Stability Indicating RP–HPLC Method for the Estimation of Acamprosate in Pure and Tablet Dosage Form

B. Thangabalan, Avinash Koya*, G. Chaitanya, N. Sunitha, S. Manohar Babu.

Department of Pharmaceutical Analysis, SIMS College of Pharmacy, Mangaldas Nagar, Guntur-522 002 (A.P.)

*Corresponding Author E-mail avinashkoyi@live.com

ABSTRACT:

A simple, fast and precise stability indicating RP – HPLC method was developed for the separation and quantification of Acamprosate in pharmaceutical dosage form. The quantification was carried out using Hypersil C18 4.6X150 mm, 5 μm enhanced polar selectivity column and mobile phase comprised of 0.2M Ammonium acetate and acetonitrile in proportion of ratio 40:60 and degassed under ultrasonication. The flow rate was 1mL/min and the effluent was monitored at 220 nm. The retention time of Acamprosate was 4.187min. The method was validated in terms of linearity, precision, accuracy, specificity, limit of detection and limit of quantitation in accordance with ICH guidelines. Linearity of Acamprosate was in the range of 5 – 30 μg/mL. The percentage recoveries of Acamprosate was 99.00% from the tablet formulation. The stability – indicating capability was established by forced degradation experiments. The proposed method is suitable for determination of Acamprosate in pharmaceutical dosage form.

KEYWORDS: Chromatography, Acamprosate, Forced degradation, Method development, Method validation.

INTRODUCTION:

Acamprosate is chemically 3-Acetamido Propane-1-Sulphonicacid. Acamprosate mechanism of action is not completely understood. Chronic alcohol exposure is thought to negatively alter the normal balance between neuronal excitation and inhibition

Figure 1: Structure of Acamprosate

The objective of this work is to develop an analytical HPLC procedure, which will serve as stability indicating method for Acamprosate. A through literature^1,2,3 search revealed none of the reported analytical procedures describe a stability indicating HPLC method for the determination of Acamprosate.

To establish the stability indicating nature of the method, forced degradation of drug substance & drug product was performed under stress conditions (thermal, acid, base and oxidative), and stress samples were analysed by proposed method⁴. In this, we describe a reverse – phase HPLC method for the assay of Acamprosate. The developed LC method is validated with respect to specificity, LOD, LOQ, linearity, precision, accuracy, and robustness as per ICH recommended conditions^5,6. From the best of our knowledge via literature search, this is the first known stability indicating RP – HPLC method.

MATERIALS AND METHODS:

Materials, reagents and chemicals:

Acamprosate is obtained as gift sample from Hetero Pharma Ltd, Hyderabad. HPLC grade acetonitrile and analytical grade Ammonium Acetate, ortho phosphoric acid, Hydrochloric acid, sodium hydroxide, hydrogen peroxide of analytical grade was purchased from Merc Chemicals Ltd, Mumbai.

Chromatographic conditions

Shimadzu HPLC with UV detector was used. The output of signal was monitored and integrated using LC – solutions 2000 software. Hypersil C18 (4.6 × 150mm, 5µm) column was selected as stationary phase. Mobile phase comprised of 0.2M Ammonium acetate and acetonitrile in proportion of ratio 40:60. Mobile phase was mixed, filtered through 0.45µmembrane filter and degassed under ultrasonication. The mobile phase was used as diluents. Injection volume was 10µl and flow rate was 1 ml/min. The column was maintained at ambient temperature and the eluent was monitored at 220 nm.

Preparation of standard solution

A stock solution (1000 µg/ml) of Acamprosate was prepared by dissolving 100 mg of drug taken in 100 ml volumetric flask, dissolved in 50ml of mobile phase, sonicated for 15 min and the volume was made up to 100 ml with mobile phase. From this 10 ml was diluted to 100 ml gives 100 µg/ml. The standard chromatogram for Acamprosate (100μg/ml) was shown in figure 2.

Preparation of working standards

The working standard solutions of Acamprosate were prepared by accurately transferring the (0.5, 1, 1.5, 2, 2.5, 3 ml) aliquots of the standard stock solution in a series of 10 ml volumetric flasks. The volume was made upto mark with mobile phase to obtain concentration of 5-30 µg/ml.

Preparation of sample solution

10 tablets were accurately weighed and powdered. Weight equivalent to 25mg was weighed and taken in 50ml volumetric flask. Mobile phase (30 ml) was added and sonicated for 15min. The solution was filtered through Whatman filter paper No.1 and the volume was adjusted up to the mark with mobile phase to 0.5ml of resulting solution was taken in 10ml volumetric flask and volume made upto mark with mobile phase to obtain 25 µg/ml of sample solution.

