UV SPECTROSCOPY:

UV-Visible spectroscopy is a valuable analytical technique used to analyze and identify substances based on their absorption or transmission of UV and visible light. This technique takes advantage of the fact that different substances absorb light at specific wavelengths due to the energy required to promote electrons to higher energy states. The relationship between the wavelength of light and its energy is crucial in understanding this process. Shorter wavelengths of light carry more energy, while longer wavelengths carry less energy. When light with the right energy level is absorbed by a substance, it promotes electrons to higher energy states, and this absorption is what UV-Visible spectroscopy detects. By analyzing the specific wavelengths at which maximum absorbance occurs, scientists can identify and quantify substances in a sample. The visible light spectrum that humans can perceive ranges from violet (around 380 nm) to red (around 780 nm). UV light has even shorter wavelengths, making it suitable for this analytical method. UV-Visible spectroscopy is widely used in various applications, such as determining the concentration of substances in a sample and identifying different compounds based on their unique absorption spectra. UV-Visible spectroscopy is indeed based on the interaction between light and matter, where the absorption of ultraviolet or visible light leads to the excitation of electrons from a ground state to an excited state. The energy difference between these states corresponds to the energy of the absorbed radiation, which is reflected in the resulting spectrum. This technique is valuable in analyzing the electronic structure of chemical compounds and determining their concentration in a sample.

WORKING METHOD OF UV-VISIBLE SPECTROSCOPY

1. Light Source This component emits light over a range of wavelengths, including ultraviolet (UV) and visible (Vis) light. Common sources include tungsten lamps for visible light and deuterium lamps for UV light.
2. Monochromator:

This is used to select a specific wavelength of light from the broad spectrum produced by the light source. It disperses the light into its constituent wavelengths and allows you to choose the desired wavelength for analysis.

3. Sample Compartment:

This is where you place your sample, which may be in a cuvette or other appropriate container. The light passes through the sample in this compartment.

4. Detector:

The detector measures the intensity of the light that passes through the sample. It is essential for quantifying the amount of light absorbed by the sample at the selected wavelength.

5. Display/Processing:

The instrument displays the data collected, usually as an absorbance spectrum showing how much light is absorbed at different wavelengths. It may also include software for data analysis and processing. These are the core components of a UV-Visible spectrophotometer, which is a versatile tool for analyzing the absorption of light by various substances, aiding in quantification and identification of  analytes in a sample.

DRUG PROFILE:

EMPAGLIFLOZIN:

IUPAC Name

2S,3R,4R,5S,6R)-2-[4-chloro-3-({4-[(3S)-oxolan-3-

yloxy] phenyl} methyl) phenyl]-6- (hydroxymethyl)oxane-3,4,5-triol

Melting Point

151-153°C

Uses:

1. Empagliflozin is a medicine used to treat type 2 diabetes.
2. Type 2 diabetes is a condition where the body does not make enough insulin, the insulin that it makes does not work properly. This can cause high blood sugar levels (hyperglycemia).

FINERENONE:

IUPAC Name

(4S)-4-(4-cyano-2-methoxyphenyl)-5- ethoxy-2,8-dimethyl-1,4-dihydro-1,6- naphthyridine-3-carboxamide

Melting Point

 554.7±50.0 °C

Uses:

Finerenone is used to lower the risk of serious kidney and heart problems (eg, kidney function decline, end-stage kidney disease, cardiovascular death, heart attack, and hospitalization for heart failure) in patients with chronic kidney disease

associated with type 2 diabetes.

MATERIALS AND METHOD:

Procurement of Drugs:

API:

Sample of Finerenone (FIN) procured from Actiza Pharmaceutical, Surat, Gujarat. Empagliflozin (EMP) as procured from Chemical Bull Pvt. Ltd., Vapi,

IR Spectra:

Drug Finerenone (FIN) and Empagliflozin (EMP) was placed in sample compartment of FT-IR instrument, where it was scanned in the range of 4000 - 650 cm-1. Principle IR peaks were observed for drug are shown and from this data it was concluded that drugs were found to be authentic.

Preparation of standard solutions:

Standard solution of Finerenone (FINE)

Accurately weighed 10 mg of Finerenone (FINE) were transferred separately in 10 ml volumetric flasks, dissolved in small volume of methanol and then volume was adjusted to the mark with methanol to obtain concentration of 1000 ?g/ml of Finerenone (FINE). These solutions were further diluted to obtain concentration of 100 ?g/ml of Finerenone (FINE). Then, five quantities of 1.0, 2.0, 3.0, 4.0 and 5.0 mL were pipetted out to 10- mL flasks and diluted with methanol to prepare a series of standard solutions of Finerenone (FINE) with concentrations of 10, 20, 30, 40, and 50 ?g/Ml

Standard solution of Empagliflozin (EMPA)

