P. Aravinda Reddy. et al.

J Pharm Res, 2016; 05 (12): 268-281

World Inventia Publishers

Journal of Pharma Research

Available online through
www.jprinfo.com

Vol. 05 Issue 12, 2016 ISSN: 2319-5622

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF BENFOTIAMINE

AND METFORMIN IN COMBINED DOSAGE FORM
P. Aravinda Reddy1, Ramya Sri. S2

1Department of Pharmaceutical analysis, Vaageswari College of Pharmacy, Karimnagar, Telangana-505527, India

2Department of Pharmacy, University College of Technology, Osmania University, Hyderabad, Telangana, 500007, India

Received on: 13-11-2016; Revised and Accepted on: 31-12-2016

ABSTRACT

A rapid and precise reverse phase high performance liquid chromatographic method has been
developed for the validation of Benfotiamine and Metformin, in its pure form as well as in tablet dosage form. Chromatography was
carried out on a Phenomenex Gemini C18 (4.6×250mm) 5µ column using a mixture of Methanol: TEA Buffer (65:35 v/v) as the mobile
phase at a flow rate of 1.0ml/min, the detection was carried out at 230nm. The retention time of the Benfotiamine and Metformin was
2.121, 3.643 ±0.02min respectively. The method produce linear responses in the concentration range of 10-50mg/ml of Benfotiamine
and 20-100mg/ml of Metformin. The method precision for the determination of assay was below 2.0%RSD. The method is useful in the
quality control of bulk and pharmaceutical formulations.

Keywords: Benfotiamine, Metformin, RP-HPLC, validation.

INTRODUCTION

Analytical chemistry1 Structural analysis is the determination of the spatial

arrangement of atoms in an element or molecule or the
Analytical chemistry is a scientific discipline used to identification of characteristic groups of atoms (functional
study the chemical composition, structure and behaviour of groups). An element, species or compound that is the subject of
matter. The purposes of chemical analysis are together and analysis is known as analyte. The remainder of the material or
interpret chemical information that will be of value to society in sample of which the analyte(s) form(s) a part is known as the
a wide range of contexts. Quality control in manufacturing matrix.
industries, the monitoring of clinical and environmental
samples, the assaying of geological specimens, and the support The gathering and interpretation of qualitative, quantitative and
of fundamental and applied research are the principal structural information is essential to many aspects of human
applications. Analytical chemistry involves the application of a endeavour, both terrestrial and extra-terrestrials. The
range of techniques and methodologies to obtain and assess maintenance of an improvement in the quality of life throughout
qualitative, quantitative and structural information on the the world and the management of resources heavily on the
nature of matter. information provided by chemical analysis. Manufacturing
industries use analytical data to monitor the quality of raw
 Qualitative analysis is the identification of elements, materials, intermediates and finished products. Progress and
species and/or compounds present in sample. research in many areas is dependent on establishing the
chemical composition of man-made or natural materials, and the
 Quantitative analysis is the determination of the monitoring of toxic substances in the environment is of ever
absolute or relative amounts of elements, species or increasing importance. Studies of biological and other complex
compounds present in sample. systems are supported by the collection of large amounts of
analytical data. Analytical data are required in a wide range of
*Corresponding author: disciplines and situations that include not just chemistry and
P. Aravinda Reddy most other sciences, from biology to zoology, butte arts, such as
painting and sculpture, and archaeology. Space exploration and
Department of Pharmaceutical analysis
clinical diagnosis are two quite desperate areas in which
Vaageswari College of Pharmacy analytical data is vital. Important areas of application include the
Karimnagar, Telangana-505527 following.
India
Email: surapharmalabs1@gmail.com Quality control (QC) in many manufacturing industries, the
DOI: chemical composition of raw materials, intermediates and
finished products needs to be monitored to ensure satisfactory

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P. Aravinda Reddy. et al. J Pharm Res, 2016; 05 (12): 268-281

