A Green Analytical Method Development, Validation and Stress Degradation Studies of Atorvastatin Calcium in Bulk and Formulation by UV-Visible spectrophotometric Method

Fig. No. 1: Chemical structure of Atorvastatin Calcium

Green Analytical Chemistry (GAC) underlines minimizing the environmental and health impacts of analytical procedures. Green analytical chemistry advocate for reduction in solvent use, replacement of toxic reagents, energy conservation, and elimination of hazardous waste. Tool for assessing the greenness of analytical methods is the AGREE which is applied in this study.^[2]

After a method has been formulated, it is crucial to evaluate its capability to produce accurate results consistently. Therefore, validating the analytical method is essential, confirming its resilience to outside factors. Validation includes specific parameters and also incorporates various statistical tools that enhance the method's credibility. Once a method has been established, thorough validation must occur before it is shared or transferred between laboratories. Validation procedures should be well-documented and conducted using properly calibrated tools and instruments. The purpose of analytical method validation is to confirm its appropriateness for the intended application. Validation of a pre-existing analytical method is necessary when modifications are made to the drug substance composition, its synthesis, or when there are alterations in the procedure.^[3]

FDS is purposeful deterioration research in which a pharmaceutical material or drug product is subjected to an extra stress to quicken its normal rate of degradation. To ascertain whether unintentional exposure to situations different from the usual When exposure damages the product, stress testing may be helpful. The main cause of impurities in medication substances or products is degradation of the active pharmaceutical ingredient (API) or impurities associated to the procedure. The primary cause of the medication material's disintegration under various conditions is instability, which happened during manufacture, isolation, drying, storage, and shipping. The API's basic chemical stability regulates it. Any substance can degrade primarily through the following processes: heat, oxidation, photolysis, and hydrolysis. The generation of every degradation product that could occur under every circumstance is aided by the stress testing.

Various analytical techniques, including UV spectrophotometry, reverse-phase high-performance liquid chromatography (RP-HPLC), ultra-performance liquid chromatography (UPLC), and high-performance thin-layer chromatography (HPTLC), have been reported for the estimation of Atorvastatin Calcium. However, the development of a simple, economical, specific UV-Visible Spectrophotometric method including combination of solvents specifically optimized for pharmaceutical dosage forms remains limited. Therefore, there is a need for a statistically optimized, accurate, precise, and robust analytical method. Accordingly, the present study is focused on the development and optimization of a simple, efficient, and reliable UV-Visible Spectrophotometric method for the estimation of Atorvastatin Calcium in bulk drug and pharmaceutical formulations using a green solvent.

1. METHODOLOGY:
   1.  Materials:

1. Instruments:

Digital weighing balance, Melting point apparatus, Sonicator, Hot air oven, Double beam UV/Visible Spectrophotometer.

2. Reagents and chemicals:

Atorvastatin calcium powder was gifted by the AUROBINDO, Gujrat, India. All other chemicals and reagents used were of analytical grade.

• 2.  Methods:
• Method development of UV-Visible Spectroscopy^[4,5]

1. Selection of solvents:

Selection of a suitable solvent is influenced by the wavelength expected to be studied.

2. Selection of wavelength 

10 mg of drug was weighed & 10 ml of solvent system (water + ethanol) (50:50) were used to dissolve 10 mg of drug. To reach a concentration of 1000 µg/ml, a 1 ml solution was taken out of 1st stock solution and then diluted using 10 ml of solvent system. To final concentration of 10 µg/ml, a 1 ml solution taken out of the standard stock solution and diluted to a volume of 10 ml with solvent system. The solution was scanned between 200 and 400 nm, and when compared to solvent system as a blank, the greatest absorbance was recorded at 246 nm.

3. Preparation of standard solution

10 mg of drug was weighed & 10 ml of solvent system (water + ethanol) (50:50) were used to dissolve 10 mg of drug. To reach a concentration of 1000 µg/ml, a 1 ml solution was taken out of 1st stock solution and then diluted using 10 ml of solvent system. Then 10 ml volumetric flasks were filled with the standard solution, increasing in size from 0.2 ml to 1 ml. Then, using solvent system the volume in each flask was adjusted to 10 ml to produce a concentration range of 2 – 10 µg/ml. After measuring the solutions' absorbance at 246 nm with respect to the blank, a calibration curve for Atorvastatin calcium was plotted.

• Method validation of UV-Visible Spectroscopy^[6-8]

The UV method developed for was validated in terms of parameters. These parameters include linearity, precision, Accuracy, ruggedness, robustness, limit of quantification (LOQ) and limit of detection (LOD). The validation was carried out according to ICH guideline Q2 (R1). Validation was conducted using predefined calibration standards to ensure the method's reliability and reproducibility across different condition.

1. 1. 1. 1. Linearity

To reach concentration of 2, 4, 6, 8, and 10μg/ml, aliquots of the standard solution were precisely transferred into several flasks and diluted to volume with distilled water. The linearity of the process was assessed.

1. 1. 1. 2. System precision

Absorbance was measured at a concentration of 6µg/ml using six replications of the sample solution on the same day. 

1. 1. 1. 3. Method Precision 

Through intra-day and inter-day precision experiments, the method's precision was established. Three identical solutions were made and examined at a single day for the intra-day precision investigation. In the meanwhile, identical solutions were prepared and analyzed for three days in order to assess the inter-day precision. In addition, the RSD was computed. 

