Novel UV Spectrophotometer Methods for Quantitative Estimation of Empagliflozin (EMPA) and Linagliptin (Lina) Using Mixed Hydrotropy Solubilization

Simple, precise accurate, novel and safe UV-Spectrophotometric simultaneous equation method developed for the simultaneous estimation of poorly water-soluble drugs Empagliflozin (EMPA) and Linagliptin (LINA) in tablet dosage form using 2M ammonium acetate: 2M sodium citrate and (50:50% W/W) as mixed hydrotropic solution and validated as per ICH guidelines. This Method involves solving of simultaneous equations based on measurement of absorbance at two wavelengths 270 nm and 294 nm (λmax of EMPA and LINA) in hydrotropic solutions. Ammonium acetate and sodium citrate solution did not show any absorbance above 240 nm and thus no interference in the estimation of drugs was seen. EMPA and LINA follow Beers law in the concentration range of 10-50g/ml and 5-25g/ml (r2 = 0.999). % Recovery for both the drugs was in the range of 98.50 to99.23 % indicating excellent accuracy. The methods were precise, with a relative standard deviation of less than 2% for both drugs. The developed methods were validated according to ICH guidelines and values of accuracy, precision and other statistical analysis were found to be in good accordance with the prescribed values. Thus, method can be used for routine monitoring of drugs in industry for the assay of bulk drugs and commercial formulation. Original Research Article Rathore and Goyal; JPRI, 33(58A): 620-627, 2021; Article no.JPRI.78974 621


INTRODUCTION
Empagliflozin (EMPA) is used as a sodium glucose cotransporter-2 (SGLT-2) inhibitor to improve glycemic control in adult patients with type 2 diabetes. SGLT-2 co-transporters reabsorb glucose from the glomerular filtrate in kidney and the glucuretic action resulting from inhibition of SGLT-2 which reduces renal absorption and lowers down the renal threshold for glucose, therefore increases glucose excretion which reduces hyperglycaemia and also helps in blood pressure reduction [1, 2]. Chemically EMPA is 1-chloro-4-(glucopyranos-1yl)-2-(4-(tetrahydrofuran-3-yloxy)benzyl)benzene and having empirical formula is C 23 H 27 ClO 7 with molecular weight 450.91 g/mole (Fig. 1A). Linagliptin (LINA) is having competitive, reversible DPP-4 inhibitory action which is responsible for DPP-4 breakdown reduction of GLP-1 and glucose-dependant insulin tropic polypeptide (GIP). From beta cells of the pancreas, GLP-1 and GIP stimulate the release of insulin during inhibiting release of glucagon from pancreatic beta cells. These effects together reduce the breakdown of glycogen in the liver and increase insulin release in response to glucose [2][3][4]. Chemically LINA is (R)-8-(3aminopiperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4methylquinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione and having empirical formula is C 25 H 28 N 8 O 2 with molecular weight 472.5422 g/mole (Fig. 1B). Literature review revealed that few methods were described for the determination of EMPA and LINA alone or in combination with other drugs from pharmaceutical dosage forms and in human plasma including spectrophotometry [5][6][7][8] [18][19][20][21][22][23][24][25][26]. Various organic solvents such as methanol, chloroform, dimethyl formamide and acetonitrile have been employed for solubilization of poorly water-soluble drugs to carry out spectrophotometric analysis. Drawbacks of organic solvents include their higher cost, toxicity and pollution. Hydrotropic solution may be a proper choice to preclude the use of organic solvents. Therefore, it was thought worthwhile to employ this mixed hydrotropic solution to extract out the drug from fine powder of tablets to carry out spectrophotometric estimation. There are no reports yet for the determination of this combination by proposed methods. The present work emphasizes on the quantitative estimation of EMPA and LINA in

Preliminary Solubility Studies of Drugs
Solubility of both drugs was determined at 25±1 0 C. An excess amount of drug was added to two screw capped 25 ml of volumetric flasks containing different aqueous systems viz. distilled water and different combination of hydrotropic agent. The volumetric flasks were shaken mechanically for 12 h at 25±1 0 C in a mechanical shaker. These solutions were allowed to equilibrate for next 24 h. and then centrifuged for 5 min at 2000 rpm. The supernatant liquid was taken for appropriate dilution after filtering through Whatman filter paper #41 and analyzed spectrophotometrically against water as blank. After analysis, it was found that the enhancement in the solubility of EMPA and LINA was found to be more than 60 to 70% in a mixture of 2 M ammonium acetate: 2M sodium citrate solution (1:1) as compared to solubility studies in other solvents.

Establishment of Stability Profile
Stability of EMPA and LINA was observed by dissolving in a mixture of 2M ammonium acetate: 2M sodium citrate (50:50 % V/V) solution used as hydrotropic agent. Solution of EMPA and LINA was scanned under time scan for 30 min. Spectra of the drug under time scan shows that drug is stable in hydrotropic solution.

Preparation of standard stock solution (Stock-A)
Standard stock solutions were prepared by dissolving separately 100 mg of each drug in 80`ml mixed hydrotropic solution containing 2M ammonium acetate: 2M sodium citrate (1:1) and the flask was sonicated for about 10 min to solubilize the drug and the volume was made up to 100 ml with mixed hydrotropic agent to get a concentration of 1000 µg/ml (Stock-A) for both drugs.

