Introduction

Rosuvastatin calcium is (E)-(3R, 5S)-7-{4-(4-flourophenyl)-6-isopropyl-2-[methyl (sulphonylamino)] pyrimidin-5-yl}-3,5-dihydroxyhepten-6-oic acid calcium, is a synthetic lipid-lowering agent which acts on plasma lipids [1]. It is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A, an enzyme responsible for the conversion of HMG-CoA to mevalonate, which was a rate-limiting step in cholesterol biosynthesis [2]. The structure of rosuvastatin calcium is given in Figure 1.

Figure 1: Rosuvastatin calcium

Figure 2: Teneligliptin hydrobromide hydrate

Teneligliptin hydrobromide hydrate belongs to an antidiabetic class. It increases incretin levels (GLP-1 and GIP), which reduces glucagon release, and ultimately increases insulin secretion, decreases gastric emptying, and blood glucose levels. Oral teneligliptin, a dipeptidyl peptidase-4 inhibitor, is indicated for the treatment of adults with type 2 diabetes (T2DM) [3, 4]. Structure of teneligliptin is given in Figure 2.

This combination is used in the treatment of a patient suffering from dyslipidemia associated with type 2 Diabetes Mellitus. Diabetes, and increasing resistance to insulin, even in persons considered to have ‘‘normal’’ insulin sensitivity, has been associated with higher concentrations of cholesterol and TG and lower concentrations of HDL cholesterol [4,5].

Various analytical methods are available to determine the rosuvastatin calcium and teneligliptin hydrobromide hydrate individually, which include HPLC method for rosuvastatin calcium[6,7], teneligliptin [8,9], HPTLC method for rosuvastatin calcium[10], HPTLC method for teneligliptin[11], LC-MS method for rosuvastatin calcium[12], LC-MS method for teneligliptin hydrobromide hydrate[13], UV method for rosuvastatin calcium[14,15] and UV method for teneligliptin[16-18]. But there is no method available that can simultaneously detect both in combination.

To the best of our knowledge, there is no UV method available for this combination. Therefore, in this study, it was decided to carry out the first-order derivative method. This method was validated to comply with the ICH(Q2R1) [19]. First-order derivative method spectroscopy was found to be more selective, accurate, precise, and simple for the estimation.

Material and methods

Chemicals and Reagents

Teneligliptin hydrobromide hydrate and rosuvastatin calcium and methanol were provided by B. K. Mody Government Pharmacy College. UV visible spectrophotometer (UV-1800 Shimadzu) was used; data were processed using UV probe (version 2.6) software. The experiment was carried out at B.K. Mody Government Pharmacy College in 2021.

Instrumentation

The proposed work was done on a Shimadzu UV-visible spectrophotometer (model UV-1800 series), which possesses a double beam double detector configuration with a 1cm quartz matched cell. All weighing was done on electronic balance (MAB 220 Wensar).

Selection of Solvents

Based on the solubility study, methanol was selected as the solvent for dissolving teneligliptin hydromide hydrate and rosuvastatin Calcium.

Preparation of standard stock solution

A standard stock solution of 100 µg/ml of rosuvastatin calcium and 100 µg/ml teneligliptin hydrobromide hydrate was prepared in methanol as diluents.

Selection of wavelength

From appropriate dilution of the working standard stock solution, 10µg/ml of Rosuvastatin calcium and teneligliptin hydrobromide hydrate were separately prepared and scanned in the UV range 200-400 nm. The overlain zero-order absorption spectra of both drugs were obtained. These absorption spectra were converted to 1^st order derivative spectra by using UV probe software. After observing overlay 1^st order derivative spectra with Δλ 16 and scaling factor 1 for Rosuvastatin calcium and teneligliptin hydrobromide hydrate, zero-crossing points of both drugs were selected. The first wavelength selected was 222.66 nm (Zero crossing of Rosuvastatin calcium), where teneligliptin hydrobromide hydrate showed considerable absorbance. The second wavelength selected was 230.03 nm (Zero crossing of teneligliptin hydrobromide hydrate), where Rosuvastatin calcium showed considerable absorbance. 

