Forced Degradation Studies of Agomelatine: Development and Validation of Stability Indicating RP-HPLC Method using Internal Standard

 

Mukthinuthalapati Mathrusri Annapurna*, Bukkapatnam Venkatesh, Angirekula Narendra

Department of Pharmaceutical Analysis and Quality Assurance, GITAM Institute of Pharmacy,

GITAM University, Visakhapatnam-530045, India

*Corresponding Author E-mail: mathrusri2000@yahoo.com

 

ABSTRACT:

Agomelatine is an anti-depressent drug. A new simple stability indicating reverse phase liquid chromatographic method has been established for the determination of Agomelatine in presence of an internal standard. Agomelatine is used for the treatment of prostate cancer. The proposed work has been performed on Shimadzu Model CBM-20A/20 Alite with Phenomenex C18 column (250 mm × 4.6 mm i.d., 5 µm particle size) using 0.1% formic acid and acetonitrile mixture as the mobile phase with flow rate 0.6 ml/min (UV detection at 205 nm).  The method was validated as per ICH guidelines and the regression equation was found to be y = 0.2084x - 0.0475. Agomelatine was subjected to acidic, alkaline, oxidation, UV and thermal stress degradations and the method was reported to be robust and specific and can be applied for the assay of pharmaceutical formulations.

 

KEYWORDS:  Agomelatine, RP-HPLC, validation, stability-indicating, Bimatoprost.

 

 


INTRODUCTION:

Agomelatine (AGM) is a melatonergic anti-depressent (Fig 1). It is chemically known as N-[2-(7-methoxynaphthalen-1-yl) ethyl] acetamide, with molecular formula C15H17NO2 and molecular weight 243.301 g/mol. AGM is a melatonin receptor agonist (Dridi, 2013). It is a novel anti-depressant approved in February 2009 for use in the European Union (European Medicines Agency, 2012). Agomelatine, a naphthalene analog of melatonin, is a newly developed selective agonist of the human melatonergic MT1 and MT2 receptors and also shows 5-HT2C receptor antagonist activity (Milan, 2003). Agomelatine has been reported to be an effective antidepressant therapy with an entirely new mechanism of action (Olie and Kasper, 2000).Very few analytical methods have been reported for the determination of Agomelatine such as HPLC (Meghana, 2014; E.L. Shaheny, 2014; HE Xue et al., 2010; Harika, 2013; Nohas Rashed, 2014; Vineela P, 2014, Mathrusri, 2015a, Mathrusri, 2015b), LC-MS/MS in human plasma (Satish R. Patil, 2012; Xiaolin Wang, 2014) and HPTLC (Joshi Hitendra, 2013).

 

So, at present the authors have developed a stability indicating RP-HPLC method for the determination of Agomelatine in presence of an internal standard (IS), Bimatoprost. Bimatoprost is an ophthalmic drug used for the reduction of elevated intraocular pressure

 

MATERIALS AND METHODS:

Instrumentation:

Chromatographic separation was achieved by using Shimadzu Model CBM-20A/20 Alite HPLC system, equipped with SPD M20A prominence photodiode array detector with Phenomenex C18 column (250 mm × 4.6 mm i.d., 5 µm particle size) maintained at 25 ºC. Isocratic elution was performed using 0.1% formic acid and acetonitrile (30:70% v/v) and the flow rate was 0.6 ml/min. The overall run time was 10 min. The detection was carried at 205nm. 20 µL of sample was injected into the HPLC system and all chromatographic conditions were performed at room temperature (25°C ± 2°C).

 

Chemicals and Reagents:

Reference standards of Agomelatine (purity >99%) was obtained from Sun Pharmaceuticals Industries Ltd (India). Agomelatine is available as tablets with brand name AGOPREX® (Sun Pharmaceuticals Industries Ltd, Mumbai, India) and AGOVIZ® (Abbott India Limited, Mumbai) with label claim of 25 mg of drug. Methanol, sodium hydroxide and hydrochloric acid, formic acid and hydrogen peroxide were purchased from Merck (India). All chemicals are of HPLC grade. All chemicals were of analytical grade and used as received. The stock solution was prepared by transferring accurately 25 mg of Agomelatine in to a 25 ml volumetric flask and diluting with mobile phase (1000μg/ml) and further dilutions were made on daily basis from the stock solution with mobile phase as per the requirement and filtered through 0.45μm membrane filter prior to injection. The stock solution of Bimatoprost internal standard (IS) was also prepared by transferring accurately 25 mg of Bimatoprost in to a 25 ml volumetric flask in acetonitrile (1000 μg/ml) and 10μg/ml this solution was employed during the study after dilution with mobile phase.

