Quantitative Estimation and Validation of Metformin Hydrochloride and Gliclazide in their Tablet Dosage Form by RP-HPLC

 

Ganesh S Shinde^*1, Godge Rahul K ¹, Dr Ravindra Jadhav²

¹Department of Pharmaceutical Chemistry, Pravara Rural College of Pharmacy, Pravaranagar. Tal-Rahata,

Dist.-  Ahmednagar.

²Department of Pharmacognosy, Pravara Rural College of Pharmacy, Pravaranagar. Tal-Rahata,

Dist.-  Ahmednagar.

 

Abstract:

A rapid and sensitive isocratic reversed phase high performance liquid chromatographic method has been developed for quantitative analysis of metformin hydrochloride and gliclazide in bulk as well as pharmaceutical dosage forms. The method was validated according to FDA, ICH and USP guidelines with respect to accuracy, precision, specificity and linearity. The method was developed by using the mobile phase comprising of methanol and water (80:20) at a flow rate of 0.9 mL/min over Grace C18 (250mm x 4.6ID, Particle size: 5 micron) at ambient temperature. The retention time obtained for gliclazide and metformin was 5.3and 3.4min respectively. The recovery was found to be more than 97% for metformin and 102% for gliclazide that demonstrated the accuracy of the protocol. Intraday and interday precisions of the new method were less than the maximum allowable limit (% RSD  2.0) specified by the  ICH and FDA. The proposed RP-HPLC method is sensitive, precise and accurate. 

 

 

 

INTRODUCTION:

Type 2 diabetes mellitus (T2DM) is a chronic progressive metabolic disorder characterized by absolute or relative insulin deficiency.¹ Metformin hydrochloride (metformin), a biaguanide reduces HbAlc, FPG and PPG concentrations in type 2 diabetes patients and getting better glycaemic control by decreasing hepatic glucosecreation, reducing intestinal absorption of glucose , and recovering insulin sensitivity and productivity by improving peripheral glucose uptake and utilisation.² The chemical name of metformin hydrochloride (metformin HCL) is 1,1-Diamethylbiaguanidehyrochloride and has the structure  (Figure1), molecular formula C₄H₁₁N₅.HCL with molecular weight 165.63 g/mol. Metformin soluble in water, methanol, practically insoluble in acetone , ether, and chloroform.³

 

 

Figure.1: Structure of Metformin HCL.

 

Gliclazide, sold under the brand name Diamicron among others, is an anti-diabetic medication used to treat diabetes mellitus type 2. It is used when dietary changes, exercise, and weight loss are not enough. It is taken by mouth.⁴ Side effect may include low blood sugar, vomiting, abdominal pain, rash, and liver problems. Use by those with significant kidney problems, liver problems, or who are pregnant is not recommended. Gliclazide is in the sulfonylurea family of medications. It works mostly by increasing the release of insulin.⁵

 

Gliclazide was patented in 1966 and approved for medical use in 1972. It is on the World Health Organization's List of Essential Medicines, the most effective and safe medicines needed in a health system. The wholesale cost in the developing world is about US$2.46–3.92 per month. In the United Kingdom a month of medication costs the NHS about 2.12 pounds. It is not available for sale in the United States. The chemical name of gliclazide is N-(hexahydrocyclopenta(c) pyrrol-2-(1H)-ylcarbamoyl)-4-methyl) benzenesulphonamide and has the structure (Figure2), molecular formula C₁₅H₂₁N₃O₃S with molecular weight 323.40g/mol. Gliclazide poorly soluble in water, and soluble in methanol, ethanol and  acetonitrile.⁶

 

 

Figure.2: Structure of  Gliclazide.

 

There are several HPLC methods either in pharmaceutical products or biological samples reported in the literature for determination of metformin alone⁷ gliclazide alone ^8,9, metformin with other agents ¹⁰

 

MATERIAL AND METHOD : 

Table 1: List of apparatus/ instruments used.

List of Instruments

 

Table 2: List of chemical used.

List of Chemicals

 

Table 3: List of API used.  

List of API used.

 

Preparation of mobile phase:

Mixed a HPLC grade Methanol :

Water  with pH 3.0  (80:20) in volumetric flask. Filter through 0.45μ filter under vaccum filtration.

