Karim et al., IJPSR, 2012; Vol.

3(9): 3170-3174 ISSN: 0975-8232

IJPSR (2012), Vol. 3, Issue 09 (Research Article)

Received on 07 May, 2012; received in revised form 13 June, 2012; accepted 20 August, 2012

DEVELOPMENT AND VALIDATION OF UV SPECTROSCOPIC METHOD FOR THE DETERMINATION OF METFORMIN

HYDROCHLORIDE IN TABLET DOSAGE FORM

Reatul Karim*, Nurunnahar Poly and Rebecca Banoo

Department of Pharmacy, State University of Bangladesh- 77, Satmasjid Road, Dhanmondi, Dhaka-1205,
Bangladesh

ABSTRACT

Keywords: A simple, economic, sensitive, precise and accurate UV spectrophotometric

Metformin hydrochloride, method was developed and validated for quantification of Metformin
Antidiabetic, hydrochloride in bulk and in tablet dosage form. Adequate drug solubility
Method validation,
and maximum assay sensitivity was found in 0.01N sodium hydroxide at
UV,
Quantitative analysis
233nm. Calibration graph constructed at 233nm was linear in concentration
Correspondence to Author:
range of 1-25μg/ml with correlation coefficient of 0.9998. The method was
validated as per ICH guidelines in terms of linearity (within 1-25µg/ml),
Reatul Karim accuracy (% recovery), precision (inter-day and intraday), specificity and
robustness. The limit of detection (LOD) and limit of quantification (LOQ)
Department of Pharmacy, State University
of Bangladesh- 77, Satmasjid Road, were found to be 0.2226µg/ml and 0.6745µg/ml respectively. Therefore, the
Dhanmondi, Dhaka-1205, Bangladesh proposed method is suitable and can be adopted for the determination
of Metformin hydrochloride from pharmaceutical dosage form in routine
E-mail: reatulkarim@gmail.com
quality control analysis.

INTRODUCTION: Metformin hydrochloride (MET) is a Indeed, MET may produce a small decrease in body
biguanide class of oral antidiabetic drug or blood weight during the initial months of treatment. This
glucose lowering agent, chemically is N, N-dimethyl cannot be attributed to a chronic decrease in food
imidodicarbonimidic diamide hydrochloride (Figure 1) consumption or increased physical activity, suggesting
1-6
. Metformin, itself produces the antidiabetic effect, that the drug increases metabolic energy expenditure.
but its hydrochloride salt is used as the salt form is The drug is found official in Merck index 10. There are
more soluble in aqueous medium. It is the first line various spectrophotometric methods developed for
drug for the treatment of type II diabetes or non- estimation of MET 11-13. Solvents used for the methods
insulin dependent diabetes mellitus (NIDDM), are comparatively of high cost. There is a need for a
particularly in overweight and obese people and those simple, rapid, cost effective and reproducible method
with normal kidney function 7, 8 and evidence suggests for assay of MET in pharmaceutical dosage forms.
it may be the best choice for people with heart failure
9
. It is also used in the treatment of polycystic ovary QUICK RESPONSE CODE IJPSR:
syndrome. Its therapeutic effect is achieved without ICV- 4.57
raising insulin concentrations, and it appears to reduce
insulin resistance. Unlike the other major blood Website:
glucose lowering agents, namely suphonylurea and www.ijpsr.com

insulin, MET does not cause weight gain.

