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International Journal of Analytical and Bioanalytical Chemistry

Universal Research Publications. All rights reserved

ISSN-2231-5012
Original Article
VALIDATED RP- HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF
METFORMIN HYDROCHLORIDE AND BENFOTIAMINE IN BULK DRUG
AND IN PHARMACEUTICAL DOSAGE FORM
Mihirkumar G. Patel, Pravin O. Patil*, Sanjay B. Bari
Department of Quality Assurance, H.R. Patel Institute of Pharmaceutical Education and Research,
Shirpur, Dist: Dhule (M.S.) India 425 405
Addresses for correspondence*
H. R. Patel Institute of Pharmaceutical Education and Research, Shirpur Dist: Dhule (M.S.) 425405 India
Tel/fax: +91- 2563-257599, Email: patilpo1980@rediffmail.com
Received 20 July 2012; accepted 28 July 2012
Abstract
A new simple, precise, accurate, selective and economical RP-HPLC method has been developed and validated for
simultaneous estimation of Metformin Hydrochloride (MET) and Benfotiamine (BEN) in tablet dosage form. The method
was carried out on a Waters column C-18 (250 mm x 4.6 mm, 5 µm) with a mobile phase consisting of water (pH 3.2
adjusted with ortho-phosphoric acid) and acetonitrile (75:25 v/v); at a flow rate of 0.8 mL min -1 with run time of 10 min.
Detection was carried out at 254 nm. The retention time for MET and BEN was found to be 2.125 and 3.881 min,
respectively. The MET and BEN followed linearity in the concentration range of 200- 600 µg mL-1 and 30- 90 µg mL-1
with r2= 0.999, respectively. The amounts of both drugs estimated by proposed method were found to be in good
agreement with label claim. The developed method was validated for precision, accuracy, sensitivity, robustness and
ruggedness. The LOD and LOQ were found to be 0.49 and 0.19 µg for MET and 1.6 and 0.65 µg for BEN respectively.
The developed method can be used for routine analysis of titled drugs in tablet formulation.
© 2011 Universal Research Publications. All rights reserved
Keywords: Metformin Hydrochloride; Benfotiamine; RP-HPLC, Validation.
Introduction 23] method for estimation of benfotiamine in
Metformin hydrochloride (MET) is an oral anti-diabetic pharmaceutical formulation. Combination product of
drug and is chemically N,N dimethylimidodicarbon metformin hydrochloride (MET) and benfotiamine (BEN)
-imidic diamide hydrochloride (1,1-dimehylbiguanide is Benforce M tablet which used as a neutraceuticals [24].
hydrochloride) [1] which acts by suppressing excessive The chemical structures of both drugs are shown in (Figure
hepatic glucose production and improving glucose 1, 2).
clearance, its predominant effect is to decrease fasting To the best of our knowledge, few literature methods were
plasma glucose [2]. It is official in Indian Pharmacopoeia reported for estimation of MET and BEN individually. but
[3], British Pharmacopoeia [4], European Pharmacopoeia no literatures have been found for simultaneous
[5] and United States Pharmacopeia [6]. A literature survey determination of MET and BEN in pharmaceutical
revealed spectrophotometry [7-9], HPLC [10-14], LC- preparations. The present paper describes a precise,
MS/MS [15] and Ion-pairing HPLC [16] methods for accurate, specific, sensitive and cost effective RP-HPLC
estimation of MET in pharmaceutical formulation. method for the simultaneous determination of MET and
Benfotiamine (BEN) is a synthetic derivative of thiamine BEN in the tablet dosage form and validation of the same
(vitamin B-1) and is chemically N-((4-amino-2-methyl- 5- as per the ICH guidelines [25-27].
pyrimidinyl) methyl)-N-(4-hydroxy-2- mercapto-1-methyl-
1 butenyl)formamide- S-benzoate dihydrogen phosphate Materials and Methods
[1]which show beneficial effects on general nerve health, Chemicals
neuropathy, retinopathy, peripheral neuropathy, general Metformin hydrochloride and Benfotiamine were obtained
ageing [17-21] A literature survey revealed RP-HPLC [22- from Rajat Pharmachem. Pvt. ltd, Ankleshwar and Akhil
International Journal of Analytical and Bioanalytical Chemistry 2012; 2(3): 196-200
196