Table 1: Optimized chromatographic parameters

Optimized Chromatographic parameters

Elution

Mobile phase

Column

Flow rate

Detection

Injection volume

Temperature

Retention time

Run time

Concentration

Isocratic

0.2M Ammonium acetate and Acetonitrile (40:60)

Hypersil C₁₈column

1ml/min

220 nm

10μl

Ambient

4.187 min

8 min

5-30 μg/ml

RESULTS AND DISCUSSION:

HPLC method development and optimization:

To optimize the chromatographic conditions, different columns, mobile phases, flow rates etc., were tested. 0.2M Ammonium acetate and acetonitrile in proportion of ratio 40:60 was preferred as mobile phase because it resulted in a greater response to Acamprosate after several preliminary investigatory runs compared with the different mobile phase combinations. The effect of the flow rate was studied in the range 0.9 to 1.2 ml/min and 1ml/min was preferred to be effective. Under these conditions, the analyte peak obtained was well-defined and free from tailing. The retention time (RT) was found to be 4.187 min. The optimized chromatographic parameters were listed in table 1.

Validation of the method:

When method development and optimization are complete, it is necessary to accomplish method validation. The validation studies include linear range (correlation coefficient), method precision (RSD, %), method accuracy (% recovery and RSD, %), sensitivity studies (LOD & LOQ), and robustness.

System Suitability:

Having optimized the efficiency of a chromatographic separation the quality of the chromatography was monitored by applying the following system suitability tests: capacity factor, tailing factor and theoretical plates. The system suitability method acceptance criteria set in each validation run were: capacity factor >2.0, tailing factor ≤2.0 and theoretical plates >2000. In all cases, the relative standard deviation (R.S.D) for the analytic peak area for two consecutive injections was < 2.0%. The system suitability test was performed using five replicate injections of standards before analysis of samples. The results are summarized in table – 2.

Table 2: System suitability parameters for Acamprosate

S.No	Parameter	Condition
1	Peak asymmetric factor	1.117
2	No. of theoretical plates	9711
3	Retention time	4.187min

Linearity:

Standard curves were constructed daily, for three consecutive days (Twice a day), using six standard concentrations in a range of 5, 10, 15, 20, 25 and 30 µg/ml for Acamprosate. The linearity of peak area responses versus concentrations was demonstrated by linear least square regression analysis. The results are summarized in table–3 and the calibration curve was shown in figure 3.

Table 3: Linearity study for Acamprosate

S.No	Concentration (µg/ml)	Area (mV.sec) (n= 6)
1	5	40.128
2	10	71.164
3	15	101.513
4	20	133.616
5	25	166.858
6	30	196.467

Concentration μg/ml

Figure 3: Calibration curve of Acamprosate

Precision:

System precision:

To study precision, six replicate standard solutions of Acamprosate (25 µg/ml) were prepared and analyzed using the proposed method. The percent relative standard deviation (% RSD) for peak responses was calculated.

Method precision:

The intraday and inter-day precision of the proposed method was determined by analyzing the corresponding responses 6 times for concentration of sample solutions of 25 µg/ml. Percentage assay was calculated and the result was reported in terms of relative standard deviation (% RSD).

Intermediate precision:

The intermediate precision of the proposed method was determined by performing the method by two analysts for concentration of sample solutions 25 µg/ml. The percent relative standard deviation (% RSD) for peak responses was calculated.

Limit of Detection & Limit of Quantification

LOD and LOQ of drug were calculated using the following equations designated by International Conference on Harmonization (ICH) guidelines.

LOD = 3.3 × σ/S

LOQ = 10 × σ/S

Where σ is the standard deviation of response

S is slope of the calibration curve

Accuracy (Recovery study):

The accuracy of the method was determined by calculating recovery of Acamprosate by the standard addition method. Known amounts of standard solutions of Acamprosate (2.5 µg/ml) were added to pre analysed sample solutions (20, 25, 30 µg/ml) of Acamprosate. The closeness of obtained value to the true value indicates that the proposed method is accurate.

% Recovery =[(Ct – Cpa)/ Cs] × 100

Where Ct = Total concentration of analyte

Cpa = Concentration of pre-analysed sample

Cs = Concentration of standard added to pre-analysed sample.

The results are summarized in table – 4.