1. 1. 1. 4. Ruggedness

The method's ruggedness was assessed for an Atorvastatin calcium concentration of 6μg/ml & by two analysts evaluate aliquots from a homogeneous slot under identical operational and environmental circumstances.

1. 1. 1. 5. Robustness  

The method's robustness was assessed by varying the wavelength of Atorvastatin calcium by ±2 nm, from 246 nm to 244 nm and 248 nm. 

1. 1. 1. 6. Accuracy

Accuracy was performed by the recovery in percent. The study was carried out at three distinct percentage: 80%, 100%, and 120%. The percent recovery and RSD were calculated.

1. 1. 1. 7. Limit of Detection (LOD) & Limit of Quantification (LOQ) 

The LOD and the LOQ were determined utilizing the SD of the intercept and the slope of the curve. 

• Forced degradation studies (FDS) of API and formulation by UV Spectrophotometry^[9-11]

Forced degradation aims to forcefully break down of the active ingredient. These investigations are utilized to assess an ability of method to quantify substance and the breakdown substances independently. FDS of selected API in bulk and formulation was carried out as per ICH guidelines Q1A (R2). It involves subjecting the drugs to factors such as heat, light, heat and acidic or alkaline conditions. This data is necessary for establishing the stability of drug across different conditions aiding in formulation development and ensuring compliance with regulatory requirements. The sample was exposed to following conditions.

1. Acid Degradation

Samples were treated with 10 ml of 0.1 M HCl & placed at room temperature for 2 hours. Then neutralize with 0.1 M NaOH solution and scanned in UV region at 246 nm. The same solution was scanned on 3th & 5th day to check the Inter-day effects.

2. Alkali Degradation

Samples were treated with 10 ml of 0.1 M NaOH & placed at room temperature for 2 hours. Then neutralize with 0.1 M HCl solution and scanned in UV region at 246 nm. The same solution was scanned on 3rd & 5th day to check the Inter-day effects.

3. Oxidative Degradation

Samples were treated with 5% H2O2 & placed at room temperature for 2 hours. Scanned in UV region at 246 nm. The same solution was scanned on 5th day to check the Inter-day effects.

4. Thermal Degradation

Sample was kept in oven at 50 °C for about 24 hours. Sample was dissolved in solvent system & scanned in UV region at 246 nm.

5. Photolytic Degradation

Sample was exposed to sunlight. On next day sample was dissolved in solvent system & scanned in UV region at 246 nm.

2. RESULTS AND DISCUSION
   1. λ max determination:

A solution comprising 10µg/ml was scanned between 200 and 400 nm, and the highest absorbance detected at 246 nm when compared to solvent system as a blank.

3.2 Analytical method development, validation & forced degradation study by using UV Spectrophotometry.

 (a) Analytical method development by using UV spectrophotometry

• Selection of solvent

Selection of a suitable solvent is influenced by the wavelength expected to be studied. The solvent thus used for present study was solvent system (water + ethanol) (50:50).

• Selection of wavelength

The final solution of 10µg/ml was scanned in the UV region of 200-400 nm with the highest absorbance at 246 nm against solvent system as a blank.

Fig. No. 2: UV spectrum of                             Fig. No. 3: Overlay spectrum of

Atorvastatin calcium                                           Atorvastatin calcium

(b) Validation of developed analytical method by using UV spectrophotometry 

1. Linearity:

The sample were scanned at concentration range of 2, 4, 6, 8, 10 µg/ml.

Table No. 1: Results of calibration for Atorvastatin calcium

Fig. No. 4: Calibration curve of Atorvastatin calcium

2. System precision:

Absorbance was measured at a concentration of 6 µg/ml using six replications of the sample solution on the same day.

Table No. 2: System Precision results of Atorvastatin calcium

3. Method Precision

Precision was carried out on Intraday and Inter-day at 3 various concentration and measurements were taken in triplicate for both studies.

Table No. 3: Intra-day precision data of Atorvastatin calcium

Table No. 4: Inter-day precision of Atorvastatin calcium

4. Accuracy

The recovery study was carried out at three distinct percentage: 80%, 100%, & 120%.

Table No. 5: Recovery data of Atorvastatin calcium

5. Ruggedness

Ruggedness was established by analyzing the sample by two distinct analysts.

Table No. 6: Ruggedness data of Atorvastatin calcium

6. Robustness

Robustness determined by subjecting change in the wavelength. The method was found to be robust and result are shown in Table No. 7.

Table No. 7: Robustness data of Atorvastatin calcium

7. LOD & LOQ:

Table No. 8: LOD & LOQ data

Where,

σ = Std. deviation of the response

s = slope of the calibration curve

c) Forced degradation of API & It’s formulation by using UV spectrophotometry

• Degradation study of API 

Table No. 9: Results for Degradation of API

Table No. 10: % of Degradation of API

Fig. No. 5: Graphical representation showing degradation of API

• Degradation study of Atorvastatin calcium formulation

Table No. 11: Results for Degradation of Atorvastatin calcium formulation

Table No. 12: % of Degradation of formulation

Fig. No. 6: Graphical representation showing degradation of Atorvastatin calcium formulation