 

Validation:

Linearity:

A series of solutions (0.05–200 μg/ml) were prepared from the AGM stock solution with mobile phase along with the internal standard (BMP) and 20 µL of each of these solutions were injected in to the HPLC system. The peak area ratio of AGM to that of the internal standard (peak area of AGM / peak area of BMP) were calculated from the chromatograms and a calibration curve was drawn by taking the concentration of the AGM solutions on the x-axis and the corresponding peak area ratio values on the y-axis. The limit of quantification and limit of detection measured as described in ICH guidelines Q2 (R1) (ICH guidelines, 2005). The limit of quantification and limit of detection were based on the standard deviation of the response and the slope of the constructed calibration curve (n=3), as described in ICH guidelines Q2 (R1) (ICH guidelines, 2005). Sensitivity of the method was established with respect to limit of detection (LOD) and LOQ for analytes.

 

Precision:

The intra-day precision of the assay method was evaluated by carrying out 9 independent assays of a test sample of AGM at three concentration levels (10, 20 and 50 μg/ml) against a qualified reference standard. The %RSD of three obtained assay values at three different concentration levels was calculated. The inter-day precision study was performed on three different days i.e. day 1, day 2 and day 3 at three different concentration levels (10, 20 and 50 μg/ml) and each value is the average of three determinations. The % RSD of three obtained assay values on three different days was calculated.

 

Accuracy:

The accuracy of the assay method was evaluated in triplicate at three concentration levels (80, 100 and 120%), and the percentage recoveries were calculated. Standard addition and recovery experiments were conducted to determine the accuracy of the method for the quantification of AGM in the drug product. The study was carried out in triplicate at 18, 20 and 22μg/ml. The percentage recovery in each case was calculated.

 

Robustness:

The robustness of the assay method was established by introducing small changes in the HPLC conditions which included wavelength (203 and 207 nm), percentage of methanol in the mobile phase (68 and 72%) and flow rate (0.9 and 0.6 ml/min). Robustness of the method was studied using six replicates at a concentration level of 10 μg/ml of AGM.

 

Forced Degradation Studies:

Forced degradation studies were performed to evaluate the stability indicating properties and specificity of the method (ICH guidelines, 2003). All solutions for stress studies were prepared at an initial concentration of 1 mg/ml of AGM and refluxed for 30 min at 80 ºC. Acidic degradation was performed by treating the AGM solution (1.0 mg/ml) with 0.1 M HCl for 30 min in a thermostat maintained at 80ºC. The stressed sample was cooled, neutralized with NaOH and then diluted with mobile phase as per the requirement. BMP (Internal standard; 5μg/ml) was added just before injecting in to the HPLC system. Alkaline degradation was performed by treating the drug solution (1.0 mg/ml) with 0.1 N sodium hydroxide for 30 min in a thermostat maintained at 80ºC. The stressed sample was cooled, neutralized with HCl and then diluted with mobile phase as per the requirement. BMP (Internal standard; 5μg/ml) was added just before injecting in to the HPLC system. Oxidation degradation was performed by treating the drug solution (1.0 mg/ml) with 30% H2O2 for 30 min in a thermostat maintained at 80ºC. The drug solution mixture was cooled and then diluted with mobile phase and 20 µl of the solution was injected in to the HPLC system after the addition of internal standard. Thermal degradation was performed by exposing the AGM solution to 80ºC. The drug solution mixture was cooled, diluted with mobile phase and internal standard was added just before injecting in to the HPLC system.

 

Assay of Agomelatine Tablets:

Two different brands procured from the local pharmacy store were transferred carefully in to two different volumetric flasks and extracted AGM with mobile phase. The contents of the volumetric flask were sonicated for 30 min, filtered and diluted with mobile phase as per the requirement. Internal standard was added just before injecting in to the HPLC system and the peak area ratio (AGM/BMP) was calculated from the respective chromatograms.