Figure 1: Chemical Structure of Metformin Hydrochloride
Figure 2: Chemical Structure of Benfotiamine.
Health care, Baroda, India as a gift sample respectively.
Ortho-phosphoric acid (AR Grade), acetonitrile (HPLC
Grade), was purchased from Merck (India) Ltd., Worli,
Mumbai, India. Tablet formulation (Benforce- M) was
purchased from Indian market, containing MET (500 mg),
BEN (75 mg). HPLC Grade water (Milli-Q) was used
throughout the experiment.
Instrumentation
Analysis was performed on chromatographic system of
HPLC LC-2010C HT Shimadzu with LC Solution
equipped with Shimadzu SPD-M20A Prominence Diode
array detector. HPLC instrument was controlled by LC
Solution software.
Chromatographic Conditions
A Waters column C18, 5 μ (250 X 4.6 mm) was used for
chromatographic separation. The mobile phase composed
of water (pH 3.2 adjusted with ortho-phosphoric acid) and
acetonitrile (75:25 v/v); at a flow rate of 0.8 mL min -1 with
run time of 10 min. Mobile phase and sample solutions
were filtered through a 0.45 µm membrane filter and
degassed. The detection of both drugs was carried out at
254 nm.
Preparation of Diluent
1000 ml of HPLC Grade water was adjusted to pH 3.2 with
o-Phosphoric acid.
Preparation of Standard Stock Solutions
Standard stock solutions of 2000 µg mL-1 of MET and 300
µg mL-1 of BEN were prepared separately using diluent.
The stock solution of MET was diluted with diluent to give
working standard solutions containing 200 - 600 µg mL-1
concentrations, similarly the BEN stock solution was
diluted with diluent to give working standard solutions in
the range 30 - 90 µg mL-1. These standard solutions were
injected into HPLC column and calibration curves were
plotted by taking drug peak areas vs concentrations.
International Journal of Analytical and Bioanalytical Chemistry 2012; 2(3): 196-200
197
Analysis of marketed tablet formulation
To determine the content of MET and BEN in tablets
(Brand name: Benforce M, label claim: MET 500 mg, BEN
75 mg per tablet), twenty tablets were weighed, their mean
weight determined and finally powdered. An accurately
weighed tablet powder equivalent to 500 mg of MET and
75 mg BEN was transfer into 100 mL volumetric flask
containing 50 mL of diluent, sonicate for 10 minute and
volume was made up to the mark with diluent, the resulting
solution was filtered using 0.45 µm filter (Mill filter,
Milford, MA). From filtrate, 8 mL of solution was
transferred into 100 mL volumetric flask and volume was
made up to mark with diluent to obtain the concentration of
400 µg mL-1 MET and 60 µg mL-1 of BEN was subjected to
propose method and the amount of MET and BEN were
determined.
Validation of Method
The HPLC method was validated in accordance with ICH
guidelines.
Precision
The precision of the analytical method was studied by
analysis of multiple sampling of homogeneous sample.
Precision was estimated by repeatability and the
repeatability was assessed by analyzing six injection of a
homogeneous sample of 400 µg mL-1 of MET and 60 µg
mL-1 of BEN. The value of precision of repeatability along
with intra-day and inter day using three different
concentrations 300 µg mL-1, 400 µg mL-1 and 500 µg mL-1
of MET and 45 µg mL-1, 60 µg mL-1 and 75 µg mL-1 of
BEN respectively, in triplicate for three consecutive days.
Accuracy
The different between theoretical added amount and
practically achieved amount is called accuracy of analytical
method. Accuracy was determined at three different level
80 %, 100 % and 120 % of the target concentration 400 µg
mL-1 of MET and 60 µg mL-1 of BEN in triplicate.
Specificity
The specificity of an analytical method may defined as the
ability to detect the analyte peak in the presence of the
analyte by product, or other inactive components, such as
dosage form excipient or impurities.
Limit of detection (LOD) and Limit of quantification
(LOQ)
Limit of detection and limit of quantification were
estimated from signal to noise ratio. LOD is the lowest
concentration resulting in a peak area of three times the
baseline noise and the equation is LOD = 3.3 x ASD/S.
LOQ is the lowest concentration that provide signal to
noise ratio more than 10 and the equation is LOQ = 10 x
ASD/S, where ‘ASD’ is the average standard deviation and
‘S’ is the slope of the line.
Robustness
Robustness was performed by deliberately changing the
chromatographic conditions. The important parameter to be
studied was the resolution factor between two peaks.
Robustness of the method was carried out by deliberately
made small variation in the flow rate, pH of mobile phase,
organic phase ratio and column oven temperature by using
400 µg mL-1 of MET and 60 µg mL-1 solution of BEN,
respectively.
Ruggedness Table 1: Linearity Studies
Ruggedness was determined between two different labs, PARAMETER MET BEN
different analyst, different instrument and columns. Linearity [µg mL-1] 200 – 600 30 – 90
Linearity Equation Y = 5481X +33007 Y = 26543X-10859
Ruggedness of the method was performed by injecting 400 Slope 5481 26543
µg mL-1 of MET and 60 µg mL-1 solution of BEN, Intercept 33007 10859
respectively. Correlation Coefficient 0.999 0.999
Results and Discussion
Selection of Chromatographic Conditions and
Optimization of Mobile Phase
Mobile phase was optimized to separate MET and BEN
using Waters column C18, 5 μ (250 X 4.6 mm). Initially,
phosphate buffer pH 3.5 and acetonitrile in the various
proportions were tried as mobile phase but the broad peaks
for both the drugs were observed. Therefore, we select
Water (pH 3.2 adjusted ortho-phosphoric acid) and
acetonitrile as a mobile phase composition in 75:25 % v/v
ratio. Good resolution and symmetric peaks were obtained Figure 4: Calibration curve of MET Correlation
for both drugs when the pH of the mobile phase was Coefficient = 0.999, Slope = 5481, Intercept = 33007
adjusted to 3.2. The flow rate of the mobile phase was 0.8
mL min-1. Under optimum chromatographic conditions, the
retention time for MET and BEN was found to be 2.125
and 3.881 respectively when the detection was carried out
at 254 nm. A typical chromatogram of two drugs is shown
in (Figure 3).