Accurately weighed 10 mg of Empagliflozin (EMPA)were transferred separately in 10 ml volumetric flasks, dissolved in small volume of methanol and then volume was adjusted to the mark with methanol to obtain concentration of 1000 ?g/ml of Empagliflozin (EMPA). These solutions were further diluted to obtain concentration of 100 ?g/ml of Empagliflozin (EMPA). Then, five quantities of 1.0, 2.0, 3.0, 4.0 and 5.0 mL were pipetted out to 10-mL flasks and diluted with methanol to prepare a series of standard solutions of Empagliflozin (EMPA) with concentrations of 10, 20, 30, 40, and 50 ?g/mL

Selection of Wavelength:

An ideal wavelength is the one that gives maximum response for the drugs that was to be detected. Scan UV spectra of Finerenone (FINE) and Empagliflozin (EMPA) with concentrations of 30 ?g/mL and 30 ?g/mL, respectively solutions individually between the ranges of 200nm-400nm using methanol as a blank. Overlay both the spectra and find iso-absorptive point if any if not, then select wavelength which have proper absorbance. An ideal wavelength is the one that gives Maximum response for the drugs that was to be detected. From the overlay spectra we are develop first derivative UV spectroscopy method for the estimation of Finerenone (FINE) and Empagliflozin (EMPA) with concentrations of 30 ?g/mL and 30 ?g/mL.

METHOD VALIDATION

Linearity:

The linearity of calibration curves in pure solution was checked over the concentration ranges of about 10-50 ?g/ml for Finerenone (FINE) and 10 – 50 ?g/ml Empagliflozin (EMPA). Appropriate aliquots from the standard stock solutions of Finerenone (FINE) were used to prepare two different sets of dilutions Series A and B stock solutions as follows. Series A consisted of different concentration of Finerenone (FINE) (10-50 ?g/ml). Aliquot from the stock solution of Finerenone (FINE) (100 ?g/ml) was pipette out in to a series of10 ml volumetric flask and diluted with methanol to get final concentration in range of 10 – 50 ?g/ml. Series B consisted of varying concentrations of Empagliflozin (EMPA) 10 – 50 ?g/ml. Appropriate volume of the stock solution of Empagliflozin (EMPA) (100 ?g/ml) was transferred into a series of 10 ml volumetric flask and the volume was adjusted to the mark with methanol to get final concentration in range of 10 – 50 ?g/ml. The calibration curve was constructed by plotting drug concentration versus the absorbance values.

Precision:

Repatibility:

The precision of the instrument was checked by repeated scanning and measurement of absorbance of solutions for Finerenone (FINE) and Empagliflozin (EMPA) with concentrations of 30 ?g/mL and 30 ?g/mL without changing the parameter of the proposed spectrophotometric method.

Intermediate Precision (Reproducibility)

The intraday and interday precision of the proposed method was determined by analyzing the corresponding responses 3 times on the same day and on 3 different days over a period of 1 Week for 3 different concentrations of standard solutions of 10, 30, 50 ?g/ml for Finerenone (FINE) and 10, 30, 50 ?g/ml Empagliflozin (EMPA). The intra and inter-day accuracy and Precision were calculated and results were presented in the table-2.

Accuracy:

To ensure the reliability of the above method recovery studies were carried out by mixing standard quantity of standard drug with the pre-analyzed sample synthetic mixture and the contents were re-analyzed by the proposed method. Recovery studies were carried out at 50,100 and 150 % level. The recovery study was performed three times at each level. The synthetic mixture which contains 10 mg Finerenone (FINE) and 10 mg of Empagliflozin (EMPA) and dissolve into 100 ml of methanol. So, the final concentration would be 100 ?g/ml and 100 ?g/ml of FINE and EMPA, respectively. Then take 0.5 ml of above solution and dilute upto 10 ml with methanol to make 20 ppm and 20 ppm FINE and EMPA respectively. Then prepare the 3 different levels solution from it as given below at 50, 100 and 150 % level

Assay of drugs in synthetic mixture:

Weigh powder equivalent to 1 tablet and dissolved in 50.0 ml of distilled water and then diluted to the mark in a 100.0ml standard flask and sonicated for 5 min., filtered and filtrate was used for validating the above-mentioned methods. Further diluted 1 ml of above solution to 10 ml volumetric flask and volume was make up to the mark with diluent. Further diluted 1 ml of above solution to 10 ml volumetric flask and volume was make up to the mark with diluent. Concentration of 30 ?g/ml and 30 ?g/ml of FINE and EMPA. Absorbance of above solution were measure using developed method.

ANALYTICAL METHOD VALIDATION:

LINEARITY AND RANGE:

       
           
       
    Fig. 6:  Overlay first-order derivative spectra of 10-50 ?g/ml for Empagliflozin (EMPA) at ZCP of 232.80nm

       
           
       
    Fig. 7:  Overlay first-order derivative spectra of 10-50 ?g/ml Finerenone (FINE), at ZCP of 304.00 nm

CALIBRATION DATA:

Table 1:  Calibration data of Empagliflozin (EMPA)

Precision:

Repeatability:

LOD and LOQ:

Table 7: LOD and LOQ data

ASSAY OF SYNTHETIC MIXTURE:

Table 8: Data of determination of Empagliflozin (EMPA) and Finerenone (FINE) in synthetic mixture