Method Development and Validation for the Simultaneous Estimation of Eplarestat and Pragabalin in Bulk and Pharmaceutical Formulation by using RP-HPLC

 

Janmajoy Banerjee, H. Padmalatha, Rahul, Ranabir Chanda*

Gyana Jyothi College of Pharmacy, Uppal Bus Dept. Hyderabad, India

*Corresponding Author E-mail: ranabirchanda@gmail.com

 

ABSTRACT:

In our present investigation we have developed an accurate procedure to estimate Epalrestat and Pregabalin in tablet dosage form. Chromatogram column was used 250x4.6 mm. Buffer: Acetonitrile 60:40 was used as mobile phase and it was pumped through at a flow rate of 0.8 ml/min. Temperature was constant at 25°C. Wavelength was selected at 274.0 nm. Maintenance time of Epalrestat and Pregabalin were observed 2.520 min and 3.745 min. Percentage of recovery was acquired as 99.87% and 99.55% for Epalrestat and Pregabalin. Limit of Detection (LOD) were found 0.153 and 0.376 respectively for Epalrestat and Pregabalin and limit of Quantitation (LOQ) were found 0.464 and 1.139 for Epalrestat and Pregabalin respectively. Results of analysis were validated statistically. The results of the study showed that the proposed RP-HPLC method is simple, rapid, precise and accurate, which is useful for the routine determination of Epalrestat and Pregabalin in tablet dosage form.

 

KEYWORDS: Epalrestat, Pregabalin, RP-HPLC, Validation.

 

 


INTRODUCTION:

Analytical chemistry is a branch of chemistry involved with the analysis of chemical substances. It encompasses the theory and practice of all means of acquiring information about the composition of matter. Pharmaceutical analysis is considered as interdisciplinary subject comprising of chemistry, pharmacy, physics, electronics, biology etc. It is used in identifying drug substances, called qualitative analysis, determining the concentration or amount of drug substances, called quantitative analysis and confirming the structures of drug substances.

 

 

Analytical techniques like chromatography, spectroscopy, titrimetry etc. play a major role in these studies. The major areas of pharmaceutical analysis are assay of the drug from bulk drugs and formulations, detection and quantification of probable impurities and metabolites of drugs, accelerated stability studies, in-vitro dissolution studies etc1.

 

Impurity is defined as any substance coexisting with the original drug. Control of toxic impurities in drug substances has received more and more attention over the past years. These are to be determined based on the maximum daily dose. According to EMEA guidelines, a TTC value of 1.5 microgram/day intake of a toxic impurity is considered to be associated with an acceptable risk. The concentration limits in ppm of permitted toxic impurity in a drug substance is the ratio of TTC in microgram/day and daily dose in gram/day2.

 

In literature no method was developed to estimate combination of Epalrestat and Pregabalin in tablet dosage form. In this investigation we have developed a optimized method to estimate such combination in tablet dosage form.

 

A detailed literature survey reveals various RP-HPLC analytical methods for the quantitative estimation of Eplarestat3,4 Pregabalin5,6 individually in bulk, plasma and in various pharmaceutical dosage forms. RP-HPLC methods are reported for the analysis of combination of various drugs7,8.

 

MATERIALS AND METHODS:

Chemical and Reagents:

Epalrestat and Pregabalin pure drugs, acetonitrile, phosphate cradle, methanol, potassium dihydrogen ortho phosphate support, ortho-phosphoric corrosive, HPLC grade water were purchased from Rankem, Hyderabad, India. Combination of Epalrestat and Pregabalin tablets (Somaflam) were purchased from local market.

 

Instrument:

HPLC analysis was performed on Waters HPLC 2695 System equipped with quaternary pumps, Photo Diode Array detector and reverse phase colomn (250x4.6 mm, 5μ). A manually operating Rheodyne injector with 10 μL sample volume was equipped with the HPLC system. The HPLC was controlled with Empower 2 Software. In addition, an electronic analytical weighing balance (0.1mg sensitivity, Shimadzu AY 220), digital pH meter (DELUX model 101), a sonicator (sonica, model 2200 MH) and UV-Visible Spectrophotometer (Shimadzu UV-1800 series, software: UV probe version 2.42) were used in this study.

 

METHOD:

Selection of Wavelength:

Suitable wavelength for the HPLC analysis was determined by recording UV spectrums in the range of 200-400 nm for individual drug solutions of Epalrestat and Pregabalin. Suitable experimental wavelength was closed to 274 nm and it was selected (Figure 1, 2).

