Development and Validation of Stability

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INTRODUCTION:

Lacosamide is a functionalized amino acid that has activity in the maximal electroshock seizure test, and is indicated for the adjunctive treatment of partialonset seizures and diabetic neuropathic pain. Recent studies indicate that Lacosamide only affects those neurons which are depolarized or active for long periods of time, typical of neurons at the focus of an epileptic seizure, as opposed to other antiepileptic drugs such as carbamazepine or lamotrigine which slow the recovery from inactivation and reduce the ability of neurons to fire action potentials.

For drug such as Lacosamide that is not intended to be absorbed into the bloodstream, Bioequivalence needs to be established by conducting a study with Pharmacodynamic endpoints. A comparison of In-Vitro dissolution profiles in different pH medium is an appropriate method for evaluating the bioequivalence of generic oral tablets.

MATERIALS AND METHODS: 

Instrumentation: The analysis of the drug was carried out on a Jasco LC system equipped with 2089 pump and photo-diode array detector (PDA) was used and a Reverse phase HPLC column Apollo Grace RP-C18 ((Make: Waters Corporation, Ireland); 250 mm x 4.6 mm I.D; particle size 5µm) was used. The output of signal was monitored and integrated using Chromnav software.

Chemicals and solvents: 

Milli-Q Water, Acetonitrile (ACN) (HPLC Grade), Orthophosphoric acid (GR Grade), Sodium dihydrogen ortho phosphate (GR Grade) was obtained from Merck, Mumbai.

Buffer preparation: 

Accurately weigh and transfer about 1.56 grams of Sodium di-hydrogen ortho phosphate in 1000 ml of purified water and mix. Adjust pH to 3.5 (±0.05) with dilute orthophosphoric acid solution. Filter the solution through 0.45µm membrane filter.

Mobile phase preparation:

Prepare a filtered and degassed mixture of Buffer and Acetonitrile in the ratio of 70:30 v/v respectively. Standard preparation Accurately

Standard preparation:

Accurately weigh and transfer about 100mg of Lacosamide into a 100 ml volumetric flask, add 60 ml of Acetonitrile, sonicate to dissolve. Cool the solution to room temperature and dilute to volume with Acetonitrile. Transfer 1.0 ml of the above solution into a 100 ml volumetric flask and dilute to volume with Acetonitrile.

Sample preparation:

(For Lacosamide Tablets 50mg) Weigh and finely powder 20 Tablets. Accurately weigh and transfer equivalent to 10 mg of Lacosamide into a 100 ml volumetric flask add about 70 ml of Acetonitrile, and sonicate for 30 minutes with intermittent shaking at controlled temperature and dilute to volume with ACN and mix. Filter the solution through 0.45µm membrane Filter. Transfer 1.0 ml of the above solution into a 100 ml volumetric flask and dilute to volume with ACN.

RESULTS AND DISCUSSION

Method development To develop a suitable and robust HPLC method for the determination of Lacosamide, different mobile phases were employed to achieve the best separation and resolution. The method development was started with Thermosil RP-C18 ((Make: Waters Corporation (Ireland); 250 mm x 4.6 mm I.D; particle size 5µm)) with the following mobile phase. Accurately weigh and transfer about 1.56 grams of Sodium di-hydrogen phosphate monohydrate in 1000 ml of purified water and mix. Adjust pH to 3.5 (±0.05) with dilute orthophosphoric acid solution. Filter the solution through 0.45µm membrane filter. Prepare a filtered and degassed mixture of Buffer and Acetonitrile in the ratio of 50:50 v/v respectively.

Lacosamide peak was eluted at void volume. For next trial the mobile phase composition was changed slightly. The mobile phase composition was Buffer and Acetonitrile in the ratio of 60:40 v/v.

In the above trail also the retention time of the peak improved but not satisfactory. Again the mobile phase composition changed slightly to Buffer and Acetonitrile in the ratio of 70:30 v/v respectively as eluent at flow rate 1.0 ml/min. UV detection was performed at 215nm. The retention time of Lacosamide was about 5.6 minutes and the peak shape was good.

The chromatogram of Lacosamide standard using the proposed method and System suitability results of the method are given as follows. Lacosamide shows significant UV absorbance at Wavelength 215nm. Hence this wavelength has been chosen for detection in analysis of Lacosamide.

Method validation The developed RP-HPLC method extensively validated for the determination Lacosamide Content using the following Parameters.

Specificity Blank interference A study to establish the interference of blank was conducted. Solvent was injected into the chromatograph in defined above chromatographic conditions and the blank chromatogram was recorded. Chromatogram of Blank solutions showed no peaks at the retention time of Lacosamide peak. This indicates that the diluent solution used in sample preparation do not interfere in estimation of Lacosamide in Lacosamide tablets. The chromatogram of Lacosamide Blank using the proposed method is shown in Figure 3.

System and Method Precision In the study of the instrumental system precision where, a RSD of 1.40% was obtained for the standard area obtained corresponding to the first day, being 1.37 % for the second day, respectively. The method precision study for six sample preparations in marketed samples showed a RSD of 0.5% and with the assay range of 99.1-101.5 with an average of 99.0. For the intermediate precision, a study carried out by the same analyst working on different day. The results calculated as inter-day RSD corresponded to 1.30 % (For Standard). The same study was carried out for different analysts (n = 6 number of samples per analyst) obtaining a RSD of 1.65 % (Intermediate Precision) and with the assay range of 99.1-100.9 with an average of 99.5. The Overall %RSD for n=12 is 1.44. Both results together with the individual results are showing that the proposed analytical technique has a good intermediate precision.

