1. INTRODUCTION:

Epalrestat is a reversible, noncompetitive aldose reductase inhibitor derived from carboxylic acids that is used to treat diabetic neuropathy. A long-term, well-tolerated medicine that effectively slows the progression of diabetic neuropathy is epalrestat and improve the symptoms that accompany the condition, especially in patients with mild microangiopathy and good glycaemic control¹.Chemically, it is [{(5Z)-5-[(2E)-2-Methyl-3-phenylprop-2-en-1-ylidene]-4-oxo-2-sulfanylidene-1,3thiazolidin-3-yl} acetic acid (figure 1). Epalrestat dissolves in methanol, acetonitrile, ethanol, and other organic solvents but is insoluble in water^2,3.

One highly sensitive analytical method utilised in method development is RP-HPLC. When approved methods are unavailable, methods are devised for new items⁴. Analytical quality by design, or AQbD, is a new technique for optimising HPLC instrument parameters like flow rate, solvent concentration, column, length, buffer, temperature, injection volume, peak area, tailing, plate count and retention time. Ensuring the quality and dependability of analytical data requires the creation and optimisation of analytical procedures^5,6. Custom design aids in identifying crucial method parameters, choosing the best experimental design, and maximising method performance. The total quality of analytical and pharmaceutical procedures is improved with the aid of AQbD⁷. The ICH Q₂R₁ guidelines is used to further validation of method. Studies on forced deterioration were conducted using both chemical and physical techniques. The Epalrestat peak was not interfered with by the physical (heat, sunlight, water) or chemical (acid, base, and peroxide) degradation peaks, and the degradation was below the limit^8,9.

Figure 1: Structure of Epalrestat³.

2. MATERIALS AND METHODS:

2.1 Materials:

Sunlight Sciences (Hyderabad) provided Epalrestat. NaH₂PO₄, HCl, NaOH, H₂O₂, and methanol of HPLC grade are acquired from "Merck Specialties Ltd.," an HPLC grading company, via Sd Fine-Chem Limited in India.

2.2 Methods:

High-performance liquid chromatography (HPLC, waters corporation, Massachusetts U.S. A) attached with a UV detector was used for the method development of Epalrestat. X bridge C₁₈ column, flow rate of 1.1ml/min at 240nm was used for chromatographic separation of Epalrestat mobile phase comprises of NaH₂PO₄ and methanol (10:90v/v) at room temperature. Custom design used to optimize the method with the help of JMP software¹⁰.

2.3 METHODOLOGY:

2.3.1 Risk assessment study:

Reputable technologies that support and ease risk assessment include the risk estimation matrix (REM) and the Ishikawa diagram. Ishikawa diagram will help to identify the origin of the problem to find the possible causes and sub-causes affecting the CAAs (critical analytical attributes) of the product. REM studies were carried out in order to determine the critical method performance (CMPs), that are high-risk factors with significant effects on the CAAs. The factors with the great risk were selected by building REM with the grade of a low, medium, and high^11,12.

2.3.2 Method development for Epalrestat:

· Mobile phase was prepared as follows: 500ml of 50% methanol and 500ml (50%) of NaH₂PO₄ buffer were combined, degassed in an ultrasonic water bath for a total of ten minutes, and then filtered through a 0.45 µm filter utilising vacuum filtration^13,14.

· Preparation of diluent: The mobile phase was used as diluent.

· Standard stock solution preparation: Comprised precisely weighing 25mg of Epalrestat working standard, placing it into a 25ml clean, dry volumetric flask, administering 5ml of diluent, sonicating it for 30 minutes, and then making up the difference with diluent. Above solution was diluted to obtain various concentrations^15,16.

2.3.3 Method development trials:

Here reverse-phase HPLC was used to develop, validate and perform forced degradation studies. Here method is detected at 240nm, injection volume 10µl, run time of 20min, sample and column temperature 25°C with different column, composition of mobile phase and flow rate given in the table 1^17,18.