 

Chromatographic Conditions:

The separation of the drugs was achieved on a Reverse phase C18 column, Enable Make C18G (250X4.6 mm, 5μ). The mobile phase consists of a mixture of potassium dihyrogen Ortho phosphate buffer and acetonitrile in the ration of 60:40 v/v. The mobile phase was set at a flow rate of 0.8 ml/min and the volume injected was 10 μl for every injection. The detection wavelength was set at 274 nm.

 

 

 

Figure 1. Individual UV spectra of Epalrestat and Pregabalin

 

 

Figure 2. Overlay UV spectra of Epalrestat and Pregabalin

 

 

Buffer Preparation:

The buffer solution is prepared by weighing 1.36 gm of potassium dihydrogen orthophosphate (KH2PO4) and transferring to 1000 ml of HPLC grade water and the mixture was degassed by sonicate the volume made with HPLC grade water then pH was adjusted at 5.4 using 0.1N potassium hydroxide solution. The buffer was then filtered through 0.45 μm nylon membrane filter.

 

Mobile Phase Preparation:

The mobile phase was prepared by mixing buffer and acetonitrile in the ratio of 60:40 v/v. and later sonicated for 10 minutes for the removal of air bubbles.

 

 

Preparation of Stock and Working Standard Solution:

15 mg of Epalrestat and 7.5 mg Pregabalin were accurately weighed and taken in 100 ml clean and dry volumetric flask and 50 mo of diluents was added and then sonicated for 10 minutes to dissolve. Then the volume was made up to make a strength of 1500 µg/ml Epalrestat and 750 µg/ml of Pregabalin. This is considered as standard stock solution. 1 ml of solution was taken out and transferred into a 10 ml volumetric flask and the volume was made up with diluents to get working standard solution, treated as 100% target concentration.

 

Preparation of Stock and Working Sample Solution9:

Sample solution containing two drugs was prepared by dissolving tablets into diluent (mobile phase). 5 tablets were weighed and the average weight of each tablet was calculated, then the weight equivalent to one tablet was transferred into a 100 ml volumetric flask, 50 ml of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered through 0.45μm nylon membrane filter to get sample stock solution. 1ml of the stock solution was pipetted out and made up to 10ml to get working sample solution, treated as 100% target concentration.

 

Diluent Preparation:

Mobile phase was used as Diluent.

 

Chromatographic Conditions:

Flow rate                          : 0.8 ml per min

Column                                             : symmetry C18 (4.6x250mm, 5µm)

Detector wavelength     : 274 nm

Column oven                   : Ambient

Injection volume            : 10 µl

Run time                           : 10 min

 

RESULTS AND DISCUSSION:

Method Development:

A Reverse phase HPLC isocratic method was developed keeping in mind the system suitability parameters i.e. resolution factor (Rf) between peaks, tailing factor (T) and number of theoretical plates (N), runtime The optimized method developed resulted in the elution of Epalrestat at 2.52 min and Pregabalin at 3.74 min. Figure 3 and Figure 4 represent chromatograms of blank solution and mixture of standard solutions respectively. The total run time is 10 minutes. System suitability tests are an integral part of method development and are used to ensure adequate performance of the chromatographic system. Retention time (Rt), number of theoretical plates (N), peak resolution (Rf) and peak Tailing factor (T) were evaluated for six replicate injections of the standards at working concentration. Results given in Table 1 were within acceptable limits.

Table 1. System Suitability Studies Results

Parameters

Required Limits

Epalrestat

Pregabalin

Retention time (min)

% RSD<1%

2.515

3.741

Resolution factor (Rf)

Not less Than 2

 

8.9

Number Of Theoretical plates (Efficiency)

Not less Than 2000

6391

10998

Tailing factor (T)

Not More Than 2

1.21

1.17

 

 

Figure 3. Typical Chromatogram of Blank Solution

 

Figure 4. Typical Chromatogram for the Mixture of Standard Solutions

 

In order to test the applicability of the method developed to a commercial formulation (marketed tablet, Somaflam), was chromatographed and it is represented in Figure 5. The sample peaks were identified by comparing the relative retention times with the standard drugs mixture, Figure 4. System suitability parameters were ideal for the chromatographed sample. Integration of separated peak area was done and each drug was determined by using the peak area‐concentration relationship obtained in the standardization step. The protocol affords reproducible quantification of the two drugs with error less than 10%, which is the standard level in any pharmaceutical quality control.

 

Figure 5. Typical Chromatogram for Sample (Tablet)

 

Method Validation10

Validation of the analytical method is the process that establishes by laboratory studies in which the performance characteristics of the method meet the requirements for the intended analytical application. RP-HPLC method developed was validated according to International Conference on Harmonization (ICH) guidelines (10) for validation of analytical procedures. The method was validated in terms of parameters like system suitability, selectivity, linearity, accuracy, precision, ruggedness, robustness, limit of detection (LOD) and limit of quantitiation (LOQ).

