^20, and LC-MS method for TENEG^21,22 UV methods for ROSU^23,24 and TENEG.^25-27

There is a one RP-HPLC method available²⁸in which were no system suitability parameter data available, concentration is not mention in repeatability and linearity table and conclusion is not sufficiently written. Thus, it is worthwhile to develop method and validated²⁹ to have sufficient data and also reliable.

MATERIAL AND METHOD:

Mille Q water, HPLC grade MeOH, ACN and AR grade KH₂PO₄of Merck were used.

Nstrumentation:

The proposed work was carried out on a TSP Thermo Separation (1700 Infinity Series), which contains a UV detector and Peak ABC software was used for data analysis and optimized condition is shown in Table no.1.

Table 1: Optimization of RP-HPLC chromatographic condition

Sr. No	Parameter	Optimize Condition
1	Flow rate	1.0ml/min
2	Detection Wavelength	245nm
3	Mobile Phase Composition	Phosphate Buffer (pH 3): Methanol: Acetonitrile (75:20:5)
4	Column	Waters C18 column (25cm x 4.6mm, 5µm)
5	Injection volume	20µl

Preparation of Solutions:

Selection of Solvents:

HPLC Grade methanol was selected.

Buffer:

In 1L of water dissolves 1.36g of KH₂PO₄and brings pH to 3 with OPA.

Mobile Phase:

Mix buffer (3 pH), methanol, and acetonitrile (75:20:5 % v/v) and filter through 0.44µm membrane filter.

Standard stock (SS) solution:

A methanol was used to prepare 100µg/mL of ROSU and TENEG.

System suitability:

The adequacy was established by injecting 5 consecutive injections of a SS and parameters were assessed based on a chromatogram.

VALIDATION:²⁹

Linearity:

The SS was diluted properly with methanol to obtain the 10-50 µg/mL for both ROSU and TENEG.

Specificity:

Six replicates of specificity testing were conducted at a concentration of 20µg/mL for both ROSU and TENEG, with and without the presence of excipients, to assess interference from excipients.

LOD and LOQ:

The LOD and LOQ were determined using the formula specified in the ICH (Q2R1) guidelines¹⁹

Accuracy:

The method's accuracy was assessed in triplicate at 3 varied % levels (±50) corresponding to 10, 20, and 30 µg/mL) for both ROSU and TENEG. The accuracy was verified through the determination of % recovery.

Precision:

Repeatability was assessed with six samples of 20 μg/mL for both TENEG and ROSU. Intra-day and inter-day variability of TENEG and ROSU was examined in (n=3) at three specific concentration levels: 50%, 100%, and 150%.

Robustness:

The robustness of the method consist of wavelength ±1 nm and flow rate ±1mL/min changes.

Assay of Synthetic Mixture:

A synthetic blend was created to simulate a dosage of 20 mg for both ROSU and TENEG, containing standard tablet excipients in appropriate quantities. This blend was then diluted with methanol to achieve a concentration of 20μg/mL for both medications.

RESULT AND DISCUSSION:

The optimized method shows retention time of TENEG and ROSU is 2.086 and 3.199min respectively mentioned in Figure 2. Also meets the criteria for system suitability parameters, as demonstrated in Table 2, indicating its acceptability.

Figure 2: optimized chromatogram of TENEG and ROSU

Table 2: System Suitability Parameter

Peak	Retention time (min)	Theoretical plate	Tailing factor	Resolution
TENEG	2.083	5791	0.91	6.5
ROSU	3.199	6286	0.87	6.5

Validation:

Linearity:

The calibration curve is in 10 to 50μg/mL, for ROSU and TENEG, with correlation coefficients of 0.997 for both, and is depicted in Figure 3. The data for LOD and LOQ is illustrated in Table 3.

Table 3: LOD and LOQ

Peak	LOD (μg/mL)	LOQ
TENEG	0.075	0.228
ROSU	0.106	0.323

Specificity:

No interference from excipients as the percentage interference was below 0.05% for both drugs. Hence, the method demonstrates specificity.

Accuracy:

This method proves its precision, as the percentage of recovery fell within the range of 98 to 102% for both medications. This information is depicted in Table 4.

Precision:

Repeatability and intermediate precision are conveyed through the RSD. Since the RSD is determined to be < 2, it signifies that the method is precise. The summarized results can be found in Table 4.

Robustness:

Adjusting the flow rate and wavelength deliberately occurred, revealing a RSD of less than 2%, indicating the method's resilience. The outcomes are presented in Table 5.

Assay of synthetic mixture

The developed method was used to analyze a synthetic blend comprising 20mg of ROSU and 20mg of TENEG., the result revealed as shown in Table no 6.

Figure 3: calibration curve of ROSU and TENEG

Table 4: Accuracy and precision Data for ROSU and TENEG

Level	Accuracy		Precision				Repeatability
	%Drug Recovery		RSD
	%Drug Recovery		Intra-Day	Inter-Day			ROSU	TENEG
	ROSU	TENEG	ROSU	TENEG	ROSU	TENEG
50	99.91	100.13	1.16	1.81	1.08	1.65
100	100.97	99.92	0.50	1.56	0.52	0.88	1.24	1.75
150	99.24	100.09	0.64	1.57	0.34	1.22

Table 5: Robustness Study of ROSU and TENEG

Parameter	ROSU			TENEG
	Normal Condition	Variable 1	Variable 2	Normal Condition	Variable 1	Variable 2
Mobile Phase	75:25:5	73:21:6	77:94:4	75:25:5	73:21:6	77:94:4
RSD	0.38	0..92	1.1	0.67	0.87	0.89
Wavelength (nm)	245	244	246	245	244	246
RSD	1.65	1.24	1.15	0.74	0.87	0.61
Flowrate (mL/min)	1.0	0.9	1.1	1.0	0.9	1.1
RSD	0.70	1.70	1.02	1.41	1.08	0.50

Table 6: Assay of Synthetic Mixture

Drugs

Dose strength

%Assay

ROSU

TENEG

20mg

99.85%

101.91%

CONCLUSION:

RP-HPLC method for ROSU and TENEG detection in synthetic mixtures. Under ICHQ2 (R1) recommendations, validation for ROSU and TENEG was performed and were observed to be within recommendation of ICH Q2R1, with the accuracy of ROSU and TENEG being in the range of 99.24%-100.97% and 99.92% - 100.13%, respectively, and the precision being less than 2% in terms of RSD. Consequently, the method proved to be precise and applicable for quantifying the concentrations of ROSU and TENEG within a synthetic blend.

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Received on 21.04.2023 Modified on 06.01.2024

Research J. Pharm. and Tech 2024; 17(9):4325-4328.

DOI: 10.52711/0974-360X.2024.00668