Table 1: Method trials

Trials no:	Composition NaH₂Po₄: MeoH(v/v)	Column	Flow rate (ml/min)
1	50:50	Cosmicsil Aster C₁₈, 150×4.6mm,5µm	0.5
2	60:40	Waters C₁₈,250×4.6mm,5µm	1
3	20:80	Waters C₁₈,250×4.6mm,5µm	0.9
4	20:80	X bridge C₁₈,250×4.6mm,5µm	1
5	10:90	X bridge C₁₈,250×4.6mm,5µm	1.1

2.3.4 Design of experiment – Custom design:

In this study, Custom Design can handle designs with difficult-to-change factors and other limitations and may be used for nearly any experimental situation, including factor screening, optimization, and mixing issues. Limits of factors and responses have taken from the method development trials. In Custom design, for the 3 factors and 3 responses, a total of 5 runs were obtained as shown in Table 3^19,20.

Table 2: Method runs by custom design

Sl. no	Factors			Responses
	Flow rate (ml/min)	Column length (mm)	Buffer	Retention time (min)	Peak area (AU)	Tailing
1	0.5	150	20	2.9	3580000	1.1
2	1.5	150	20	3.1	2729000	1.3
3	0.5	250	1	2.6	3984300	1.2
4	1.5	250	1	2.9	3809000	1.9
5	0.5	250	20	2.6	3799000	1.1

2.3.4.1 Statistical optimization of selected responses of method development:

It involves carefully selecting and adjusting attributes such as solvent composition, column type, flow rate, temperature, and detection wavelength to optimize the efficiency of the method. Optimization of method can be achieved through two approaches such as numerical optimization and graphical optimization²¹.

2.3.5 Validation of the method-preparation of solutions:

2.3.5.1 Preparation of Epalrestat standard stock solution: Put 100mg of the working grade Epalrestat, precisely weighed, into a 100ml dry volumetric container. After pouring 30ml of diluent and sonicating for 30 minutes, diluent was added to make up the leftover volume. Multiple concentrations of Epalrestat were obtained by diluting the stock solution via diluents ²².

2.3.5.2 Blank: The selected Mobile phase should be injected to check whether any peak appearing in the chromatogram²³.

a. System suitability: This test goal is to examine whether the overall testing system (RP-HPLC system) is effective for the specified use.

b. Specificity: For identification, purpose specificity is ability to discriminate between closely related compounds or in other means comparison to known reference sample or impurity. The blank should be injected to check whether any peak appearing in the chromatogram^24,25.

c. Accuracy: Percent recovery by the assay of known added amounts of analytic solution is frequently utilised to describe accuracy. Accuracy 50%, 100%, 150% solutions were prepared along with concentrations of 50µg/ml, 100µg/ml and 150µg/ml are prepared and inject into HPLC²⁶.

d. Precision: 100µg/ml concentration is prepared, prepare 6 vials and inject each one time²⁷.

e. Linearity and concentration range: Different concentrations like 50µg/ml, 75µg/ml, 100µg/ml, 125µg/ml and 150µg/ml were prepared from 50%-150% and all these concentrations were filled in a vial, each vial one injection²⁸.

f. Detection and Quantification Limit: From the stock solution, 100µg/ml concentration is prepared. Then pipette out 0.2ml for LOD and 0.6ml for LOQ from above solution into 10ml volumetric flask make up with diluent and injected to find best signal to noise ratio. A typical signal to noise ratio is 3:1 for LOD and A typical signal-to-noise ratio is 10:1 for LOQ^29,30.

g. Robustness: Robustness studies were performed by changing method parameters in system such as Flow rate (±0.1%), Ph (±0.1) and Composition of mobile phase (±5%)^31,32.