 

Specificity:

Figures 3-5 for blank, mixture of standard drug solution and sample reveal that the peaks observed in mixture of standard solution and sample solution are only because of the drugs as blank has no peaks at the retention times of Epalrestat and Pregabalin. Accordingly, the method developed is said to be specific.

 

Precision:

System Precision

Six replicate injections of the mixture of standard solution at working concentration showed % RSD (Relative Standard Deviation) less than 2 concerning peak areas for the two drugs, which indicates the acceptable reproducibility and thereby the precision of the system. System precision results are tabulated in Table 2.

 

Table 2. System Precision Table of Epalrestat and Pregabalin

S. No

Area of Epalrestat

Area of Pregabalin

1.

833462

305200

2.

834331

306239

3.

834945

305922

4.

835387

306092

5.

833420

305032

6.

834364

305075

Mean

834318

305593

S.D

784.5

549.9

% RSD

0.1

0.2

Method Precision:

Method precision was determined by performing calculating % of RSD of samples under the tests of repeatability (Intra day precision) and Intermediate precision (Inter day precision) during 3 days at working concentration.

 

Repeatability (Intra Day Precision):

Six consecutive injections of the sample at working concentration showed % RSD less than 2 concerning peak areas for all the two drugs which indicate the method developed is method precise by the test of repeatability and hence the method should give consistently reproducible results (Table 3).

 

Table 3. Repeatability Table of Epalrestat and Pregabalin

S. No

Area of Epalrestat

Area of  Pregabalin

1.

836001

302111

2.

842315

302814

3.

838677

306444

4.

833196

302969

5.

834603

307530

6.

833154

301257

Mean

836324

303854

S.D

3587

2525

%RSD

0.4

0.8

 

Ruggedness (Intermediate Precision/Inter Day Precision):

Six consecutive injections of the sample solution at working concentration on three consecutive days for two drugs, showed % RSD less than 2 concerning peak areas for all the drugs, which indicate the method developed is inter day precise/rugged (Table 4).

 

Table 4. Interday Precision Results of Epalrestat and Pregabalin

S. No

Area of  Epalrestat

Area of Pregabalin

1.

809650

309650

2.

813874

313874

3.

809241

319241

4.

807578

307578

5.

809120

309120

6.

810767

310767

Mean

810038

311705

S.D

2141

4250

%RSD

0.3

1.4

 

Linearity:

Standard solutions of Epalrestat and Pregabalin of different concentrations level (25%, 50%, 75%, 100%, 125% and 150%) were prepared in triplicate. Calibration curves were constructed by plotting the % concentration levels of drugs versus corresponding mean peak area. The results show an excellent correlation exists between mean peak area and % concentration levels of drugs within the concentration range of  Epalrestat (1500 µg/ml) and Pregabalin (750 µg/ml) and the results were given in table 5 and figure 6-11. The correlation coefficients of Epalrestat and Pregabalin are greater than 0.999, which meet the method validation acceptance criteria and hence the method developed is said to be linear in the range of Epalrestat (1500 µg/ml) and Pregabalin (750 µg/ml).

 

Table 5. Linearity of the Chromatography System of Epalrestat and Pregabalin

Epalrestat

Pregabalin

Conc (μg/mL)

Peak area

Conc (μg/mL)

Peak area

0

0

0

0

37.5

232143

18.75

85326

75

429782

37.5

155006

112.5

649942

56.25

236005

150

854183

75

311447

187.5

1038992

93.75

378466

225

1235617

112.5

461691

 

 

Figure 6. Linearity 25% Chromatogram of Epalrestat and Pregabalin

 

 

Figure 7. Linearity 50% Chromatogram of Epalrestat and Pregabalin

 

 

Figure 8. Linearity 75% Chromatogram of Epalrestat and Pregabalin

 

Figure 9. Linearity 100% Chromatogram of Epalrestat and Pregabalin

 

 

Figure 10. Linearity 125% Chromatogram of Epalrestat and Pregabalin

 

 

Figure 11. Linearity 150% Chromatogram of Epalrestat and Pregabalin

Accuracy:

Accuracy was determined by means of recovery experiments, by the addition of active drug to preanalyzed sample at different spiked levels (50-150%). At each level, three determinations were performed and results obtained. The accuracy was calculated from the test results as the percentage of the analyte recovered by the assay. The amounts recovered, values of percent mean recovery were calculated as shown in Tables 6 and 7. The accepted limits of recovery are 98.28 %-101.79 % and all observed data are within the required range that indicates good recovery values and hence the accuracy of the method developed. Mean % of recovery is 99.87 % for Epalrestat and 99.55% for Pregabalin.