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Therefore, it was thought of interest to develop MET was dissolved in 0.01 N NaOH, shaken for about
simple, accurate, fast and cost effective method for the 10 minutes and filtered through filter paper. The
analysis of MET in its tablet formulation. This paper filtered solution was further diluted to make the final
describes development and validation of simple, concentration of working sample equivalent to 100%
specific, sensitive, accurate and precise Ultraviolet of target concentration (10µg/ml).
spectroscopic method for the estimation of MET in
bulk and its formulation. Determination of λmax: Standard solution containing
10µg/ml of MET was scanned using 0.01N NaOH as
CH3 blank in the range of 200-400 nm to determine the
H wavelength of maximum absorption (λmax) of the
N N NH2 . HCl
drugs. MET showed absorbance maxima at 233 nm.
H3C
Development of Equation for Assay: 10 ml of the stock
NH NH solution was diluted to 100 ml with 0.01N NaOH to
FIGURE 1: STRUCTURE OF METFORMIN HYDROCLORIDE
obtain a 10 µg/ml MET reference standard solution.
MATERIALS AND METHODS: The absorbance of the sample solutions and the
reference standard solution was measured at 233 nm
Materials: All chemicals and reagents were of using 0.01N NaOH as blank. The amount of MET per
analytical grade unless stated otherwise. MET standard tablet and respective potency (%) in the marketed
was a gift from Beximco Pharmaceuticals Ltd, Dhaka. brands were determined using the following
Sodium hydroxide (NaOH) used was purchased from E. equations-
Merck, Darmstadt, Germany. Water was deionized and
double distilled. Marketed tablet formulations Amount of MET (mg) per tablet,
containing 500mg of MET were purchased from local
A Ws 100  10 P
drug stores in Dhaka city after checking their Z    Wt 
manufacturing license numbers, batch numbers, As 100  10 W 100
production and expiry dates.
Potency; (%) =
Instrumentation: A double beam Shimadzu (Kyoto,
Z A Ws 100  10 P 100
Japan) UV Visible spectrophotometer, Model UV mini-  100%     Wt   %
1700, equipped with 1 cm quartz cells, with a fixed slit Wc As 100  10 W 100 Wc
width (1 nm), wavelength accuracy of ±0.5 nm (with
Where, A= absorbance of sample solution; As=
automatic wavelength correction) was used. The drug
absorbance of reference standard solution; Ws=
analyses data were acquired and processed using UV
weight of reference MET powder (mg); W = weight of
Probe software (Version 2.0, Shimadzu, Japan) running
generic powder sample (mg); Wt= average weight of
under Windows XP on a Pentium PC. For scanning, the
tablet (mg); Wc= weight of drug claimed per tablet
wavelength range selected was from 400 nm to 200
(mg), P = potency of reference MET powder.
nm with medium scanning speed.

Preparation of Standard Solutions: Stock solution of Method Validation: Present study was conducted to
obtain a new, affordable, cost-effective and
MET was prepared by dissolving 10 mg drug in 100 ml
0.01N NaOH. Several aliquots of standard solutions of convenient method for spectroscopic determination of
MET (100µg/ml) were diluted to get standard solutions MET in solid dosage form. The method was validated
across the range of 1-25µg/ml. for the parameters like linearity, accuracy, precision
and robustness as per ICH guidelines 14.
Preparation of Sample Solutions: Average weight of
Specificity: Specificity of the method was determined
MET tablets of each brand was calculated. Then the
tablets were grinded to fine powder with the help of by comparing the spectrum of standard MET with that
of market product.
mortar and pestle. Then, powder containing 10 mg

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Linearity Study: The linearity of an analytical method is Limit of detection (LOD) and Limit of quantification
its ability to elicit that test results are proportional to (LOQ): Limit of detection (LOD) and Limit of
the concentration of analyte in samples within a given quantification (LOQ) for the assay were calculated
range. This was determined by means of calibration using the following equations 14;
graph using increasing amounts of standard solutions
(1-25µg/ml). Calibration curves were constructed and LOD= 3.3xSo/b and LOQ= 10xSo/b
the proposed method was evaluated by its correlation
coefficient and intercept value calculated in the Where So and b are the standard deviation and the
corresponding statistical study. Characteristic slope of the calibration line respectively.
parameters for regression equation of the method
Analysis of Marketed Products: This was carried out
were obtained by least squares treatment and of the
using the developed and validated method.
results and these parameters were used to confirm the
good linearity of the method. Statistical Analysis: Where applicable, results were
expressed as mean ± SD and analyzed statistically.
Intraday and Inter-day Precision Study: Intraday
precision (reproducibility) was determined by RESULTS AND DISCUSSION:
performing three repeated analysis of the five
standard MET solutions (1, 2, 10, 20, 25µg/ml) on the Specificity: UV spectroscopic method for MET analysis
same day, under the same experimental conditions. was found specific as spectrum of standard MET
Inter-day precision (ruggedness) of the method was coincide with that of market product indicating that
assessed by carrying out the analysis of standard excipients has no noticeable effect on the
solutions on three different days in the same effectiveness of the method.
laboratory. Measurement of absorbance was in
triplicate manner and the mean, standard deviation Linearity: Linearity of the method was evaluated from
and relative standard deviation (% RSD) was the correlation coefficient of calibration curves that
determined in order to assess the precision of the were constructed from average absorbance of drugs at
method. different concentration level (1-25µg/ml). The linearity
parameter (Table 1 and Figure 2) and the
Accuracy Study: This study was carried out using corresponding regression data indicated excellent
preformulated placebo granules prepared according to linear relationship (R2= 0.9998) over the working
a common formulation. 8 mg, 9 mg, 10 mg, 11 mg, and concentration range (1-25 μg/ ml). Table 2 shows
12 mg pure MET powder were then transferred statistical data of the calibration curve.
respectively in to five 100 ml volumetric flasks and
fixed amount of placebo granules were added to each TABLE 1: ABSORBANCE AND CORRESPONDING CONCENTRATION
OF STANDARD MET
of the volumetric flasks. These were then dissolved
Concentration (µg/ml) Absorbance
and then diluted up to 100 ml with 0.01N NaOH. Same 0 0.000
dilution pattern as sample solution was followed to 1 0.081
obtain five concentrations, 80%, 90%, 100%, 110%, and 2 0.149
5 0.381
120% of sample solution respectively. The solutions 10 0.752
were then analyzed for the content of MET using the 15 1.151
proposed method with reference solution (10μg/ml of 20 1.548
25 1.943
pure Metformin hydrochloride). All analyses were
carried out in triplet. TABLE 2: STATISTICAL DATA OF THE CALIBRATION CURVE
Parameter Criteria Values
Robustness Study: The robustness of the method was Mean + SD 0.0776 + 0.0004
Slope
assessed by altering the solvent composition of the RSD (%) 0.5135
experiment. Intercept Mean + SD -0.0059 + 0.0052
2 Mean + SD 0.9998 + 0.0001
R
RSD (%) 0.0057

