INTRODUCTION:

Epalrestat (EPT) is a carboxylic acid derivative, a noncompetitive and reversible aldose reductase inhibitor used for the treatment of diabetic neuropathy, which is one of the most common long-term complications in patients with diabetes mellitus. It reduces the accumulation of intracellular sorbitol which is believed to be the cause of diabetic neuropathy, retinopathy and nephropathy. It is well tolerated, with the most commonly reported adverse effects being gastrointestinal issues such as nausea and vomiting, as well as increase of certain liver enzymes.It has been proven in animal experiments that there is an improvement in sorbitol levels and Na⁺/K⁺ ATPase activity leading to improved nerve conduction velocity. It is chemically, 2-[(5Z)-5-[(E)-2-methyl-3-phenylprop-2-enylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]acetic acid. It has an empirical formula of C₁₅H₁₃NO₃S₂and molecular weight is 319.401 g/mol.

Pregabalin (PRN) is a medication used to treat epilepsy, neuropathic pain, fibromyalgia, and generalized anxiety disorder. Its use for epilepsy is as an add-on therapy for partial seizures with or without secondary generalization in adults. Some off-label uses of pregabalin include restless leg syndrome, prevention of migraines, social anxiety disorder, and alcohol withdrawal. It is chemically, (S)-3-(aminomethyl)-5-methylhexanoic acid.It has an empirical formula of C₈H₁₇NO₂and molecular weight is 159.23 g/mol.

Various analytical methods have been described in the literature for the estimation of EPT and PRN by LC-MS/MS^1-2, RP-HPLC^3-26,HPTLC²⁷and UV-Spectrophotometer^28-35.

In this study a simple, isocratic UPLC method was developed for the simultaneous determination of EPT and PRN in tablet dosage form and to quantify the content of EPT and PRN in dosage form. The method was validated according to ICH guidelines³⁶.

Figure 1: Structure of Epalrestat

Figure 2: Structure of Pregabalin

MATERIALS AND METHODS:

Generic product of fixed dose combination of EPT150 mg and PRN 150 mg (Prealdonil 150 mg Tablet) was purchased from the local market. Reference standards EPT&PRN were obtained as gift sample from Hetero Labs, Nakkapally. Acetonitrile, (UPLC Lichrosolv) and orthophosphoric acid was purchased from Fisher Scientific, Mumbai.

Instrumentation:

The UPLC system(watersAcquity) comprised of quaternary pump, auto sampler, PDA 2996 detector, controlled by EMPOWER 2 Software,HSS column (100mm x 2.1 mm, 1.8m) was used for analysis.An electronic balance (Sartorius), hot air oven, ultra sonicator and millipore Vacuum Filtration Assembly were also used in the study.

Preparation of solutions:

Mobile phase:

Accurately measured1 ml of orthophosphoric acid was transferred into a 1000ml of volumetric flask containing 900ml of milli-Q water and finally made up the volume with water. Mobile phase was prepared by mixing o-phosphoric acid (OPA) buffer (0.1%)and acetonitrile in the ratio of 55:45% v/v, sonicated for 10 mins and filtered through 0.45µm membrane filter using vacuum filtration assembly.

Reference and Sample solution:

Standard stock solutions were prepared by dissolving 15 mg of EPT and 15 mg of PRN working standard separately into a 10 ml clean dry volumetric flasks and 5 ml of diluent was added,sonicated for 5 minutes and final volume made with diluent. From the above stock solutions, 1 ml was pipetted out into a 10ml volumetric flask and then made upto the final volume with diluent. Sample solution of tablet dosage form was prepared by weighing suitable quantity of tablet powder equivalent to 150 mg of EPT and 150 mg of PRN into a 100 ml volumetric flask containing diluent.Further dilution was made to obtain a composite sample stock solution containing EPT and PRN were 150 µg/ml.

Assay:

Standard and sample solutions were injected into the chromatographic system and the areas of peaks for EPT and PRNwere measured and the % assay was calculated and chromatograms of standard and sample were given in Fig 3 & 4 respectively.

Figure 3: Standard chromatogram of Epalrestat and Pregabalin

Figure 4: Sample chromatogram of Epalrestat and Pregabalin

METHOD VALIDATION :

The analytical method was validated properly according to the ICH guidelines for linearity, accuracy, precision, specificity, robustness and ruggedness.

