Research J. Science and Tech.

11(3): July –September, 2019

ISSN 0975-4393 (Print) Available online at

2349-2988 (Online) www.anvpublication.org
DOI:

Vol. 11| Issue-03|

Research Journal of Science and Technology
July-September | 2019 Home page www.rjstonline.com

REVIEW ARTICLE

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
1
Department of Pharmaceutical Chemistry, Pravara Rural College of Pharmacy, Pravaranagar. Tal-Rahata,
Dist.- Ahmednagar.
2
Department of Pharmacognosy, Pravara Rural College of Pharmacy, Pravaranagar. Tal-Rahata,
Dist.- Ahmednagar.
*Corresponding Author E-mail: shinde.ganesh904@gmail.com

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.
KEY WORDS: Metformin, Gliclazide, RP-HPLC, Validation.

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

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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
Sr. No. Name Model Manufacturer/Supplier
1. Weighing balance PGB 100 Wenser High Precision Balance
Max : 100gm
Min : 0.001gm
2. Digital PH Meter PICO+ Lab India pvt ltd.
4. Sonicator WUC-4L Capacity -4 liter Wenser Ultra Sonicator
5. Magnetic stirrer Remi Equipment
6. HPLC HPLC 3000 Series Analytical Technologies Ltd.

Table 2: List of chemical used.

List of Chemicals
Name Supplied by Grade
Distilled water In House Double distilled
Methanol Merck HPLC
O-Phosphoric acid Lobachemie AR
Potassium dihydrogen phosphate Analab Fine Chemicals AR

Table 3: List of API used.

List of API used.
Sr.No. Name of Drugs Drug supplies & Manufacturer
1 Metformin HCL INDOCO remedies Ltd,Mumbai, India.
2 Gliclazide INDOCO remedies Ltd,Mumbai, India.

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.

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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:

Parameters Values
Column Grace C18 (250mm x 4.6ID, Particle size: 5 micron)
Mobile Phase Methanol :Water (80:20)
pH 3
Wavelength 234nm
Flow rate 0.9 ml/min
Injection volume 20µl
Run time 8.20min
Retention time 3.670min MET 6.541min GCL

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

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mV

3.670'
240

210

6.541'
180

150

120

90

60

30

-30
2 4 6 8 10 12 14 16 18 min
Time Area Resolution T.plate Asymmetry
3.670 1592021 13.34 5997 1.20
6.541 135512 0.00 12465 1.10
Fig. No. 4 : Chromatogram of standard solution (Gliclazide and metformin HCL)

mV
600

540

3.366'
480

420

360

300

240

180

120

60
6.042'

-60 2 4 6 8 10 12 14 16 18 min

Time Conc Area Resolution T.plate Asymmetry

3.366 20ppm 3564580 4.87 6193 0.96
6.042 20ppm 289597 0.00 11798 1.12
Fig. No. 5 Chromatogram for drug sample of Metformin HCL and Gliclazide

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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
Concentrations Area
10 1511680
20 3564580
30 5730756
40 7769094
50 9745534

Fig No.6 Linearity graph for Metformine HCL

Table No. 5. Linearity data for Gliclazide
Concentrations Area
10 155308
20 289597
30 424115
40 581456
50 702245

Fig No.7. Linearity graph for Gliclazide

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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
Metformin HCL Gliclazide
Morning Area Morning Area
5730756 424115
5731545 424396
5738815 427774
Evening 5736500 Evening 423863
5739063 426573
5747839 425662
Mean 5737420 Mean 425397
Standard deviation Standard Deviation
%RSD 0.11% %RSD 0.37%

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
Metformin HCL Gliclazide
Day 1 Area Day1 Area
5730756 424115
5731545 424396
5738815 427774
Day2 5733498 Day2 424081
5738201 423689
5740954 424386
Mean 5740954 Mean 424386
Standard deviation Standard Deviation
%RSD 0.07% %RSD 0.36%
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
Parameter Metformin HCL Gliclazide
LOD (µg/ml) 0.20 0.76
LOQ (µg/ml) 0.63 2.31
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:-
Drug Sample Flow rate(ml/min) Area Mean SD %RSD
MetformimnHCL 0.9 3564580 3563375 1047.40
1.1 3562680 0.029
0.8 3562866
Gliclazide 0.9 289597 290520 1133.23 0.39
1.1 290179
0.8 291785

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

Change in Wavelength:-
Drug Sample Wavelength Area Mean SD %RSD
Metformin HCL 234 3564580 3565219 2123.862284 0.05957172
232 3567589
236 3563488
Gliclazide 234 289597 290992 1543.398523 0.53039208
232 290729
236 292650

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.
Drug Level weight taken (mg) Mg Recovered % recovery % mean recovery
Metformin HCL 50 125 122 97.1 97
100 250 240 96.5
150 375 370 97
Gliclazide 50 20 51.18 102.1.7 102
100 40 40.81 100.9
150 60 62.51 102.6

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 06.12.2019 Modified on 02.01.2019

Accepted on 08.02.2019 ©A&V Publications All right reserved
Research J. Science and Tech. 2019; 11(3):.
DOI:

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