 

Diluent preparation:

Use mobile phase as diluent.

 

 

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.

 

Preparation of Sample solution:

Procedure:

20 tablets were weighed and powdered, tablets powder equivalent to 10 mg of Metformin HCL and   Gliclazide was transferred 100 ml volumetric flask, sufficient amount of mobile phase was added and dissolved by 20minutes ultrasonication. Then made the volume up to the mark with the mobile phase and filtered with 0.45µ filter paper. Pipette out respective solution from above solution and diluted to 10 ml mobile phase and use for sample injection.

 

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.

 

 

Fig. 3. Wavelength of Metformin HCL and Gliclazide

 

Optimized Chromatographic Conditions:

 

Following are the optimized chromatographic conditions for RP-HPLC method.

 

 

Fig. No. 4 : Chromatogram of standard solution (Gliclazide and metformin HCL)

 

 

Fig. No. 5 Chromatogram for drug sample of Metformin HCL and Gliclazide

 

RESULT AND DISCUSSION :

Validation of RP-HPLC:

I.        Linearity and Range:

Linearity of an analytical method is its ability to elicit test results that are directly proportional to the concentration of analyte in samples within a given range.The calibration curves exhibited linear relationship of peak area to concentration in the range 10-50 µg/mL for Metformine HCL and Gliclazide.  The graph of concentration Vs area was plotted which is showing straight line passing through all points. So as per ICH guidelines, the proposed  method for the determination of Metformine HCL and Gliclazide was found to be linear

 

Table No. 4. Linearity data for Metformine HCL     

               

 

Fig No.6 Linearity graph for Metformine HCL

 

Table No. 5.  Linearity data for Gliclazide

 

 

Fig No.7. Linearity graph for Gliclazide

II.     Precision :

The precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision study was carried out by injecting a sample into HPLC without changing the assay procedure and the result are shown the %RSD is less than 2% for Meclizine hydrochloride and Caffeine.

 

1.       Repeatability (intraday):

This study was performed with a minimum of three replicate measurement of sample solution at morning and evening in a same day.

 

Table No. 6. Intra-day variability for metformin HCL and Gliclazide

 

2. Intermediate precision (inter-day):

Intermediate precision was performed by injecting the sample solution in to HPLC at two different day.

Table No. 7. Inter-day variability for metformin HCL and Gliclazide

III. 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. The LOD were 0.022 µg/mL and 0.039 µg/mL for Metformine HCL and Gliclazide, respectively. The LOQ were found to be 0.06 µg/mL and 0.12µg/mL respectively.

 

Table No. 8. LOD and LOQ data for Metformine HCL and Gliclazide

 

IV. 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.  . %RSD of change in wavelength was found 0.059% for Metformin hydrochloride and 0.530% for Gliclazide %RSD of change in pH was found 0.54% for Metformin hydrochloride and 0.39% for Gliclazide. The result of robustness were found to be satisfactory within range.

 

Table No.9.  Data for Robustness (At Different Flow Rate)

1.        Change in Flow Rate:-

Table No. 10.Data for Robustness (At different wavelength)

Change in Wavelength:-

 

V. 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)

 

Table No. 11.Accuracy data of Metformin  HCL and Glicazide.

 

CONCLUSION:

The developed RP-HPLC method offers several advantages such as rapidity, usage of simple mobile phase and easy sample preparation steps. From the present study, it can be concluded that the proposed method is simple, specific, sensitive, precise, accurate and reproducible. Results of validation parameters demonstrated that the analytical procedure is suitable or appropriate for its intended purpose. Further, improved sensitivity makes it and reliable specific for its intended use. Hence, this method can be applied for the analysis of pharmaceutical dosage forms and pure drug.

 

ACKNOWLEDGEMENT :

The authors express their gratitude to the Pravara Rural College of Pharmacy, Loni for providing all the facilities and INDOCO remedies Ltd, Mumbai, India, for providing me the gift samples of Metformin HCL and Gliclazide.

 

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Received on 14.06.2019       Modified on 21.06.2019 Accepted on 16.07.2019      ©A&V Publications All right reserved Research J. Science and Tech. 2019; 11(3):201-207. DOI: 10.5958/2349-2988.2019.00030.5