 

Table 6. Accuracy Table of Epalrestat

%Level

Amount Spiked (μg/mL)

Amount recovered (μg/mL)

%Recovery

Mean %Recovery

50%

75

75.20

100.26

99.87%

75

74.17

98.89

75

75.01

100.01

100%

150

150.43

100.29

150

151.78

101.18

150

147.74

98.49

150%

225

224.66

99.85

225

225.64

100.29

225

224.06

99.58

 

Table 7. Accuracy Table of Pregabalin

%Level

Amount Spiked (μg/mL)

Amount recovered (μg/mL)

% Recovery

Mean%Recovery

50%

37.5

37.37

99.64

99.55%

37.5

36.86

98.28

37.5

37.15

99.06

100%

75

75.51

100.68

75

74.81

99.75

75

110.64

99.46

150%

112.5

114.52

98.34

112.5

111.34

101.79

112.5

110.64

98.97

 


Robustness:

The robustness of an analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. It is concluded that the method is robust as it is found that the % RSD is less than 2 concerning flow rate (1.1ml/min-1.3ml/min), mobile phase ((-) 35B:65A-(+) 45B:55A) and temperature ((-) 25°C-(+) 35°C). The robustness results were given in table 8.

 

Table 8. Robustness Data for Epalrestat and Pregabalin

Sl.no

Condition

%RSD of Epalrestat

%RSD of Pregabalin

1

Flow rate (-) 1.1ml/min

0.3

0.2

2

Flow rate (+) 1.3ml/min

0.1

0.1

3

Mobile phase (-) 35B:65A

0.2

0.0

4

Mobile phase (+) 45B:55A

0.0

0.1

5

Temperature (-) 25°C

0.2

0.2

6

Temperature (+) 35°C

0.2

0.1

Sensitivity:

The sensitivity of measurement of Epalrestat and Pregabalin by use of the proposed method was estimated in terms of LOQ) and LOD. The limit of detection (LOD) was obtained as 0.153 μg/ml for Epalrestat and 0.376 μg/ml for Pregabalin. The limit of quantitation (LOQ) was obtained as 0.464 μg/ml for Epalrestat and 1.139 μg/ml for Pregabalin. The sensitivity results were given in table 9.

 

Table 9. Sensitivity Table of Epalrestat and Pregabalin

Molecule

LOD (μg/ml)

LOQ

Epalrestat

0.153

0.464

Pregabalin

0.376

1.139

 

Assay:

Somaflam, bearing the label claim Epalrestat 150 mg, Pregabalin 75 mg. Assay was performed with the above formulation. Average % of assay for Epalrestat and Pregabalin obtained was 100.14% and 99.33% respectively. The results were given in table 10 and 11.

Table 10. Assay Data of Epalrestat

Sl.no

Standard Area

Sample area

%Assay

1

833462

836001

100.101

2

834331

842315

100.858

3

834945

838677

100.422

4

835387

833196

99.766

5

833420

834603

99.934

6

834364

833154

99.761

Avg

834318

836324

100.14

Stdev

784.5

3587

0.43

%RSD

0.1

0.4

0.43

 

Table 11. Assay Data of Pregabalin

S.no

Standard Area

Sample area

% Assay

1

305200

302111

98.762

2

306239

302814

98.991

3

305922

306444

100.178

4

306092

302969

99.042

5

305032

307530

100.533

6

305075

301257

98.482

Avg

305593

303854

99.33

Stdev

549.9

2525

0.825

%RSD

0.2

0.8

0.83

 

CONCLUSION:

A reverse phase HPLC isocratic method developed has been validated as per ICH guidelines in terms of specificity, accuracy, precision, linearity, ruggedness, robustness, limit of detection and limit of quantitiation, for the simultaneous quantitative estimation of Epalrestat and Pregabalin in tablets. A good linear relationship was observed for the two drugs between concentration ranges of 750 µg/ml and1500 µg/ml. The correlation coefficients were greater than 0.999 for two drugs. The inter day and intraday precision results were goodenough to say that the method developed is precise and reproducible. Accuracy studies revealed that mean recoveries after spiking experiments were between 99.87% and 99.55%, an indicative of accurate method. Accordingly it can be concluded that the developed reverse phase HPLC isocratic method is accurate, precise, linear, rugged and robust. Accordingly, the method can be used for the routine analysis of Epalrestat and Pregabalin in tablets.

 

ACKKNOWLEDGEMENT:

We cordially acknowledge authority of Gyana Jyothi College of Pharmacy, Hyderabad, India for providing laboratory facility for our research work.

 

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Received on 13.06.2018       Accepted on 26.07.2018     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2018; 8(3): 174-180.

DOI: 10.5958/2231-5675.2018.00032.7