 

RESULTS AND DISCUSSION:

Method Optimization:

The authors have established a validated stability indicating RP-HPLC method (isocratic mode) for the determination of Agomelatine using Bimatoprost as an internal standard. Initially the stressed samples were analyzed using a mixture of 0.1% formic acid: acetonitrile (50: 50% v/v) with a flow rate of 1.0 ml/min in which the peak was obtained at Rt 17.27 mins and also the resolution and peak symmetry were not satisfactory. The mobile phase ratio was changed to 45:55% v/v and the drug sample was injected in to the loop where a sharp peak was eluted at 12.11 mins with tailing. Finally the mobile phase composition was modified as 30:70% v/v and a sharp Agomelatine peak was eluted at 4.732 ± 0.03 min and that of the internal standard, Bimatoprost at 6.434 ± 0.02 (UV detection at 205 nm). The chromatogram of the mobile phase (blank) as well as the Agomelatine standard in presence of internal standard were shown in Fig 2 and 3 respectively.

 

Method Validation:

The method was validated for system suitability, linearity, limit of quantization (LOQ), limit of detection (LOD), precision, accuracy, selectivity and robustness (ICH, 2005).

 

Linearity:

AGM shows linearity over a concentration range of 0.05–200μg/ml (Table 1) and the chromatographic response was shown in Fig 3. The linear regression equations were found to be y = 0.288 x + 0.048 (r2 = 0.9990).

 

Accuracy:

The method accuracy was proved by the recovery test at three different concentrations (80, 100 and 120 %). A known amount of AGM standard (5 μg/ml) were added to aliquots of sample solutions and then diluted to yield the total concentrations of 9, 10 and 11 μg/ml as described in Table 2. The % RSD was found to be 0.07-0.16 (<2.0 %) with a recovery of 98.7-99.4%.

 

Precision:

The intra-day precision of the method was determined by assaying three samples of each at three different concentration levels (10, 20 and 50 µg/ml) on the same day. The inter-day precision was calculated by assaying three samples of each at three different concentration levels (10, 20 and 50 µg/ml) on three different days. The % RSD for intra-day precision was found to be 0.21-0.53 whereas the inter-day precision was found to be 0.58-0.93 (Table 2).

 

Robustness:

Slight changes in flow rate, detection wavelength, mobile phase composition etc. affects the chromatographic response such as retention time, tailing factor and theoretical plates etc and the results were given in Table 3.   The % RSD obtained was 0.044-0.539 for Agomelatine (< 2.0%) indicating that the proposed method is robust.

 

Forced degradation studies:

The stability indicating capability of the method was established from the separation of AGM peak from the degraded samples. The degradation of AGM was found to be very similar for both the marketed formulation and standard. Typical chromatograms obtained following the assay of stressed samples are shown in Fig 4 (a-c). A slight decomposition (< 20 %) was observed when AGM drug was exposed to acidic (5.32 %), alkaline (8.08 %), oxidative (14.34 %) and thermal (0.03 %) conditions (Table 4). The system suitability parameters for all the degradation studies were shown in Table 4. The number of theoretical plates (N) is used to determine the performance and effectiveness of the column. The efficiency of a column can be measured by the number of theoretical plates per meter. It is a measure of band spreading of a peak. Smaller the band spread, higher is the number of theoretical plates, indicating good column and system performance. Columns with theoretical plates ranging from 4,000 to 100,000 plates / meter are ideal for a good system. The theoretical plates were found to be more than 4000 and the tailing factor was less than <1.5–2 or <2 indicating that the method is more selective and specific.

 

Analysis of commercial formulations:

The proposed method was applied for the determination of Agomelatine in marketed formulations available (AGOPREX® and AGOVIZ®). The % recovery was found to be 99.25-99.89 (Table 5).


 

Table.1. Linearity of Agomelatine

Conc. (μg/mL)

IS mean peak area

AGM mean peak area

BMT mean peak area/ I.S mean peak area

%RSD

0.05

803408

15843

0.019719744

0.41

0.1

838491

93737

0.111792494

0.23

10

840398

1533554

1.824794919

0.17

20

830558

3049204

3.671271603

0.11

50

792289

8336821

10.52244951

0.31

100

791907

16682423

21.06613908

0.40

150

832441

26542445

31.88507654

0.22

200

790245

32421447

41.02708274

0.34

*Mean of three replicates

Table.2. Precision and accuracy studies of Agomelatine

Conc.

