INTRODUCTION:

Epalrestat^1-2 is a 2-[(5Z)-5-[(E)-2-methyl-3-phenylprop-2-enylidene]-4-oxo-2-sulfanylidene-1, acetic acid 3-thiazolidin-3-yl]. It is a carboxylic acid derivative that inhibits aldose reductase, a rate-limiting enzyme of the polyol pathways. In hyperglycaemia, Epalrestat, an uncompetitive aldose reductase inhibitor, significantly reduces intracellular sorbitol accumulation. Pregabalin^3-4is an antiepileptic and analgesic and chemically known as (3S)-3-(amino methyl)-5-methylhexanoic acid. It’s mechanism of action differs from several known mechanisms of action of other antiepileptic and analgesic medicinal products. Pregabalinis an analogue of the mammalian neurotransmitter gamma-aminobutyric acid (GABA). It interacts with an auxiliary subunit (α2-δ protein) of voltage-gated calcium channels in the central nervous system, potently displacing [3H]-gabapentin. Binding to the α2-δ site is required for analgesic, anticonvulsant and anxiolytic activity in animal models. Pregabalin reduces the release of several neurotransmitters, including glutamate, noradrenaline, and substance P.

Diabetic peripheral neuropathy⁵ is a major long term problem allied with diabetes that can cause serious disability and also death. It is caused by damage to any nerve in the peripheral nervous system. Fifty to seventy five percent of all ulcerations and non trauma amputations are a consequence of diabetic neuropathy. Epalrestat and Pregabalin are widely used to overcome neuronal damage.

Ultra-performance liquid chromatography (UPLC) methods are used over the HPLC methods, as they provide leverage of time, efforts, and resources saving. Hence author made efforts to develop a stability-indicating RP-UPLC method for the simultaneous quantification of Epalrestat and Pregabalin in presence of interaction/degradation products. This method has advantages of less sample volume, shorter retention time and higher selectivity compared to reported UPLC method.

Figure 1: Structure of (a) Epalrestat and (b) Pregabalin

In the literature Madhavi⁶et al., has developed a stability indicating ultra pressure liquid chromatographic method and validated for the simultaneous estimation of Epalrestat and Pregabalin in bulk and tablet dosage form. In this study HSS column (100mm x 2.1 mm, 1.8µ) was used to separate the analytes, with a mobile phase consisting of 0.1% o-phosphoric acid buffer (55% v/v) and acetonitrile (45%v/v) at a flow rate of 0.3ml/min. The detection was carried at 210 nm and the retention times were found to be 1.704 and 1.084 min for Epalrestat and Pregabalin respectively. A detailed Literature survey reveals that there are various analytical methods have been described for the estimation of Epalrestat and Pregabalin in combination with other drugs^6-11 and estimation of Epalrestat¹² and Pregabalin^13-23by RP-HPLC.

The author attempts to demonstrate a stability indicating UPLC method for separation and quantification of Epalrestat and Pregabalin with shorter run time, retention times were observed as 0.979 and 1.280 minutes respectively. In the present study author has explored a mass compatible mobile phase and diluent which facilitates to identify the degradation products. Method is linear, accurate, specific and forced degradation studies indicate that the stability indicating nature of method.

MATERIALS AND METHODS:

Reagents and materials

Analytical grade reagents such as Acetonitrile, Formic acid and HPLC grade water were procured from Merck India. Epalrestat and Pregabalin working standard were procured from Vivan life sciences, (India).

Preparation of 0.1% formic acid: Accurately transferred 1mL of Formic acid in a 1000mL of volumetric flask and added about 900mL of milli-Q water. Made up the volume with water up to 1000 mL and mixed well to form homogenous mixture.

Preparation of mobile phase: Measured 60% formic acid (0.1%) and 40% Acetonitrile and mixed well. filtered through 0.22µ membrane filter.

Diluent preparation: Mixed 0.1% Formic acid and Acetonitrile in the ratio 50:50% v/v.