Table 4: Recovery data for Acamprosate

S.No

Pre Analysed Sample Concentration (µg/ml)

Spiked amount (µg/ml)

% Recovery

Mean % Recovery

% RSD

2.5

100.8

102

100.8

100.4

98.8

99.2

100.8

98.8

101.2

99.46

99.6

0.680

0.836

1.063

Robustness:

The robustness study was performed to evaluate the influence of small but deliberate variation in the chromatographic condition. The robustness was checked by changing parameters like flow rate of mobile phase and detection wavelength. After each change, sample solution was injected and checked for % RSD of Area. The results are summarized in table – 5.

Table 5: Robustness study of Acamprosate

Parameter

Area (mV.sec) n =5

% RSD

Flow rate (ml/min)

0.9ml/min

1.1ml/min

Wavelength (nm)

218 nm

222 nm

159.171

164.211

195.987

167.425

0.054

0.331

0.11

0.26

Assay of pharmaceutical formulation

The proposed validated method was successfully applied to determine Acamprosate in their tablet dosage form. The results are summarized in table – 6.

Table 6: Assay of Acamprosate

S. No	Dosage	Sample estimated	%Assay
1	333 mg	330.28mg	99.24

Stability studies

In order to demonstrate the stability of both standard and sample solutions during analysis, both solutions were analyzed over a period of 24hr at room temperature. The results show that for both solutions, the retention time and peak area of Acamprosate remained almost similar (% R.S.D. less than 2.0) and no significant degradation within the indicated period, thus indicated that both solutions were stable for at least 24hr, which was sufficient to complete the whole analytical process. Further forced degradation studies were conducted indicating the stability of proposed method. The results are summarized in table – 7.

Acid degradation sample

10 tablets were accurately weighed and powdered. Weight equivalent to 25mg of Acamprosate was taken in a 50mL clean dry volumetric flask add about 30mL of mobile phase and sonicate to dissolve it for about 30minutes with intermittent shaking at controlled temperature. Then add 5mL of 0.1N acid (Hydrochloric acid), refluxed for 60minutes at 80°C, then cooled to room temperature, neutralize with 0.1N base (Sodium hydroxide) and dilute to volume with mobile phase. Filter about 5mL of the above sample solution through 0.45μ membrane filter. Pipette 0.5 mL of the above filtered sample solution into a 10 mL volumetric flask and dilute to volume with mobile phase

Base degradation sample

10 tablets were accurately weighed and powdered. Weight equivalent to 25mg of Acamprosate was taken in a 50mL clean dry volumetric flask add about 30mL of mobile phase and sonicate to dissolve it for about 30minutes with intermittent shaking at controlled temperature. Then add 5mL 0.1N base (Sodium hydroxide), refluxed for 60minutes at 80°C, then cooled to room temperature, neutralize with 0.1N acid (Hydrochloric acid) and dilute to volume with mobile phase. Filter about 0.5 mL of the above sample solution through 0.45μ membrane filter. Pipette 1 ml of the above filtered sample solution into a 10 mL volumetric flask and dilute to volume with mobile phase.

Table: 8 Summary of validated parameters for proposed method

Parameter

Result

Linearity range

Regression equation

Slope (m)

Intercept (C)

Correlation coefficient(r²)

System precision (% RSD, n=5)

Method precision (% RSD, n=5)

Intermediate precision (% RSD, n=5)

LOD (µg/ml)

LOQ (µg/ml)

% Recovery (Accuracy, n =3)

% Assay (% Assay, n=5)

5 – 30 µg/ml

y = 6.290x + 8.203

6.290

8.203

0.999

0.308

0.379

0.414

0.027

0.081

0.836

99.24

CONCLUSION:

Thus the proposed Stability indicating RP-HPLC method for the determination of Acamprosate in tablet dosage form was accurate, precise, linear, reliable, simple, economic and robust. The method has several advantages, including simple mobile phase, rapid analysis, simple sample preparation and improved selectivity as well as sensitivity. The method can be used for routine analysis of pharmaceutical formulation of Acamprosate in tablet formulation.

ACKNOWLEDGEMENT:

The authors are very thankful to SRINI Pharmaceuticals Ltd. for providing gift sample of Acamprosate and to SIMS College of Pharmacy for provision of facilities for this research work.

REFERENCES:

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Received on 25.10.2013 Accepted on 10.12.2013

Asian J. Pharm. Ana. 3(4): Oct. - Dec. 2013; Page 141-146