Figure 5: Calibration curve of BEN Correlation

Figure 3: Typical Chromatogram of MET and BEN
Linearity
The linearity was determined at five levels over the range
of 50 % to 150 % of standard concentration in a diluent and
calibration curve constructed by plotting peak area against
the respective concentrations. The linearity of MET and
BEN followed in the concentration range of 200 - 600 µg
mL-1 and 30 - 90 µg mL-1, respectively. Each sample
solution was chromatographed in triplicate and the mean
peak area for MET and BEN calculated. The results are
shown in Table 1, (Figure 4,5).
Coefficient = 0.999, Slope = 26543, Intercept = 10859
Precision
The precision of this method was evaluated by calculating
the % RSD of the peak area of six replicate injections. The
RSD for repeatability of MET and BEN was found to be
0.55 and 1.27 %, respectively. The RSD values for intra-
day precision for MET and BEN was found to be in the
range 0.11-0.31 and 0.75 -1.29 %, respectively and the
inter-day precision for MET and BEN was found to be in
the range 0.18 - 0.25 and 0.67- 1.3 %, respectively. The
assay method precision acceptance criteria set in the
validation was RSD ≤ 2.0%. Results of precision study are
shown in Table 2.

Table 2: Results of Precision studies

Conc.
Conc. [g/mL] MET BEN
[g/mL]
Amount Found Amount Found
in µg mL-1 [n= 3]  % RSD in µg mL-1 [n= 3]  %RSD
SD SD
Intra – day Precision
300 300.27  0.51 0.17 45 44.86  0.57 1.29
400 400.40  0.45 0.11 60 59.85  0.63 1.04
500 499.85  0.59 0.31 75 75.15  0.005 0.75
Inter – day Precision
300 299.44  0.65 0.21 45 44.93  0.60 1.3
400 399.99  0.74 0.18 60 60.02  0.48 0.8
500 499.18  1.26 0.25 75 74.85  0.50 0.67

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Table 3: Results of Recovery Studies
Drugs Label claim (mg) Amount of standard drug added (%) % DrugRecovered [ n = 3] % R.S.D.
80 99.96 0.35
MET 500 100 99.65 0.20
120 100.15 0.43
80 99.94 0.80
BEN 75 100 100.20 0.65
120 100.08 1.02
Table-4: Assay Result of Marketed Formulation
Tablet Amount Content taken [µg/mL] Label claim (mg) % Label claim [n = 6] %RSD
MET 400 500 99.91 0.97
BEN 60 75 104.36 1.14
Specificity and Selectivity calculated for standard solutions. The results obtained from
The specificity of the method was checked for the validation of the methods and system suitability studies are
interference of retention time of tablet formulation and summarized in Table 5.
standard drug under optimized condition. Specificity of the Table 5: Summary of system suitability study
method was determined by comparing the chromatogram
Parameter MET BEN
obtained from tablet formulation and standard drug. As
there was no interference of excipient and the retention Retention time [tR] 2.125 3.881
time of standard drugs and tablet formulation were same, Theoretical plates [N] 2736 7742
so method was specific and selective. Resolution [Rs] - 7.93
Accuracy Asymmetry [T] 0.93 0.94
Accuracy was evaluated as percentage relative error
between the found mean concentration and added Conclusion
concentration for MET and BEN. The result obtained for The proposed new RP-HPLC method provide simple, fast,
three different concentration levels i.e. 80 %, 100 % and accurate, precise, reproducible and economical approach
120 % showed acceptable % recoveries in the range of for the simultaneous identification and quantification that
99.65% - 100.15 % for MET and 99.94% - 100.08 % for can be used for the quality control of the metformin
BEN which suggests that accuracy is excellent for both hydrochloride and benfotiamine in tablet formulation in
drugs. The results are shown in Table 3. routine quality control laboratories and the method was
Sensitivity validated as per ICH guidelines.
The limit of detection (LOD) and limit of quantification Acknowledgement
(LOQ) for MET and BEN was found to be 0.01 and 0.42 The authors wish to express their gratitude to the Principal
µg and 0.03 and 1.28 µg, respectively. The low values of and management, H.R. Patel Institute of Pharmaceutical
LOD and LOQ indicates high sensitivity of the method. Education and Research, Shirpur (M.S.), India for
Robustness and Ruggedness study providing the necessary facilities to carry out this research
The Robustness of the method evaluated by changing the work.
chromatographic condition and results were examined. References
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Source of support: H.R. Patel Institute of Pharmaceutical Education and

Research, Shirpur (M.S.), India; Conflict of interest: None declared

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