Preparation of Standard stock solution

Accurately Weighed and transferred 75 mg of Epalrestat and 37.5 mg of Pregabalin working Standards into 100 clean dry volumetric flasks, added about 70mL of diluent, sonicated for 5 minutes in ultra sonicator and made up to the final volume with diluent. (750µg/ml of Epalrestat and 375µg/ml of Pregabalin).

Preparation of Standard solution

5mL from each stock solution was pipetted out and taken into a 50mL volumetric flask and made up with diluent. (75µg/ml of Epalrestat and 37.5µg/ml Pregabalin)

Chromatographic conditions

The UPLC system used for method development, degradation studies, and validation was Waters H-Class UPLC (Model 2695) consisting of Quaternary pump plus autosampler, auto injector; SM4 E 07 SM 4094 A (Singapore), online degasser, column oven, and SPD-20A photo diode array (PDA) detector. The output signal was monitored and processed using Empower software, Waters Corporation, Milford, USA (Database Version 6.10.01.00). An Agilent Zorbax SB C18 (2.1 x 100mm, 1.8µm) column was used for separation and to develop the SIAM (Stability Indicating Assay Method). The analytes were separated in isocratic mode with flow rate of 1.0 mL/min. details are represented in Table 1 & 2. The column temperature was maintained at 30°C, and the detection was monitored at 226 nm. The injection volume was 0.5 µL.

RESULTS AND DISCUSSION:

Method validation parameters

The optimized HPLC method was validated in accordance with the ICH Q2 (R1) guidelines and reported.

Specificity

The results of forced degradation studies of each drug in the presence of their degradation products indicated a high degree of specificity of this method for Epalrestat and pregabalin. No interference was observed with blank, placebo and degradants with the analyte peaks. Typical chromatograms of Blank, standard and placebo are presented in Figure. 2, Figure. 3 and Figure. 4.

Figure 2: Representative chromatogram of blank.

Figure 3: Representative chromatogram of standard.

Figure 4: Representative chromatogram of placebo.

Linearity and Range

The linearity was established over the range of 10-200% level of standard concentration for Epalrestat and Pregabalin. Correlation coefficients (𝑅²) were found not less than 0.999 for the two analytes. Typically, the regression equations were y = 16215x + 11780 and y = 16363x + 3640.7 for Epalrestat and Pregabalin respectively and results are represented in Table 1 and Table 2. Linearity Plots are presented in Figure. 5 and Figure. 6.

Table 1. Linearity results for Epalrestat.

% Linearity level	Concentration (ppm)	Response
0	0	0
10	7.5	124186
20	15	246790
50	37.5	625560
100	75	1262807
150	112.5	1860687
200	150	2407893

Figure 5: Linearity plot of Epalrestat.

Table 2. Linearity results for Pregabalin.

% Linearity level	Concentration (ppm)	Response
0	0	0
10	3.75	61513
20	7.5	128177
50	18.75	305877
100	37.5	640398
150	56.25	912813
200	75	1228890

Figure 6: Linearity plot of Pregabalin.

Accuracy

Accuracy of the method was determined by performing the recovery studies by spiking both analytes to placebo at 50% level, 100% Level and 150% Level of sample concentration. The recoveries of Epalrestat and Pregabalin were found within acceptable ranges of 100 ± 2%. The results are presented in Table 3.

Table 3. Results of % Recovery of Epalrestat and Pregabalin

S.No.	Level	% Mean Recovery of Epalrestat	% Mean Recovery of Pregabalin
1	50 %	99.78	99.77
2	100 %	101.04	100.55
3	150 %	99.99	100.35

Precision

Precision was studied to find out intra and interday variations of the proposed method and results were interpreted by statistical analysis by calculating % RSD values and all the results were within the acceptance criteria of not more than 2% and the results were tabulated in the Table 4. The intraday precision of the test method was conducted by assay in six samples. The average % assay of Epalrestat and Pregabalin was found to be 100.99 % and 100.77% and the % RSD is 0.63 and 0.81 the results were given in the Table 4. Six replicate analysis of the samples through the complete analytical procedure from sample preparation (inter day) was performed. The % RSD of system precision and method precision were found to be 100.10 % and 100.25% and the % RSD was 0.52 and 0.64, respectively and were shown in the Table 4.