quality and consistency. Virtually all consumer products from Preparation of mobile phase:
automobiles to clothing, pharmaceuticals and foodstuffs,
electrical goods, sports equipment and horticultural products Accurately measured 350 ml (35%) of TEA buffer and
rely, in part, on chemical analysis. The food, pharmaceutical and 650 ml of HPLC Methanol (65%) were mixed and degassed in a
water industries in particular have stringent requirements digital ultrasonicater for 10 minutes and then filtered through
backed by legislation for major components and permitted 0.45 µ filter under vacuum filtration.
levels of impurities or contaminants. The electronic industry
needs analyses at ultra-trace levels (parts per billion) in relation Diluent Preparation:
to the manufacture of semi-conductor materials. Automated,
computer-controlled procedures for process-stream analysis The Mobile phase was used as the diluent.
are employed in some industries.
VALIDATION PARAMETERS
MATERIALS AND METHODS
SYSTEM SUITABILITY
Benfotiamine Provided by Sura labs, Metformin Provided by
Accurately weigh and transfer 10 mg of Benfotiamine and
Sura labs, Water and Methanol for HPLC from LICHROSOLV
Metformin working standard into a 10ml of clean dry
(MERCK), Acetonitrile for HPLC from Merck. volumetric flasks add about 7mL of Diluents and sonicate to
dissolve it completely and make volume up to the mark with the
HPLC METHOD DEVELOPMENT:
same solvent. (Stock solution)
TRAILS
Further pipette out 0.3 ml of Benfotiamine and 0.6ml of
Metformin from the above stock solutions into a 10ml
Preparation of standard solution:
volumetric flask and dilute up to the mark with Methanol.
Accurately weigh and transfer 10 mg of Benfotiamine and
Procedure:
Metformin working standard into a 10ml of clean dry
volumetric flasks add about 7ml of Methanol and sonicate to
The standard solution was injected for five times and measured the
dissolve and removal of air completely and make volume up to
area for all five injections in HPLC. The %RSD for the area of five
the mark with the same Methanol.
replicate injections was found to be within the specified limits.
Further pipette 0.3 ml of Benfotiamine and 0.6ml of Metformin
SPECIFICITY STUDY OF DRUG:
from the above stock solutions into a 10ml volumetric flask and
dilute up to the mark with Methanol.
Preparation of Standard Solution:
Procedure: Accurately weigh and transfer 10 mg of Benfotiamine and
Metformin working standard into a 10ml of clean dry
Inject the samples by changing the chromatographic conditions
volumetric flasks add about 7ml of Diluents and sonicate to
and record the chromatograms, note the conditions of proper
dissolve it completely and make volume up to the mark with the
peak elution for performing validation parameters as per ICH
same solvent. (Stock solution)
guidelines.
Further pipette out 0.3 ml of Benfotiamine and 0.6ml of
Mobile Phase Optimization:
Metformin from the above stock solutions into a 10ml
volumetric flask and dilute up to the mark with Diluent.
Initially the mobile phase tried was methanol: Water,
Methanol: Phosphate buffer and ACN: Water with varying
Preparation of Sample Solution:
proportions. Finally, the mobile phase was optimized to TEA
buffer (pH 4.0), Methanol in proportion 65:35 v/v respectively. Take average weight of one Tablet and crush in a mortar by
using pestle and weight 10 mg equivalent weight of
Optimization of Column:
Benfotiamine and Metformin sample into a 10mL clean dry
volumetric flask and add about 7mL of Diluent and sonicate to
The method was performed with various C18columns
dissolve it completely and make volume up to the mark with the
like Symmetry, X terra and ODS column. Phenomenex Gemini
same solvent. Filter the sample solution by using injection filter
C18 (4.6×250mm) 5µ was found to be ideal as it gave good peak
which contains 0.45µ pore size.
shape and resolution at 1ml/min flow.
Further pipette out 0.3 ml of Benfotiamine and 0.6ml of
VALIDATION
Metformin from the above stock solutions into a 10ml
PREPARATION OF BUFFER AND MOBILE PHASE: volumetric flask and dilute up to the mark with Diluent.

Preparation of Triethylamine buffer (pH-4.0): Procedure:

Take 6.0ml of Triethylamine in to 750ml of HPLC water in a Inject the three replicate injections of standard and sample
1000ml volumetric flask and mix well. Make up the volume up solutions and calculate the assay by using formula:
to mark with water and adjust the pH to 4.0 by using
Orthophosphoric acid, filter and sonicate.

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P. Aravinda Reddy. et al. J Pharm Res, 2016; 05 (12): 268-281

Accuracy:

Procedure:

Inject the Three replicate injections of individual concentrations

(50%, 100%, 150%) were made under the optimized
conditions. Recorded the chromatograms and measured the
peak responses. Calculate the Amount found and Amount added
PREPARATION OF DRUG SOLUTIONS FOR LINEARITY: for Benfotiamine and Metformin and calculate the individual
recovery and mean recovery values.
Accurately weigh and transfer 10 mg of Benfotiamine and
Metformin working standard into a 10ml of clean dry ROBUSTNESS:
volumetric flasks add about 7ml of Diluents and sonicate to
The analysis was performed in different conditions to find the
dissolve it completely and make volume up to the mark with the
variability of test results. The following conditions are checked
same solvent. (Stock solution)
for variation of results. .
Procedure:
For preparation of Standard solution:
Inject each level into the chromatographic system and measure
Accurately weigh and transfer 10 mg of Benfotiamine and
the peak area.
Metformin working standard into a 10ml of clean dry
volumetric flasks add about 7mL of Diluents and sonicate to
Plot a graph of peak area versus concentration (on X-axis
dissolve it completely and make volume up to the mark with the
concentration and on Y-axis Peak area) and calculate the
same solvent. (Stock solution)
correlation coefficient.
Further pipette out 0.3 ml of Benfotiamine and 0.6ml of
PRECISION Metformin from the above stock solutions into a 10ml
REPEATABILITY volumetric flask and dilute up to the mark with Diluent.

Preparation of Benfotiamine and Metformin Effect of Variation of flow conditions:

Product Solution for Precision: The sample was analyzed at 0.9 ml/min and 1.1 ml/min instead
Accurately weigh and transfer 10 mg of Benfotiamine and of 1ml/min, remaining conditions are same. 10µl of the above
Metformin working standard into a 10ml of clean dry sample was injected twice and chromatograms were recorded
volumetric flasks add about 7ml of Diluents and sonicate to
dissolve it completely and make volume up to the mark with the Effect of Variation of mobile phase organic composition:
same solvent. (Stock solution)
The sample was analyzed by variation of mobile phase i.e.
Further pipette out 0.3 ml of Benfotiamine and 0.6ml of Methanol: TEA buffer 4pH was taken in the ratio and 60:40,
Metformin from the above stock solutions into a 10ml 70:30 instead of 65:35 remaining conditions are same. 10µl of
volumetric flask and dilute up to the mark with Diluent. the above sample was injected twice and chromatograms were
recorded.
The standard solution was injected for five times and measured the
area for all five injections in HPLC. The %RSD for the area of five RESULTS AND DISCUSSION
replicate injections was found to be within the specified limits.
Optimized Chromatogram (Standard)
INTERMEDIATE PRECISION: Mobile phase ratio : Methanol: TEA Buffer (65:35 v/v)
To evaluate the intermediate precision (also known as Column: Phenomenex Gemini C18 (4.6×250mm) 5µ
Ruggedness) of the method, Precision was performed on
Column temperature: 40ºC
different days by maintaining same conditions.
Wavelength: 230nm
Procedure:
Flow rate: 1ml/min
DAY 1: Injection volume: 10µl
The standard solution was injected for six times and measured the Run time: 6minutes
area for all six injections in HPLC. The %RSD for the area of six
replicate injections was found to be within the specified limits.

DAY 2:

The standard solution was injected for six times and measured the
area for all six injections in HPLC. The %RSD for the area of six
replicate injections was found to be within the specified limits.

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P. Aravinda Reddy. et al. J Pharm Res, 2016; 05 (12): 268-281

Fig 1: Optimized Chromatogram (Standard)

Table 1: Optimized Chromatogram (Standard)

Resolution
S.no USP Plate
Name RT Area Height USP Tailing
Count

1 Benfotiamine 2.121 406433 77644 1.2 4009

9.8
2 Metformin 3.643 1592811 251532 1.1 7849

Observation:

Optimized Chromatogram (Sample)

Fig 2: Optimized Chromatogram (Sample)

Table 2: Optimized Chromatogram (Sample)

S.no Name Rt Area Height USP Tailing USP Plate Count Resolution

1 Benfotiamine 2.142 403871 77464 1.2 4136

2 Metformin 3.649 1573821 259361 1.1 7812 10.3

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P. Aravinda Reddy. et al. J Pharm Res, 2016; 05 (12): 268-281

VALIDATION

System suitability:
Table 3: Results of system suitability for Benfotiamine

Area Height
S.No Peak Name RT (µV*sec) (µV) USP Plate Count USP Tailing

1 Benfotiamine 2.152 382726 70725 5271 1.2

2 Benfotiamine 1.2
2.157 382621 70625 5928

3 Benfotiamine 1.2
2.141 389172 70617 5283
Benfotiamine 1.2
4 2.133 384152 70718 5763
Benfotiamine 1.2
5 2.166 389721 70172 6222
Mean 385678.4

Std. Dev. 3497.932

% RSD 0.906956

Table 4: Results of system suitability for Metformin

Area Height
S.No Peak Name RT (µV*sec) (µV) USP Plate Count USP Tailing Resolution

1 1.1
Metformin 3.674 1562821 227365 5827 10.1

2 Metformin 1.1 10.1

3.631 1562726 226748 6183

3 Metformin 1.1 10.1

3.625 1567361 227163 5029
Metformin 1.1 10.1
4 3.692 1562811 226948 4920
Metformin 1.1 10.1
5 3.629 1563816 226452 5183
Mean 1563907

