A Comprehensive Review on the Method Development and Analytical Estimation of Metformin and Gliclazide

Analytical chemistry:

Introduction to Pharmaceutical Analysis:

Analytical chemistry termed as science of determining the components of materials in terms of the elements or compound contained., Analytical techniques proved in assuring and maintain the quality of substance and are critical components of QA and QC. The analytical chemistry consists of,

1. Qualitative analysis
2. Quantitative analysis

Selection of analytical method:

1. As accurate and precise as required.
2. Most productive, economical and convenient.
3. As simple as possible.

Classification of Analytical methods:

Analytical methods classified into two categories; Classical methods and instrumental methods: - 

1. Classical methods

Classical methods of analysis is also known as wet chemical analysis. These methods use no mechanical or electronic instruments beyond a balance. They are traditionally used for both qualitative and quantitative analysis.

They are also divided into two sub-type :

1. Volumetric method
2. Gravimetric method

B) Instrumental methods

These methods are based upon the measurement of some physical properties as conductivity, electrodes potential, light absorption or emission, mass to change ratio and Fluroscence of substance. There are many techniques available for the analysis of analytes which can be broadly classified as,

1. 1. Spectroscopic techniques  
   2. Electrochemical techniques
   3. Chromatographic techniques
   4. Miscellaneous techniques
   5. Hyphenated techniques

Amongst all the techniques mentioned above UV-Visible spectrophotometry and High Performance Liquid Chromatography (HPLC) are the most widely used techniques for quantitative analysis of pharmaceutical substances. HPLC is most widely used of all the analytical techniques HPLC gives high sensitivity high resolution ready adaptability to accurate quantitative determinations and it is non-destructive and may be applied to thermally labile compounds (unlike GC); it is also very sensitive technique since it incorporates a wide choice of detection methods.

Figure: HPLC system

MATERIALS AND METHODS:

Materials and Reagents:

Procurement of Drug Samples:

Drug sample suppliers and Manufacturer

Reagents and chemicals:

Following reagents and chemicals were used in whole experimental procedures.

Reagents and chemicals used.

Instrument Used:

List of apparatus/ instruments used

HPLC Instrument Information:

METHODS :

Solubility Studies: 

These studied was carried out with a view to find an ideal solvent in which drugs were completely soluble and stable. Various solvents were tried for checking solubility of Metformin HCL and Gliclazide. From solubility studies it was concluded that the drug is soluble in distilled water and methanol so these were selected as solvent for further analysis.

Selection of Analytical Wavelength:

A stock solution of drug was prepared in distilled water and UV spectrum of 100 μg/ml solutions of Metformin HCL and Glicazide was taken. For RP-HPLC analysis, spectra of drug were taken. Wavelength selected for analysis was such, that the drug, exhibited good absorbance.

HPLC Analysis

Optimization of HPLC method:

Initially different mobile phases used such as: 

Methanol: water in different proportions with adjustment of pH 6, 5.5 and 4.0 were tried. Then trials with methanol, acetonitrile and buffers were performed. Different concentrations of water with mixture of methanol and acetonitrile were tried in order to determine the best conditions for the effective separation of Metformin HCL and Gliclazide. The mobile phase consisting of Methanol; water pH 3 adjusted with ortho-phosphoric acid (80:20 % v/v) with 0.9 ml/min as flow rate was optimized as it was found to give best system suitability parameters.

Standard solutions:

An Accurately weighed quantity of about 10mg of Metformin HCL and   Gliclazide was taken in 100 ml volumetric flask dissolved in sufficient quantity of mobile phase, then sonicated for 15 min and diluted to 100ml with the same solvent so as to get the concentration of 10μg/ml. 1 ml of above solution transferred in 10 ml volumetric flask and the volume was made with diluents. The conc. of Drug is 10μg/ml.

Determination of λmax of Metformin HCL and Gliclazide

Standard stock solution of Metformin HCL and Gliclazide was diluted separately with diluents to obtain final concentration of 10μg/ml. solution was scanned using UV-Visible Spectrophotometer in the spectrum mode between the wavelength ranges of 400 nm to 200nm.

Validation of RP-HPLC:

1. Specificity

Specificity studies were conducted to estimate the actual separation of analyte from placebo, and all other related peaks. The placebo solution usually consists of all excipients employed for dosage form manufacturing.

2. System suitability

Two replicates of the sample and six replicates of standards were injected to verify the precision and accuracy of the chromatographic system. Different parameters of system suitability which include retention time, resolution, column theoretical plates, and tailing factor

3. Linearity and Range:

Linearity of the method was studied by injecting six concentrations of the drug prepared in the mobile phase in the range of 10-50 μg/ml for MET and 10-50 μg/ml Gliclazide in triplicate into the RP- HPLC system keeping the injection volume constant. The peak areas were plotted against the corresponding concentrations to obtain the calibration curves. 

4. Precision

Precision of the method was verified by repeatability studies. The repeatability of sample application and measurement of peak area for active compound were expressed in terms of RSD (relative standard deviation). Repeatability studies were performed by analyses of concentrations of 20,30,40 ug/ml Metformin and 20,30,40 ug/ml Gliclazide and  for RP-HPLC.  

5. Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The standard deviation of Y-intercept and slope of the calibration curves were used to calculate the LOD and LOQ for both the drugs using the following formulae.

LOD= 3.3(S)/s

LOQ = 10 (S)/ s

Where,

S =   Standard Deviation and

S =   Slope of the line

6. Robustness:

To evaluate robustness of HPLC method, few parameters were deliberately varied. The parameters included variation of flow rate, percentage of buffer in the mobile phase and pH of mobile phase.

7. Accuracy: 

Accuracy may often be expressed as percent recovery by the assay of a known amount of analyte added. The ICH documents recommend that accuracy should be assessed using a minimum of nine determinations over a minimum of three concentration levels, covering the specified range (i.e. three concentrations and three replicates of each concentration).

DRUG PROFILE

METFORMIN HCL:

GLICLAZIDE

RESULT

The proposed simultaneous estimation method was found to be simple, precise, accurate and rapid for the determination of Metformin HCL and Gliclazide. The mobile phase is simple to prepare and economical. The sample recoveries in all the formulations were in good agreement with their respective label claim and their suggestive not interference of formulation excipients in the estimation. Hence this method can be conveniently adopted for routine analysis of Metformin HCL and Gliclazide in the pharmaceutical dosage form.

1.Metformin HCL

2.Gliclazide