Accuracy The accuracy of the method was determined on three concentration levels by recovery experiments. The recovery studies were carried out in triplicate preparations on composite blend collected from 20 tablets of Lacosamide and analyzed as per the proposed method. Linearity of detector response The standard curve was obtained in the concentration range of 5-15µg/ml. The linearity of this method was evaluated by linear regression analysis. Slope, intercept and correlation coefficient [r2 ] of standard curve were calculated and given in Table.

Linearity of detector response The standard curve was obtained in the concentration range of 5-15µg/ml. The linearity of this method was evaluated by linear regression analysis. Slope, intercept and correlation coefficient [r2 ] of standard curve were calculated and given in Table.

Forced Degradation

Acid Degradation Sample: Accurately weigh and transfer equivalent to 50 mg of Lacosamide into a 100 ml volumetric flask add about 70 ml of solvent, and sonicate for 10 minutes with intermittent shaking at controlled temperature. Then add 10ml of 0.1N acid, refluxed for 30min at 60°C, then cooled to room temperature, neutralize with 0.1N NaOH and dilute to volume with diluent and mix. Filter the solution through 0.45µm membrane Filter. Transfer 5.0 ml of the above solution into a 100 ml volumetric flask and dilute to volume with diluent. Base Degradation Sample Accurately weigh and transfer equivalent to 100 mg of Lacosamide into a 100 ml volumetric flask add about 70 ml of

Base Degradation Sample Accurately weigh and transfer equivalent to 100 mg of Lacosamide into a 100 ml volumetric flask add about 70 ml of solvent, and sonicate for 30 minutes with intermittent shaking at controlled temperature. Then add 10ml of 0.1N Base (NaOH), refluxed for 30min at 60°C, then cooled to room temperature, neutralize with 0.1N Acid (HCl) and dilute to volume with diluent and mix. Filter the solution through 0.45µm membrane Filter. Transfer 1.0 ml of the above solution into a 100 ml volumetric flask and dilute to volume with diluent. Peroxide Degradation Sample Accurately weigh and transfer equivalent to 10 mg of Lacosamide into a 100 ml volumetric flask add about 70 ml of solvent, and sonicate for 10minutes with intermittent shaking at

Peroxide Degradation Sample Accurately weigh and transfer equivalent to 10 mg of Lacosamide into a 100 ml volumetric flask add about 70 ml of solvent, and sonicate for 10minutes with intermittent shaking at controlled temperature. Then add 2ml of 5% Peroxide, refluxed for 30min at 60°C, then cooled to room temperature and dilute to volume with solvent and mix. Filter the solution through 0.45µm membrane Filter. Transfer 1.0 ml of the above solution into a 100 ml volumetric flask and dilute to volume with solvent.

Thermal Degradation Sample Drug Powder exposed to heat at 105°C for about 5days. Accurately weigh and transfer equivalent to 10 mg of Lacosamide into a 100 ml volumetric flask add about 70 ml of solvent, and sonicate for 30 minutes with intermittent shaking at controlled temperature and dilute to volume with solvent and mix.

Photolytic Degradation

Drug Powder exposed to normal temperature for about 5days. Accurately weigh and transfer equivalent to 10 mg of Lacosamide into a 100 ml volumetric flask add about 70 ml of solvent, and sonicate for 30 minutes with intermittent shaking at controlled temperature and dilute to volume with solvent and mix. 

Filter the solution through 0.45µm membrane Filter. Transfer 1.0 ml of the above solution into a 100 ml volumetric flask and dilute to volume with solvent.

CONCLUSION We have developed a fast, simple and reliable analytical method for determination of Lacosamide in pharmaceutical preparation using RP-LC. As there is no interference of blank at the retention time of Lacosamide. It is very fast, with good reproducibility and good response. Validation of this method was accomplished, getting results meeting all requirements. The method is simple, reproducible, with a good accuracy and precision. It allows reliably the analysis of Lacosamide in bulk, its pharmaceutical dosage forms.

Acknowledgement: Ranbaxy limited, Gurgaon (Haryana)

REFERENCES

1. www.druginfo.nlm.nih.gov (Lacosamide)
2. www.rxlist.com (Lacosamide)
3. Clare Greenaway, Neville Ratnaraj, Josemir W. Sander, Philip N. Patsalos. A High-Performance Liquid Chromatography Assay to Monitor the New Antiepileptic Drug Lacosamide in Patients with Epilepsy. Therapeutic Drug Monitoring, 32(4), 448, (2010).
4. Peter Halasz, Reetta Kalviainen, Maria MazurkiewiczBeldzinska, Felix Rosenow, Pamela Doty, David Hebert, Timothy Sullivan, on behalf of the SP755 Study Group. Adjunctive Lacosamide for partial-onset seizures: Efficacy and safety results from a randomized controlled trial.
5. Practical HPLC Method Development, Second Edition, Lloyd R. Snyder, Joseph J. Kirkland, Joseph I. Glajch, (1997).
6. United States Pharmacopeia, USP 34-NF 29, (2011).
7. ICH Guidelines on Validation of Analytical procedure: Text and Methodology Q 2 (R1), (2011).

Posted by: Sunil Nana Patil. in Science | Date: 22/01/2016

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