Robustness

Robustness of the method was checked by small deliberate changes made in the method parameters such as Organic composition (±2%), flow rate (±0.1 mL) and temperature (40°C ±2°C) and these changes did not affect the method results. The results are presented in Table 5 and 6.

Table 4. Results of precision.

Injec tion Number	% Assay
	Intraday		Inter day
	Epalrestat	Pregabalin	Epalrestat	Pregabalin
1	101.06	100.05	100.32	100.01
2	100.48	100.82	100.81	99.21
3	101.60	101.80	99.21	100.98
4	99.98	100.65	100.08	100.35
5	101.29	101.58	100.21	100.07
6	101.53	99.73	99.99	100.85
Mean	100.99	100.77	100.10	100.25
SD	0.64	0.82	0.52	0.64
% RSD	0.63	0.81	0.52	0.64

Table 5. Results of robustness of Epalrestat.

Chromatographic conditions	Epalrestat
Chromatographic conditions	% Assay	Theoretical Plates	Asymmetry	Retention time
Formic acid (0.1%) and acetonitrile (65 : 35v/v)	99.85	2920	1.27	0.985
Formic acid (0.1%) and acetonitrile (55 : 45v/v)	99.06	3048	1.18	0.971
Flow rate 0.9 mL/min	99.79	3045	1.12	1.011
Flow rate 1.1 mL/min	98.77	2887	1.12	0.939
Column oven temperature 38°C	101.02	2296	1.29	0.979
Column oven temperature 42°C	98.78	3026	1.12	0.980

Table 6. Results of robustness of Pregabalin.

Chromatographic conditions	Pregabalin
Chromatographic conditions	% Assay	Theoretical Plates	Asymmetry	Retention time
Formic acid (0.1%) and acetonitrile (65 : 35v/v)	99.85	3413	1.25	1.337
Formic acid (0.1%) and acetonitrile (55 : 45v/v)	99.06	3106	1.22	1.234
Flow rate 0.9 mL/min	99.79	2964	1.25	1.323
Flow rate 1.1 mL/min	98.77	3127	1.3	1.235
Column oven temperature 38°C	101.02	3185	1.26	1.287
Column oven temperature 42°C	98.78	3219	1.31	1.265

Estimation of Epalrestat and Pregabalin in Marketed formulation:

The assay for the marketed formulation was found to be within the limits as listed in Table 7. The low %RSD indicated that the suitability of the method for routine analysis of Epalrestat and pregabalin in pharmaceutical dosage forms. Concentration of Epalrestat is 750 ppm and Pregabalin is 375 ppm. Weighed and finely powder 20 tablets, transfer accurately weighed portion of a powder, equivalent to one tablet into a 100 mL volumetric flask, added about 70 mL of diluent, sonicated for 10 minutes with intermediate shaking. Cool to room temperature, diluted up to volume with diluent and mixed well. Centrifuged the above solution at 4000RPM for 10 minutes and then filtered and collected the supernatant solution through 0.45µm syringe filter. Further dilute 5mL of the above filtered solution to 50 mL with diluent. Representative chromatogram of sample is presented in Figure. 7 and results were tabulated below as table 7.

Table 7. Assay results of marketed formulation.

Injection Number	% Assay
Injection Number	Epalrestat	Pregabalin
1	99.85	99.51
2	99.97	99.99
3	100.02	100.08
Mean	99.95	99.86
SD	0.09	0.31
% RSD	0.09	0.31

Figure 7: Representative chromatogram of sample.

Stability Studies

The International Conference on Harmonization (ICH) guideline²⁴entitled stability testing of new drug substances and products requires that stress testing be carried out to elucidate the inherent stability characteristics of the active substance. In this study, the Epalrestat and pregabalin were exposed to different chemical and physical degradation conditions such as 2 N HCl (acid hydrolysis), 2 N NaOH (base hydrolysis), 0.3% H2O2 (oxidation), heat (thermal decomposition) and UV-light (radiation decomposition) for specified time, and then diluted as similar as standard dilution, and then chromatograms were obtained under the similar chromatographic conditions, the percent of degradation was calculated from the peak area of the chromatograms.