Intra-day precision

Inter-day precision

* measured conc. ± SD

%RSD

SEM

* measured conc. + SD

%RSD

SEM

10

9.95 ± 0.03

0.25

0.0145

9.93 ± 0.07

0.69

0.0393

20

19.74 ± 0.04

0.21

0.0238

19.82 ± 0.18

0.93

0.1061

50

49.58 ± 0.26

0.53

0.1513

49.73 ± 0.29

0.58

0.1679

Accuracy

Spiked conc.

Total theoretical conc.

*Conc. Found ± SD

%RSD

SEM

%Recovery

08 (80 %)

18

17.89 ± 0.0134

0.07

0.043

99.4

10 (100 %)

20

19.74 ± 0.0324

0.16

0.0936

98.7

12 (120 %)

22

21.79 ± 0.0350

0.16

0.0919

99.05

*Mean of three replicates

 

Table.3. Robustness study of Agomelatine

Parameter

*%Assay ± SD

%RSD

SEM

*Retention time ± SD

%RSD

SEM

Flow rate (± 0.05 mL/min)

0.55

100.07 ± 0.1580

0.1579

0.0912

6.540±0.003

0.044

0.0017

0.65

99.30± 0.153

0.1540

0.0883

6.040±0.003

0.048

0.0017

Detection wavelength (± 2 nm)

203

101.19±0.111

0.1095

0.0640

6.457±0.025

0.380

0.0142

207

98.46±0.099

0.1003

0.0570

6.455±0.014

0.220

0.0082

Mobile phase composition (±2 v/v)

32:68

100.15±0.214

0.2135

0.1235

6.333±0.034

0.539

0.0197

28:72

98.13±0.233

0.2377

0.1347

6.651±0.021

0.316

0.0121

*Mean of three replicates

 

Table.4. Forced degradation studies of Agomelatine

Stress Conditions

*Drug recovered (%)

*Drug decomposed (%)

Theoretical plates

Tailing factor

Standard drug (Untreated)

100

-

13777.379

1.245

Acidic degradation

94.68

5.32

14017.254

1.234

Alkaline degradation

91.92

8.08

13737.378

1.301

Oxidative degradation

85.66

14.34

13926.269

1.347

Thermal degradation

99.97

0.03

13786.378

1.274

*Mean of three replicates

 


Table.5. Analysis of Agomelatine Tablets

Formulation

Labelled claim

(mg)

Amount found* (mg)

Recovery* (%)

AGOPREX®

10

9.92

99.25

AGOVIZ®

10

9.99

99.89

* Mean of three replicates

 

 

Fig 1: Chemical structure of Agomelatine (AGM)


 

 


 

Fig 2: Typical chromatograms of blank

 

Fig 3: Typical chromatograms of Agomelatine (10μg/ml) with internal standard

 

Fig 4: Calibration curve of Agomelatine

 

Fig 5: Typical chromatograms of [a] Agomelatine (10 μg/ml) and [b] acidic [c] alkaline [d] oxidative

[e] thermal degradations in presence of internal standard.

 

CONCLUSION:

The proposed stability-indicating HPLC method was validated as per ICH guidelines and applied for the determination of Agomelatine in pharmaceutical dosage forms and can be successfully applied to perform long-term and accelerated stability studies of Agomelatine formulations. It was observed that Agomelatine is more sensitive towards oxidation in comparison to others during the stress degradation studies.

 

ACKNOWLEDGEMENT:

The authors are grateful to M/s GITAM University, Visakhapatnam for providing the research facilities and to Sun Pharmaceuticals Industries Ltd for supplying the gift samples of Agomelatine. There is no conflict of interest.

 

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Received on 25.01.2017             Modified on 05.02.2017

Accepted on 23.02.2017           © RJPT All right reserved

Research J. Pharm. and Tech. 2017; 10(3): 703-707.

DOI: 10.5958/0974-360X.2017.00132.9