Preparation of sub stock solution (Stock-B)
Aliquots of 2.5 ml withdrawn with help of pipette from standard stock solution A of EMPA and LINA and transferred into 25 ml volumetric flask separately and diluted up to 25 ml with RO Water that gave concentration of 100 µg/ml (Stock-B).

Preparation of working standard solution
 Aliquots of 1.0 ml, 2.0 ml, 3.0 ml, 4.0 ml and 5.0 ml withdrawn with help of pipette from standard stock solution (Stock-B) separately in 10 ml volumetric flask and volume was made up to 10 ml with RO Water. This gave the solutions of 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml and 50 µg/ml respectively for EMPA.  0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml and 2.5 ml from sub stock solution (Stock-B) were taken separately in 10 ml volumetric flask and volume was made up to 10 ml with RO Water. This gave the solutions of 5 µg/ml, 10 µg/ml, 15 µg/ml, 20 µg/ml and 25 µg/ml respectively for LINA.

Selection of Wavelength for Linearity
Solutions of 5g/ml of EMPA and 10g/ml LINA were prepared separately. Both the solutions were scanned in the spectrum mode from 200 nm to 400 nm. The maximum absorbance of EMPA and LINA was observed at 270.0 nm and 294.0 nm, respectively. EMPA and LINA showed linearity in the concentration range of 10-50g/ml and 5-25g/ml at their respective maxima. Calibration curve was plotted, absorbance versus concentration Figs. 2, 3.

Study of Overlay Spectra
Working standard solution from the standard stock solution prepared in concentration 5μg/ml of EMPA and 10 μg/ml of LINA were scanned in the spectrum mode over the range of 200-400 nm against RO Water as blank and the overlain spectra of the two were recorded. EMPA showed an absorbance peak at 270.0 nm, whereas LINA at 294.0 nm. The overlain spectra also showed isoabsorptive points at 280.0 nm. Due to difference in absorbance maxima and having no interference with each other so both drug can be simultaneously estimated by simultaneous equation method Fig. 4.

Vierordt's Simultaneous Equation Method
Simultaneous equation method is based on the absorption of drugs (X and Y) at the wavelength maximum of the other. Two wavelengths selected for the method are 270.0 nm and 294.0 nm that are λ max of EMPA and LINA respectively. The absorbance was measured at the selected wavelengths and absorptivities (A 1%, 1cm ) for both the drugs at both wavelengths were determined as mean of five independent determinations. Concentrations in the sample were obtained by using following equations.
Where, A 1 and A 2 are absorbance of mixture at 270 nm and 294 nm respectively, ax 1 and ax 2 are absorptivities of EMPA at λ 1 (270.0 i.e. λ max of EMPA) and λ 2 (294.0 i.e. λ max of LINA) respectively and ay 1 and ay 2 are absorptivities of LINA at λ 1 and λ 2 respectively. C LINA and C EMPA are concentrations of EMPA and LINA respectively. Figure represent the overlain spectra of both the drugs in 1 :2 ratio and the criteria for obtaining maximum precision [i.e. absorbance ratio (A 2 /A 1 )/ax 2 /ax 1 and ay 2 /ay 1 ] by this method were calculated and found to be outside the range of 0.1-2.0 which is satisfied for both the EMPA and LINA [27].

Methods Validation
Validation of the method was carried out in accordance with the International Conference on Harmonization Q2B guidelines 2005 [28].

Linearity
The linearity of analytical method was carried out to check its ability to elicit test results that are proportional to the concentration of analyte in sample within a given range. Different levels of standard solutions were prepared and estimate into the UV and the results was recorded. The results of linearity are reported in Table 1.

Accuracy
The validity and reliability of proposed methods were assessed by recovery studies. The recovery of added standards (80%, 100% and 120%) was found at three replicate and three concentrations level. The value of % means just close to 100, SD and % RSD are less than 2 indicate the accuracy of method. Result of recovery study shown in Table 2.

Precision
Precision was determined by repeatability and Intermediate precision of drug. Repeatability result indicates the precision under the same operating condition over short interval time. The intermediate precision study is expressed within laboratory variation on different days and analyst to analyst variation by different analyst. The value of SD and % RSD are less than 2 indicate the precision of method. Result of precision shown in Table 3.

Analysis of tablet sample
Twenty marketed tablets of EMPA and LINA were weighed and ground to a fine powder; amount equal to 10mg of EMPA was taken in 10 ml volumetric flask. The LINA present in this amount of tablet powder was 5mg. Then 8 ml of 2M ammonium acetate: 2M sodium citrate (1:1) solution was added and the flask was sonicated for about 10 min to solubilize the drug present in tablet powder and the volume was made up to the mark with hydrotropic solution. After sonication filtration was done through Whatman filter paper No. 41. Filtrate was collected and further diluted with RO Water to get the final concentrations of both drugs in the working range. The absorbance of final dilutions was observed at selected wavelengths and the concentrations were obtained from simultaneous equation method. The procedure was repeated for five times Table 4.

CONCLUSION
There was no interference of 2M ammonium acetate: 2M sodium citrate solution (50:50% W/V) in the estimation and hence the Vierordt's simultaneous equation UV spectrophotometric methods were found to be simple, accurate, economic and rapid for simultaneous estimation of EMPA and LINA in bulk and tablet dosage forms. The proposed method can be successfully employed for the routine analysis of EMPA and LINA containing dosage forms.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.