Linearity:

For linearity assessment six solutions (n=3) with known concentrations of EPT and PRN (37.5, 75, 112.5, 150, 187.5 & 225 µg/ml) were analysed. Calibration curve was plotted between peak response on y-axis & concentration on x-axis. The relation between concentration and response was evaluated by least square linear regression method y = mx + c , where m is slope, y= peak area , c = intercept and x = concentration.

Accuracy:

The accuracy of method was established by analysing the solutions of 3 concentrations (n=3) at 3 different levels (50%, 100%, 150%) of target assay concentration. The % recovery and relative standard deviation (RSD) were evaluated against acceptable limits of ± 2%.

Precision:

Repeatability and intermediate precision were checked by analyzing replicate composite reference solutions (n = 6) of known concentrations. The overall % RSD for peak response on day 1 and day 2 was checked against acceptable limits of ± 2% for repeatability and intermediate precision.

Specificity:

Specificity is an important task to check the interference of excipients on analytes area. A placebo solution was prepared from all tablet excipients except the active ingredient in same diluent. The solution was analyzed under the same chromatographic conditions and the base line was evaluated for peak response. Placebo interference was determined by spiking reference solution with appropriate level of excipients and evaluating for additional peaks other than peaks of EPT and PRN.

Robustness:

Robustness of method was assured by analyzing replicates (n=6) of solution used for precision with small changes in chromatographic conditions such as composition of mobile phase, flow rate etc.The influence of variables is determined by evaluating % RSD against acceptable limit of ± 2% for peak response and R_t of each analyte.

LOD and LOQ:

Limit of Detection (LOD), Limit of Quantification (LOQ) were established based on Signal to Noise (S/N) ratio method.

Forced Degradation Studies:

These studies were conducted under acid, alkaline, peroxide (oxidation), water, thermal and photolytic conditions. The sample solutions were exposed to these conditions and the main peak was studied for peak purity to indicate that the method effectively separates the degradation products from pure active ingredient. Degradation studies can be performed in solid state or solution for both drug substance and drug product.

Acid Degradation:

Acid degradation was carried out by placing 1 ml of sample stock solution in 10ml volumetric flask and 1ml of 2N HCl was added. The flask was refluxed for 30 minutes at 60°C and then 10ml with diluent and then a composite sample stock solution containing EPT and PRN of about 150 µg/ml was prepared for analysis.

Alkaline Degradation:

Alkaline degradation was performed by adding 1ml of 2N NaOH to 1 ml of sample stock solution in a 10ml volumetric flask and refluxed for 30 minutes at 60°C. The solution is neutralized with 2N HCl and diluted to 10ml with diluent. It is further diluted to obtain a composite sample stock solution containing EPT and PRN were 150 µg/ml.

Oxidative degradation:

Oxidative degradation was done by using 20% v/v H₂O₂ solution. To 0.4 ml of sample solution 1ml of 20% v/v H₂O₂ solution was added and allowed to stand for 30 minutes at room temperature. Then the solution was made upto 10ml with diluent.Then itwas made to obtain a composite sample stock solution containing EPT and PRN were 150 µg/ml and used for analysis.

Thermal degradation:

Thermal degradation was simply carried out by placing the flask containing the drug solution in an hot air oven at 105°C for 6hrs and diluted with diluent to 10ml.Further dilution is made to obtain a composite sample stock solution containing EPT and PRN were 150 µg/ml.The solution was then used for analysis.

Photo Degradation:

Photo degradation of sample solution was checked by exposing the sample solution to UV Light by keeping the solution in UV Chamber for 7 days. The solution was then diluted to 10ml with diluent. The stock solution containing composite sample of EPT and PRN (150 µg/ml) was prepared by further dilution to use for analysis.

RESULTS AND DISCUSSION:

System Suitability:

The optimized chromatographic conditions i.e., a combination of OPA buffer and acetonitrile (55:45% v/v) at a flow rate of 0.3ml/min over HSS column (100 mmx2.1mm, 1.8µ) were that checked for the system suitability parameters such as Retention time(R_t), Theoretical plates(N), Peak area(A)and Resolution. The statistical data of parameters were calculated using Empower 2.0 software. The results in Table.1 showed the performance parameters of the developed analytical method comply with USP requirements of system suitability.

