The absorbance ratio method allows for the simultaneous estimation of two components by utilizing the property that the ratio of absorbances at any two wavelengths remains constant regardless of the concentration or path length. This method involves measuring the absorption at two selected wavelengths, one of which is an iso-absorptive point and the other being the ?max of one of the two components.1 Hypolipidemic drugs, or lipid-lowering medications, are used to reduce high levels of lipids, such as cholesterol and triglycerides, in the blood.2,3  Rosuvastatin calcium (ROS) is a potent statin used as a hypolipidemic drug. It is  Chemically known as calcium (E)-7-[4-(4-fluorophenyl)-6-isopropyl-2 [methyl(methylsulfonyl)amino]pyrimidin-5 yl](3R,5S)-3,5-dihydroxyhept-6-enoate. It is an HMG-CoA reductase inhibitor that reduces cholesterol synthesis in the liver.4 This results in lower LDL cholesterol, increased HDL cholesterol, and reduced triglycerides. With a molecular formula of C44H54CaF2N6O12S2, rosuvastatin calcium is effective in managing dyslipidaemia, although monitoring for side effects like muscle pain and liver enzyme changes is necessary.5

       
           
       
    Fig 1: Structure of Rosuvastatin Calcium

Ezetimibe (EZE) is a cholesterol absorption inhibitor used to lower cholesterol levels by preventing cholesterol absorption from the small intestine. Its chemical name is (3R,4S)-1-(4-fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl) azetidin-2-one, with a molecular formula of C24H21F2NO3 and a molecular weight of 409.43 g/mol.6 Ezetimibe inhibits the NPC1L1 protein, reducing intestinal cholesterol delivery to the liver and lowering plasma cholesterol levels. It is used to treat primary hyperlipidaemia, homozygous familial hypercholesterolemia, and homozygous sitosterolaemia, and is often combined with statins. Common side effects include headache and diarrhoea, while serious ones include myopathy and hepatitis.7

       
           
       
    Fig 2: Structure of Ezetimibe

Ezetimibe combined with rosuvastatin considerably reduces LDL cholesterol levels by leveraging complementary mechanisms: rosuvastatin reduces cholesterol synthesis in the liver, whereas ezetimibe limits cholesterol absorption in the intestine. This synergy results in greater LDL-C lowering and better cardiovascular outcomes than monotherapy. Furthermore, the combination can reduce the requirement for higher statin doses, aiding individuals with statin intolerance, and has the potential to improve adherence through single-pill formulations.8,9 A detailed survey of analytical literature revealed that several analytical methods like High-performance Thin Layer Chromatography, High-performance Liquid Chromatography, and UV Spectrophotometric determination of Rosuvastatin calcium and ezetimibe either alone or from combined tablet dosage form are available. Still, the existing UV methods use costly solvents for the estimation. The main objective of the present work is to develop and validate a simple, sensitive, reproducible, and cost-effective UV spectrophotometric Q absorption ratio method for the simultaneous determination of Rosuvastatin Calcium and Ezetimibe in combined tablet dosage form.

Reagents and Chemicals:

Standard solutions of calibration curves for spectrophotometric measurements were prepared by dissolving ROS and EZE in isopropyl alcohol (IPA): water mixture (50:50) to obtain the concentration of 1mg/ml for each compound. For calibration, the above solutions were prepared containing ROS and EZE OF 5 - 25 µg/ml by diluting the stock standard solution with IPA: Water in standard volumetric flasks(10ml). The solutions were scanned in the wavelength range of 200-400nm.

Selection of wavelength

After initially stabilizing the instrument for 30 minutes, the blank correction was done using an Isopropyl alcohol: water mixture (50:50 v/v). Then 10 µg/ml solution of Rosuvastatin calcium and Ezetimibe were scanned separately in UV region ranging from 200 nm to 400 nm. The absorption spectra were observed with maximum absorption at 232 nm and 244 nm for ROS and EZE respectively. From the overlay spectra, two wavelengths were selected one at 240 nm which was the iso-absorptive point and the other at 244 nm, ?max of Rosuvastatin calcium. Statistical parameters like slope, intercept, coefficient of correlation and SD were determined.

Analysis of Standard Drug Mixture

Weighed accurately 20 mg of Rosuvastatin calcium and 10 mg of Ezetimibe and transferred to a 10 ml standard flask. The drug mixture was then allowed to dissolve in a sufficient quantity of Isopropyl alcohol: water mixture (50:50 v/v) and then made up the volume to 10 ml with the same. From this a final solution was prepared have a concentration of 20 µg/ml Rosuvastatin calcium and 10 µg/ml Ezetimibe and the absorbance was measured at 240nm and 244nm.

Analysis of tablet formulation

Twenty tablets of ROZAVEL EZ 20 were weighed and powdered. A quantity of powder equivalent to 20 mg was weighed and transferred to a glass stoppered flask. The powder was extracted with a 5 ml Isopropyl alcohol: water mixture (50:50 v/v) by sonication for 10 minutes and filtered made up to 10ml in a standard flask. From the above solution, accurately pipetted out 1ml and transferred to a 100 ml standard flask. The volume was made up to the mark using the same solvent to obtain a concentration of 20 µg/ml of ROS and 10 µg/ml of EZE. Six different mixtures were prepared as above and the absorbance of final solutions was measured at 240 nm and 244 nm. The results are tabulated in Table 1.

Validation of proposed method

 The developed method was validated according to ICH guidelines Q2(R2).14

Accuracy

The accuracy of the developed method was determined by calculating the percentage recoveries of ROS and EZE by the standard addition method. A known amount of standards of ROS and EZE (80%, 100%, and 120%) were added to the pre-analysed sample solutions of tablet dosage forms. The amounts of ROS and EZE were estimated and the results are shown in Table 3. The values proved that the method is accurate.

Precision

Precision studies were done at two levels, Repeatability and Intermediate precision. The precision of the developed method was checked by repeatedly scanning (n = 6) standard solutions of ROS (20 ?g/ml) and EZE (10 ?g/ml). The % RSD values were found to be below 2% which indicates that the proposed methods are repeatable (Table 3). The intermediate precision for the proposed method was determined by estimating the same standard solution of ROS and EZE for three different days(interday). The results are reported in terms of % relative standard deviation (% RSD). The % RSD values were found to be below 2% which indicates that the proposed method is precise (Table 3).

Linearity

Calibration curves were constructed by plotting absorbance vs. concentrations of ROS and EZE, at their respective ?max and the regression equations were calculated. The calibration curves were plotted over the five different concentrations in the range of 5-25 ?g/ml for both ROS and EZE (Fig. 6 and Fig. 7). The optical parameters and statistical parameters are depicted in Table 2.

LOD & LOQ

The LOD and LOQ were estimated from five calibration curves drawn for each drug in their respective linearity range and calculated by the following equation.

LOD = 3.3 (?/S) LOQ= 10(?/S)

Where, ?= Standard deviation of y-intercepts of regression lines S= Slope of Calibration curves.

RESULTS AND DISCUSSION