Figure 3,4: Wavelength selection of rosuvastatin calcium and teneligliptin hydrobromide hydrate

Both drugs are highly overlapping so, 1^st order derivative method was performed

Method validation

Linearity

The standard stock solution was diluted with methanol properly to obtain the concentration of 1, 6, 12, 18, 24, 30, 36, and 42 µg/ml for both rosuvastatin calcium and teneligliptin hydrobromide hydrate.

Specificity

Specificity was performed under 6 replicates at 15 µg/ml of rosuvastatin calcium and teneligliptin hydrobromide hydrate with and without the addition of excipients to check the interference of excipients.

Accuracy

The accuracy of the method was performed in triplicate at three different concentration levels of 80%,100% and 120% (12, 15, and 18 µg/ml) for both drugs rosuvastatin calcium and teneligliptin hydrobromide hydrate. The accuracy was performed by spiking of the stated concentration in assay concentration. The accuracy of the method was checked by calculating percentage recovery.

Precision

Repeatability was performed under 6 replicates at a concentration of 15μg/ml of Teneligliptin hydrobromide hydrate and rosuvastatin calcium. Intra-day and inter-day variations of Teneligliptin hydrobromide hydrate and rosuvastatin calcium were performed in triplicate at three different concentration levels 80%, 100%, 120% (12, 15, and 18μg/ml). The results are expressed in the form of RSD.

Robustness

The robustness of the method was carried out by introducing a small change in experimental conditions like a wavelength. The changes made in wavelength ± 1 nm (229.30, 230.03, and 231.03 nm) for rosuvastatin calcium and (221.66, 222.66, and 223.66) was for teneligliptin hydrobromide hydrate.

LOD and LOQ

The limit of detection (LOD) and limit of quantification (LOQ) were calculated by the formula. The calibration curve was repeated and five deviations (SD) of the intercepts were calculated.

LOD = 3.3 × Standard deviation / Slope

LOQ = 10 × Standard deviation / Slope

Assay of a synthetic mixture

Synthetic mixture was prepared by equivelant to take 20mg for both ROSU and TENELI with common tablet excipent in adequate amount. This synthetic mixture was diluted with methanol to make concentration 15 μg/mL for both drugs.

Result and Discussion

Linearity          

Linear responses were obtained in the concentration range of 1-42 µg/ml for both drugs. The data for linearity is shown in Tables 1 and 2. The calibration curve for rosuvastatin calcium and teneligliptin hydrobromide hydrate at 230.03 and 222.66 nm is given in Figure 5 and 6.

Figure 5: Calibration curve of ROSUVA                        

Figure 6: Calibration curve of TENELI

Figure 7: Calibration curve of rosuvastatin              

Figure 8: Calibration curve of teneligliptin

Specificity

Excipient interference is not observed at the working wavelength of 230.03nm for Rosuvastatin calcium and 222.66 for teneligliptin hydrobromide hydrate. The UV spectroscopic method presented in this study is specific for rosuvastatin calcium and teneligliptin hydrobromide hydrate.

Table 3: Specificity data of ROSUVA and TENELI

Accuracy

When used for evaluation of recovery at three concentration levels, 80%, 100%, and 120% after spiking with standard, the proposed method showed % recovery between 98% – 102% for both drugs.

Table 4: Recovery study of ROSUVA and TENELI

Precision

Repeatability and intermediate precision expressed in terms of RSD revealed that the proposed method provided an acceptable intraday and interday variation.

Table 5: Intraday and interday precision of ROSUVA and TENELI

Table 6: Repetability of ROSUVA and TENELI

LOD and LOQ

LOD and LOQ of rosuvastatin calcium and teneligliptin hydrobromide hydrate were determined by equation according to ICH guideline. LOD of ROSUVA and TENELI was found to be 0.2131 and 0.1204 respectively. And LOQ was found to be 0.646 and 0.3648 for ROSUVA and TENELI, respectively.

Robustness study

Deliberate change in different parameter wavelengths showed a relative standard deviation of absorbance less than 2%, indicating that the method was robust.

Table 7: Robustness study of ROSUVA and TENELI

Assay of synthetic mixture:

% Drug content of synthetic mixture of rosuvastatin and teneligliptin was found between 95.55-100.2778%.

Table 8: Assay of ROSUVA and TENELI mixture