The % RSD of peak areas (A) and retention time(Rt) for both analytes was less than 2.0.Resolution between two analytes was more than 3.0, tailing factor was less than 2.0 and the number of theoretical plates was more than 2000. The method was found to be suitable for simultaneous determination of EPT and PRN after successful application of their estimation in tablet dosage form.

Table 1: System suitability results

Parameters	EPT	PRN
% RSD	0.6	0.5
Retention time(Rt)	1.704	1.081
Theoretical plates(N)	3905	2230
Resolution	6.1	NA

Method Validation:

Linearity:

The statistical analysis of linearity data in Table.2 showed a good correlation between concentration and response. The linear regression equations for linearity of EPT and PRN were Y=4378x + 1311, Y= 2544 x + 1409 respectively and the correlation coefficient for both analytes is 0.999.

Table 2: Calibration curve statistical data

Parameters	EPT	PRN
Correlation coefficient	0.999	0.999
Slope	4378	2544
Intercept	1311	1409

Accuracy and recovery:

The sample solutions of three concentrations (n=3) at three different levels of 50%, 100% and 150% of target assay concentration were analyzed and the results were given in Table 3. The overall recovery of EPT and PRN at each concentration is 100±1% and % RSD was less than 2. The method was suitable for the assay of EPT and PRN in tablet dosage form and the results indicate that the method is accurate.

Table 3: Results of Recovery studies

Recovery (% level)	EPT		PRN
Recovery (% level)	% Recovery	% RSD	% Recovery	% RSD
50	99.25	0.5	99.32	0.6
100	100.34	0.3	100.21	0.2
150	100.61	0.4	100.56	0.5

Precision and Intermediate precision:

The results of precision and intermediate precision studies (Table 4) showed that the method was precise and repeatable within acceptable limits. The %RSD for peak response of six replicates (n=6) for both analytes was less than 2.

Table 4: Results of Precision and Intermediate precision

Precision data	EPT			PRN
Precision data	Peak Area	% Assay	% RSD	Peak Area	% Assay	% RSD
Method Precision	656530	99.3	0.4	379366	99.6	0.5
Intermediate Precision	645523	98.09	1.1	375461	98.6	0.5

Specificity:

The specificity studies revealed the absence of significant peaks at the given retention times of both analytes EPT and PRN in placebo.

Robustness and Ruggedness:

The chromatographic conditions (flow rate) were deliberately variated to obtain the results of robustness, shown in Table 5. The change in chromatographic conditions did not influence the results of EPTand PRN.

Ruggedness of method was carried out by different analyst on different days. The results indicate that the method is robust and rugged. The %RSD values were less than 2.0.

Table 5: Robustness Results

Robustness	EPT		PRN
Robustness	R_t	USP Tailing	R_t	USP Tailing
Flow rate (0.27 mL/min)	1.94	1.1	1.32	1.0
Flow rate (0.33 mL/min)	1.65	1.2	1.02	1.1

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The LOD values were 0.11 µg/ml and 0.02 µg/ml and that of LOQ values were 0.33 µg/ml and 0.06 µg/ml for EPT and PRN respectively.

Forced Degradation studies:

The results of forced degradation studies carried on drug sample indicate that the % degradation was within the limit of 5% to 20%. The degradants formed under stress conditions were separated from the active ingredients (Fig. 5 - 10).

Table 6: Results of forced degradation studies

Stress condition	% Assay of active ingredient
Stress condition	EPT	% Net Degradation	PRN	% Net Degradation
Acid	96.07	3.23	96.30	3.3
Base	96.57	2.73	95.79	3.81
Peroxide	97.30	2	97.92	1.68
Water	99.35	NA	99.70	NA
Thermal	97.46	1.84	97.29	2.31
Photolytic	98.48	0.82	98.20	1.4

CONCLUSION:

The proposed UPLC analytical method was validated according to ICH guidelines. The test results of method validation comply with the acceptance criteria. The validated method was successfully applied for the simultaneous determination of EPT and PRN in combined dosage form. It was concluded that the method can be applied for routine quality control analysis of EPT and PRN in dosage form.

Figure-5: Acid degradation

Figure-6: Base degradation

Figure-7: Peroxide degradation

Figure-8: Photolytic degradation

Figure-9: Thermal degradation

Figure-10: Water degradation

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Received on 01.08.2018 Modified on 10.10.2018

Research J. Pharm. and Tech 2019; 12(2):489-494.

DOI: 10.5958/0974-360X.2019.00086.6