Development and validation of Related substances method with Gas

Chromatography for Memantine hydrochloride drug substance.

Vishnu.G, Chhabda P.J1,.K. Ramakrishna 2, *, V.Srinivasa rao3,

Abstract

Memantine is one of the first novel class medications for treatment of Alzheimer's disease. In
this study a simple, rapid and precise gas chromatographic method has been developed for the
Memantine hydrochloride, using HP-5, 30m,0.32mm,0.25µm column and nitrogen as a carrier
gas at a flow rate of 3 ml/min.The oven temperature was programmed at 50°C for 1 min,with a rise
of 10°C /min up to 250°(Hold for 25 min).The injector and detector port temperatures were
maintained at 120°C and 280°C.Detection was carried out using Flame ionization detector. Results
of assay and recovery studies were statistically evaluated for its accuracy and precision.

Keywords: Memantine Hydrochloride; Carrier gas; Gas chromatography

Article Outline:

1. Introduction

2. Experimental

2.1 Instrumentation

2.2 Solvents and chemicals

2.3 Chromatographic conditions

2.4 Standard stock solution

2.5 Procedure for recovery studies

3. Results and discussion

3.1 System suitability

 3.2 Linearity, limit of detection and limit of quantification

3.4 Accuracy and precision

4. Conclusion

1. INTRODUCTION

Memantine (1-amino-3,5-dimethyladamantane) (Fig.1.) is a tricyclic amine

structurally and pharmacologically related to the antiviral amantadine. The drug is used
to treat Parkinson’s disease, movement disorders and dementia syndrome. Memantine
(MEM) acts as a non-competitive inhibitor of the N-methyl-D-aspartate (NMDA) receptor
complex. MEM was first synthesized in the 1960s and was found in the 1970s to affect the
CNS. In 1989 MEM was found to inhibit NMDARs with an IC5 0 of approximately 1 µM
(which corresponds well with its therapeutic concentration range). The principal
mechanism of action of Memantine is believed to be the blockade of current
through channels of N-methyl-D- aspartate (NMDA) receptors-a glutamate receptor
subfamily broadly involved in brain function.
N
H

Figure 1. Chemical structure of Memantine

A review of literature revealed that derivatization is commonly employed with high

performance
Liquid chromatography (HPLC) to make Memantine sensitive to UV
detection.Alternatevely,
referactive index detection is also used for Memantine detection. But the above
techniques may not be robust enough. Also, the other mentioned methods are
elaborate, time-consuming
On the other hand , ionization detector. Boiling point of 239° makes
Memantine amenable to gas chromatography. Therefore method development
experiments were initiated using gas chromatography with flame ionization
detector.
2. EXPERIMENTAL

2.1 Instrumentation

For all Experiments ‘Agilent gas chromatograph (Model: 6890) with liquid injector 7683B
apparatus with flame ionization detector was used. The stationary phase was HP-5,30 m x 0.25
mm x 0.25µm (Make: J &W scientific, Part No:19091L-413)
2.2 Solvents, chemicals and materials

All experiments were performed using ‘A’ class volumetric glassware and an in-house
standard of Memantine hydrochlorie.HPLC grade Touene(Rankem,india) and highly pure
HPLC grade Milli Q water(Millipore,Bedford,MA,USA) were used in preparation of standard
and samples.

2.3 Chromatographic conditions

Gas chromatographic method with Flame ionization detection was selected for method of
analysis.The stationary phase procedure utilized a HP-5 column,(30 m x 0.32mmx0.25µm)
and detection at 280°.This temperature was selected because it provides enough sensitivity
needed for quantification of low level impurities, and nitrogen as a carrier gas at a flow rate of 3
ml/min for 50 min.The oven temperature was programmed at 50°C for 1 min, with a rise of 10°C
/min up to 250°(Hold for 25 min) with split ratio 1:5.The injector port temperatures were
maintained at 120°C .

2.4 Standard Preparation

Results And Discussions

Drugs solubility and solution stability are important properties in the study the first
approach was to compare buffers of different pH against purified water after setting the
solubility parameters the chromatographic parameters were optimized.
During the development the major problem observed is of the peak response and the
peak shape. There were different trials taken for optimization of GC column and as a result a
good result with tailing of about 1.2 – 1.3 was achieved which was well within the acceptance
criteria of <1.5 as per various pharamacopoiea.

Evolution of validation data

Specificity
The aim of specificity study is to assess the non interference of components. The
specificity of method was checked per diluent interference as well as all the other excipients
interference. Diluent and subsequenty placebo mixture(in duplicate) were injected to check
any interference. No peak due to placebo was detected at retention time of analyte peak. The
study proves that the test method is specific for quantification.

Precision

System suitability
System suitability shall be checked for the conformance of the suitability &
reproducibility of chromatographic system for analysis. System suitability was checked by
injecting six replicate injections of standard solution. % RSD for standard peak shall be not
more than 2.0
Method Precision
The purpose of this experiment is to prove the repeatability of the results obtained by
this quantification methodology. To confirm this six sample solutions were injected and
%RSD of the results was observed. The resulting RSD was 2.2% and well within the
acceptance limit and showed the method precise.

Intermediate