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Intraday and Inter-day Precision: Intra-day precision

and accuracy of the proposed method were evaluated
by replicate analysis (n= 3) of calibration standards at
five concentration levels (1, 2, 10, 20, 25 µg/ml). Inter-
day precision and accuracy were determined by
assaying the calibration standards at five concentration
levels (1, 2, 10, 20, 25µg/ml) on three consecutive
days. % RSD was calculated for various runs. The
method is highly precise as % RSD for Intraday and
Inter-day Precision study was less than 2% (table 3 &
4).
FIGURE 2: CALIBRATION CURVE FOR MET
TABLE 3: INTRADAY PRECISION AND ACCURACY STUDY OF STANDARD MET
Declared Conc. Calculated Conc. (µg/ml) Mean ± SD Accuracy RSD
(µg/ ml) 1 2 3 (µg/ml) (%) (%)
1 1.0168 1.0425 1.0296 1.0296 + 0.0129 102.9639 1.2516
2 1.9704 1.9961 1.9446 1.9704 + 0.0258 98.5180 1.3080
10 9.7668 9.8441 9.9085 9.8398 + 0.0710 98.3978 0.7213
20 19.9472 19.7925 19.7410 19.8269 + 0.1073 99.1345 0.5412
25 25.0245 24.3028 25.0116 24.7796 + 0.4130 99.1186 1.6666

TABLE 4: INTER-DAY PRECISION AND ACCURACY STUDY OF STANDARD MET

Declared Conc. Calculated Conc. (µg/ml) Mean ± SD Accuracy RSD
(µg/ ml) Day 1 Day 2 Day 3 (µg/ml) (%) (%)
1 1.052 1.044 1.018 1.038 + 0.018 103.788 1.724
2 2.028 2.006 2.058 2.031 + 0.026 101.530 1.276
10 9.819 9.716 9.888 9.808 + 0.087 98.076 0.882
20 20.468 20.043 19.901 20.137 + 0.295 100.686 1.466
25 25.586 24.718 25.032 25.112 + 0.440 100.449 1.750

Accuracy: Accuracy is generally assessed by analyzing found satisfactory for MET with % RSD values below
samples with known concentration and comparing the 2.0%. All the results indicate that the method is highly
measured value with the true value. The measured accurate (table 5).
values were obtained by recovery test. % recovery was
TABLE 5: ACCURACY STUDY OF STANDARD MET
Declared Conc. Calculated Conc. (µg/ml) Mean ± SD Accuracy
RSD (%)
(µg/ ml) 1 2 3 (µg/ml) (%)
8 8.145 7.998 8.123 8.089 + 0.079 101.108 0.980
9 9.210 9.003 9.016 9.076 + 0.116 100.848 1.277
10 9.819 9.716 9.888 9.808 + 0.087 98.077 0.883
11 11.346 11.210 11.243 11.266 + 0.071 102.421 0.630
12 11.989 11.875 12.011 11.958 + 0.073 99.653 0.610

Robustness: Robustness study was performed by Low limit of quantification and limit of detection
making slight variations in solvent composition. No makes this method suitable for use in quality-control
significant effect was observed in the recovery of testing.
drugs. % recovery was 98% to 102%. So we can say
that the method is robust. Extinction Coefficient of MET: In addition, the
reliability of the proposed method was also evaluated
Limit of Detection (LOD) and Limit of Quantification by means of the determination of the extinction co-
(LOQ): Limit of detection (LOD) and Limit of efficient of MET using Beer-Lambert’s Law. Average
quantification (LOQ) were calculated as 0.2226µg/ml value was found 12579.1 M-¹cm-¹ with standard
and 0.6745µg/ml respectively. deviation of 245.294 M-¹cm-¹, RSD was 1.95%.