In the study of acid or base hydrolysis, working sample solution was prepared (750µg/ml of Epalrestat and 375µg/ml of Pregabalin). 5mL of working sample solution and 5mL 2N HCl/ 2N NaOH were mixed well and allowed for 30 min on water bath at 60°c and neutralized with 2N HCl/ 2N NaoH and added about 30 mL of diluent, sonicated for 10 minutes with intermediate shaking. Cooled to room temperature, diluted up to volume with diluent and mixed well. In case of peroxide and water degradation 5mL of sample working solution was transferred into 50 mL of Volumetric flask, added 5 mL of freshly prepared 0.3% H₂O₂/water and kept on water bath for 30mins at 60°c added about 30 mL of diluent, sonicated for 10 minutes with intermediate shaking. Cooled to room temperature, diluted up to volume with diluent and mixed well. In the study of thermal or UV-light degradation, 5mL of solution was transferred into 50 mL flask and subjected to stress (oven at 105˚C for 48 hours or UV cabinet-254 nm). Then removed from stress conditions and allowed to stand for some time to attain room temperature, diluted up to volume with diluent and mixed well. Injected into UPLC and chromatograms were obtained under optimized conditions. A study of forced degradation was carried out to evaluate the stability of the drugs in formulations and the results of degradation and stability of drugs are presented in Table 8 and 9, and representative chromatograms were presented as Figure no:8-13.

Table 8. Degradation data of Epalrestat.

Degradation conditions	Epalrestat
	%Assay	% Degradation	Purity Angle	Purity Threshold
Unstressed Sample	99.90%	N/A	0.179	0.542
2 N HCl on water bath for 30 min at 60°c	94.90%	5.10%	0.081	0.112
2 N NaOH at water bath for 30 min at 60°c	93.75%	6.25%	0.437	0.803
Thermal 105°C for 6 hours	96.74%	3.26%	0.784	1.047
UV Light at 254nm	97.98%	2.02%	0.110	0.374
water on water bath for 30 min at 60°c	99.35%	0.65%	0.171	0.770
0.3% H₂O₂ on water bath for 30 min at 60°c	94.35%	5.65%	0.169	0.542

Table 9. Degradation data of Pregabalin

Degradation conditions	Pregabalin
	%Assay	% Degradation	Purity Angle	Purity Threshold
Unstressed Sample	99.90%	N/A	0.101	0.506
2 N HCl on water bath for 30 min at 60°c	94.85%	5.15%	0.094	0.401
2 N NaOH at water bath for 30 min at 60°c	93.88%	6.12%	0.804	1.210
Thermal 105°C for 6 hours	98.02%	1.98%	0.009	0.720
UV Light at 254nm	98.42%	1.58%	0.215	0.801
water on water bath for 30 min at 60°c	99.29%	0.71%	0.150	0.711
0.3% H₂O₂ on water bath for 30 min at 60°c	97.37%	2.63%	0.095	1.205

Figure 8: Representative chromatogram of Acid degradation sample.

Figure 9: Representative chromatogram of base degradation sample.

Figure 10: Representative chromatogram of peroxide degradation sample.

Figure 11: Representative chromatogram of thermal degradation sample.

Figure 12: Representative chromatogram of Water degradation sample.

Figure 13: Representative chromatogram of UV degradation sample.

CONCLUSION:

The present developed Stability Indicating RP-UPLC method was found to be trouble-free, high throughput, cost effective, accurate and specific for the determination of Epalrestat and Pregabalin in tablet dosage forms. Finally, the simplicity of sample preparation and the shorter chromatographic runtime gives the method capability for high sample throughput. Forced degradation data is evident that the method demonstrates the stability indicating nature and from the results of all the validation parameters we can conclude that the present method can be used for routine analysis of these drugs in bulk and marketed formulations.

ACKNOWLEDGEMENTS:

The authors are thankful to Vivan life sciences for providing the standards as gift samples. The authors declare no conflict of interest.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 27.08.2021 Modified on 29.09.2021

Research J. Pharm. and Tech 2022; 15(1):206-212.

DOI: 10.52711/0974-360X.2022.00034