2.3.6 Forced degradation studies -preparation of solutions:

· Stock B: From the stock solution, pipette out 5ml and diluting to 50ml with diluent.

a. Acid: From stock B, pipette out 1ml into 10ml volumetric flask then add 5ml of 0.1N HCl mix properly make up with diluent and sonicate for 30mins, inject into the HPLC³³.

b. Base: From stock B, pipette out 1ml into 10ml volumetric flask then add 5ml of 0.1N NaOH mix properly make up with diluent and sonicate for 30mins, inject into HPLC.

c. Hydrolysis: From stock B, pipette out 1ml into 10ml volumetric flask then add 5ml of water mix properly make up with diluent and sonicate for 30mins, inject into HPLC.

d. Peroxide: From stock B, pipette out 1ml from into 10ml volumetric flask then add 5ml of 1% H₂O₂ mix properly make up with diluent and sonicate for 30mins, inject into HPLC³⁴.

e. Heat: From stock B, pipette out 1ml into 10ml volumetric flask make up with diluent that is heated at 60°C for 30 mins and inject into HPLC.

f. Sunlight: From stock B, pipette out 1ml diluting agent in a 10ml volumetric flask that is exposed to sunlight for 6 days and inject into HPLC. that is exposed to sunlight for 6 days, inject into HPLC^35,36.

3. RESULTS AND DISCUSSION:

3.1 Method development:

Sodium dihydrogen phosphate buffer and methanol make up the mobile phase (10:90v/v), X bridge C₁₈(250mm×4.6mm,5µm) column the flow rate of 1.1ml/min, at 240nm wavelength the method has developed with 2.877min retention time, 1.24 tailing and 15145 of plate count, Epalrestat chromatogram is shown in the figure 2.

Figure 2: Epalrestat chromatogram

3.2 Statistical optimization of selected responses of HPLC method and Prediction profiler of optimized method

Figure 3: Prediction profiler with maximized desirability

Numerical optimization represents the prediction profiler figure 3 displays a continuous correlation between various parameters and multiple responses. This shows that the greatest global desirability value of 76.48% provides the chance of achieving the desired goal for all three responses. figure 3 is demonstrating the significance of bar representing flow rate, column length, and buffer. The data from all 5 runs was incorporated into the design to test model fit. The obtained data were statistically analysed by fitting multiple regression models with the intercept set to zero. That shows R² = 0.93 and p = 0.0001 for response retention time, R² = 0.87 and p = 0.0007 for response peak area, and R² = 0.74 and p = 0.0103 for response tailing are statistically significant at a significance level of p<0.05.

3.3 Contour Profiler:

Figure 4: Contour Profiler showing the effect of method components on the responses

Graphical optimization represents the white region in the contour profiler shown in Figure 4 provides the optimized method operational design space showing the effect of method components on the responses. The statistical parameters obtained to ensure that the CMAs factored in the design have a significant effect on the CAAs and it was found to be significant enough for optimization and prediction of the goal of the experiment.

Analytical method validation:

a. Specificity: By examining the impact of blanks and other contaminants during Epalrestat's retention period, the specificity of the suggested approach was ascertained. As a result, no additional peak was discovered in the blank, revealing an elevated degree of specificity for the suggested technique.

b. System suitability: SST parameters passes for the number of USP plate count was found to be 15145, USP tailing factor 1.24, %RSD was found to be 0.2% and retention time was found to be 2.877 mins, as given in table 3.

c. Accuracy:

Accuracy was determined by injecting a concentration of 50%, 100%, 150% with each three injections. Accuracy at three different concentrations were in the limit of 97-103%, so the developed method passes this parameter as shown in table 4.

Table 3: System suitability studies

S. No	Sample name	Peak name	RT (min)	*Area (µVsec)**	USP Plate Count	USP Tailing
1	STD-2	EPALRESTAT	2.858	3186270	5988	1.26
2	STD-2	EPALRESTAT	2.854	3167385	5748	1.26
3	STD-2	EPALRESTAT	2.852	3175173	5624	1.27
4	STD-2	EPALRESTAT	2.849	3168360	5507	1.29
5	STD-2	EPALRESTAT	2.849	3172060	5191	1.28
Mean				3173849.6
%RSD				0.2