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Analysis of Marketed Products: After the validation of Among the different marketed brands supplied, the
the UV Spectrophotometric method, the potency test potency of all the brands was found to be within the
of four marketed tablet products were performed by limit of 98.597-101.790%.
the proposed validated method.
TABLE 6: EXTINCTION COEFFICIENT OF MET
Conc. Molar Absorbance Extinction Mean + SD
- - - - RSD (%)
(µg/ml) Conc. (M) (λmax=233nm) Coefficient, € (M ¹cm ¹) (M ¹cm ¹)
1 6.04x10-6 0.075 12422.3
2 1.21x10-5 0.149 12339.4
10 6.04x10-5 0.752 12447.1 12579.1 + 245.294 1.95%.
20 1.21x10-4 1.548 12815.6
25 1.51x10-4 1.943 12871.1

TABLE 7: POTENCY DETERMINATION OF THE MET MARKETED PRODUCTS

Code for brand Label claimed Calculated amount
Serial no. Potency (%)
name (mg) (mg)
1 P-a 500 508.95 101.790
2 P-b 500 492.99 98.597
3 P-c 500 499.07 99.814
4 P-d 500 499.93 99.986

CONCLUSION: The validation study shows that the 4. Ross MS. Dried blood spot liquid chromatography assay for therapeutic
drug monitoring of Metformin. J Chromatogr A. 1977; 133: 408-10.
developed UV method is linear, accurate, rapid, 5. Aburuz S, Millership J and McElnay J. The development and validation
precise, robust and inexpensive with acceptable of liquid chromatography method for simultaneous determination of
Metformin and glipizide, gliclazide, glibenclamide in plasma. J
correlation co-efficient, RSD (%) and standard Chromatogr B. 2005; 817: 277-82.
deviations which make it versatile and valuable for 6. Wang Y, Tang Y, Gu J, Fawcett JP and Bai X. Rapid and sensitive liquid
chromatography-tandem mass spectrometric method for quantitation
determination of Metformin hydrochloride in bulk or of Metformin in human plasma. J Chromatogr B. 2004; 808: 215-9.
pharmaceutical dosage forms. The advantages lie in 7. Clinical Guidelines Task Force. International Diabetes Federation.
"Glucose control: oral therapy". Global Guideline for Type 2 Diabetes.
the simplicity of sample preparation and the low costs Brussels: International Diabetes Federation. 35–8. Retrieved on
of reagents used. The proposed method is simple and November 6, 2007.
8. National Institute for Health and Clinical Excellence, Clinical guideline
do not involve laborious time-consuming sample 66: Diabetes - type 2. London, 2008.
preparation. So this method can be used in the quality 9. Eurich DT, McAlister FA, and Blackburn DF. Benefits and harms of
antidiabetic agents in patients with diabetes and heart failure:
control department for potency and dissolution study. systematic review. BMJ. 2007; 335: 497.
10. Budavari S. The Merck Index. ed. Merck and Co Inc. NJ. 13. 1997: 5792.
11. Arayne MS, Sultana N, Zuberi MH and Siddiqui FA. Spectrophotometric
ACKNOWLEDGEMENTS: The authors are thankful to quantitation of Metformin in bulk drug and pharmaceutical
Beximco Pharmaceuticals Ltd for providing the API as formulations using multivariate technique. Indian J Pharm Sci. 2009;
71(3): 331-335.
gift. 12. Chaturvedi PK and Sharma R. Simultaneous spectrophotometric
estimation and validation of three component tablet formulation
REFERENCES: containing pioglitazone hydrochloride, Metformin hydrochloride and
Glibenclamide. Analytical Letters.2008; 41(12): 2133-2142.
13. Rohokale BS, Jadhav VM and Kadam VJ. Studies in prospective process
1. Budavari S: The Merck Index. Merck and Co. NJ, USA. 2001. 790.
validation of Metformin HCl tablet Dosage formulation. Int J
2. Song-Jin T, Jing-Zheng S, Hai-Feng C and Zeng-Pei S. Determination of
PharmaTech Res. 2010; 2(3):1673-1678.
Metformin in plasma by capillary electrophoresis using field amplified
14. ICH: Proceedings of the International Conference on Harmonization of
sample stacking technique. J Chromatogr B. 1998; 708: 277-81.
Technical Requirement of Registration of Pharmaceuticals for Human
3. Ojala-Karlsoon P, Rouru J and Koulu M. Determination of Metformin in
Use (ICH Harmonized Tripartite Guidelines). Validation of Analytical
plasma by high performance liquid chromatography. J Chromatotgr A.
Procedures: Methodology, Q2B, Geneva, Switzerland: 1996.
1992; 583: 270-5.

How to cite this article:

Karim R, Poly N, Banoo R: Development and Validation of UV
Spectroscopic Method for the determination of Metformin
Hydrochloride in Tablet Dosage Form. Int J Pharm Sci Res, 2012;
Vol. 3(9): 3170-3174.

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