Table 4: Accuracy studies or % Recovery studies

Spiked level	Sample Weight	Sample Area	µg/ml added	µg/ml found	% Recovery	Accuracy
50%	48.00	1564919	24.750	24.60	99	Mean= 100
50%	48.00	1563408	24.750	24.58	99
50%	48.00	1574661	24.750	24.76	100
100%	96.00	3156875	49.500	49.63	100	SD= 0.707
100%	96.00	3156382	49.500	49.63	100
100%	96.00	3165092	49.500	49.76	101
150%	144.00	4741454	74.250	74.55	100	%RSD= 0.7
150%	144.00	4757848	74.250	74.80	101
150%	144.00	4745844	74.250	74.62	100

Table 5: Robustness studies by changing flow, pH and composition of mobile phase

S. no	Sample name	Peak name	RT (min)	*Area (µVsec)**	USP Plate Count	USP Tailing
1	FLOW-1	EPALRESTAT	2.371	2421326	5158	1.28
2	FLOW-2	EPALRESTAT	3.515	3643547	5837	1.30
3	pH-1	EPALRESTAT	2.851	2963623	5415	1.27
4	pH-2	EPALRESTAT	2.850	2962649	5545	1.26
5	COMP-1	EPALRESTAT	2.371	2421326	5158	1.28
6	COMP-2	EPALRESTAT	3.133	3225632	5450	1.29

d. Precision: The concordance of data between the series of measurements was studied to determine the precision of a system and approach. Six injections at 100% concentration were used in this instance. With a precision % RSD of 0.19%, the suggested approach offered a high level of precision.

e. Linearity and range: Epalrestat solution in the concentration range of 50 to 150 µg/ml were injected at 240nm wavelength. From the graph it shows that R² was found to be 1 this states that the Epalrestat passes linearity over the range of 50% to 150% as shown in figure 5.

Figure 5: Linearity graph

f. Detection and Quantitation limit: LOD and LOQ were determined by S/N ratio, that was found to be 3.8 and 10.1 respectively. The minimal amount of sample that can be detected has been identified using LOD, and it was discovered to be 0.082 µg/ml. The minimal quantity of sample that was capable of being quantified was discovered to be 0.2µg/ml, or LOQ.

g. Robustness: All of the results meet system suitability requirements, demonstrating the new method's robustness and lack of substantial deviations even when flow rate, pH, and mobile phase composition were slightly altered. given in table 5.

3.4 Studies on forced degradation:

These were conducted under a number of stressful circumstances, such as acid, base, peroxide, heat and sunlight to study the degraded products will interfere with study. From the degradation data, it was found that Epalrestat was found to be showing some degradation in stress condition, such as acid (10%), base (7.8%), peroxide (5.33), heat (9.11% and sunlight (5%) but degradation was not above 20% of limit in all conditions (table 6). Additionally, it was discovered that deteriorated products did not obstruct the primary peak.

Table 6: Forced degradation studies

Stress	Sample Area	% Assay	% Degraded
Acid stress	2800225	90	10
Base stress	2916317	92.13	7.87
Peroxide stress	2996560	94.67	5.33
Heat stress	2877043	90.89	9.11
Photo stress	2980642	94.16	5.84
Hydrolysis stress	3142857	99.29	0.74

4. CONCLUSION:

The efficacy of analytical quality by design concept in optimising the HPLC chromatographic method for Epalrestat analysis is well demonstrated in the current study. Risk assessment was conducted using the Ishikawa and risk estimation matrix to prevent method interactions. The method was developed by HPLC using an X bridge C18 column at 240nm with a flow rate of 1.1ml/min that was statically optimised using an experiment design. Method was optimized by prediction profiler for maximum desirability and counter plot for MODR which confirms the robustness of method Validation parameters were used to confirm accurate, precise and robust method in accordance with ICH Q₂R₁. Studies on the degradation of Epalrestat in bulk are conducted using acid, base, heat, sunlight, water, and peroxide.

5. ACKNOWLEDGEMENT:

The authors thank Krupanidhi College of Pharmacy, Bengaluru., Karnataka for the support extended for the successful completion of this review work.

6. CONFLICT OF INTERESTS:

The authors declare that there are no conflicts of interest related to this article.

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A new RP-HPLC Method Development, Validation and Stability-Indicating Studies of Epalrestat in Bulk Drug by Analytical Quality by Design Approach