Std. Dev. 1982.03

% RSD 0.126736

SPECIFICITY

Assay (Standard):

Table 5: Peak results for assay standard of Benfotiamine

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P. Aravinda Reddy. et al. J Pharm Res, 2016; 05 (12): 268-281

S. No Name RT Area Height USP Tailing USP Plate Count Injection

1 Benfotiamine 2.152 406538 77074 1.2 4009 1

2 Benfotiamine 2.198 409975 76001 1.2 4136 2

3 Benfotiamine 2.179 402283 76048 1.2 5263 3

Table 6: Peak results for assay standard of Metformin

S. No Name RT Area Height USP Tailing USP Plate Count Injection

1 Metformin 3.646 1609924 251956 1.1 7849 1

2 Metformin 3.604 1601840 246020 1.1 7819 2

3 Metformin 3.610 1602832 248287 1.1 7826 3

Assay (Sample):
Table 7: Peak results for Assay sample of Benfotiamine

S. No Name RT Area Height USP Tailing USP Plate Count Injection

1 Benfotiamine 2.152 406538 77074 1.2 4009 1

2 Benfotiamine 2.150 409975 76001 1.2 4136 2

3 Benfotiamine 2.187 402911 77823 1.2 5173 3

Table 8: Peak results for Assay sample of Metformin

S. No Name RT Area Height USP Tailing USP Plate Count Injection

1 Metformin 3.646 1609924 251956 1.1 7849 1

2 Metformin 3.651 1601840 246020 1.1 7819 2

3 Metformin 3.601 1603821 240291 1.1 6812 3

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P. Aravinda Reddy. et al. J Pharm Res, 2016; 05 (12): 268-281

LINEARITY Table 10: Chromatographic Data for Linearity Study of

Metformin
Table 9: Chromatographic Data for Linearity Study of
Benfotiamine Concentration Level Concentration Average
(%) g/ml Peak Area
Concentration Average
Concentration Level 33 20 469094
(%) 66 40 1149397
g/ml Peak Area
100 60 1657592
33 10 135005 133 80 2150412
166 100 2748444
66 20 277120

100 30 405128 3000000

133 40 534643
Metformin
2000000
166 50 672357

Area(AU)
1000000 y = 27563x - 15679
R² = 0.998
1000000 0
Benfotiamine
Area(AU)

0 50 100 150
500000 -1000000
Concentration(ppm)
0
0 20 40 60
y = 13396x + 2467.9 Figure 4: calibration graph for Metformin
Concentration(ppm)
R² = 0.9998

Figure 3: calibration graph for Benfotiamine

REPEATABILITY
Table 11: Results of repeatability for Benfotiamine:
Height USP Tailing
Retention USP Plate
S. No Peak name Area(µV*sec)
time Count
(µV) %Assay

1 Benfotiamine 2.157 70717 99%

400459 1.2 4987
2 Benfotiamine 2.159 402118 71819 99.4%
1.2 5019
3 Benfotiamine 2.186 405412 73930 100%
1.2 5126
4 Benfotiamine 2.160 406506 73333 100%
1.3 4999
5 Benfotiamine 2.170 407673 72623 100%
1.2 5214
Mean 404433.6

Std.dev 2716.809

%RSD 0.671757

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P. Aravinda Reddy. et al. J Pharm Res, 2016; 05 (12): 268-281

Table 12: Results of repeatability for Metformin:

Height USP Tailing

Retention USP Plate
S. No Peak name Area(µV*sec)
time Count
(µV) %Assay

1 Metformin 3.603 226985 98.7%

1617864 1.1 7045
2 Metformin 3.608 1618493 234764 98.8%
1.1 7399
3 Metformin 3.600 1628262 227712 99.4%
1.2 7159
4 Metformin 3.696 1615796 235459 98.6%
1.1 7896
5 Metformin 3.629 1619626 242158 98.8%
1.1 7965
Mean 1620008

Std.dev 4310.623

%RSD 0.266086

Intermediate precision:

Day 1:

Table 13: Results of Intermediate precision for Benfotiamine

Area Height %Assay

S.No Peak Name RT (µV*sec) (µV) USP Tailing
USP Plate count

1 Benfotiamine 100%
2.198 405262 70572 5672 1.2

2 Benfotiamine 100%
2.196 405637 70516 5639 1.2

3 Benfotiamine 100%
2.160 405628 70572 6183 1.2
Benfotiamine 2.160 100%
4 405647 70372 5923 1.2
Benfotiamine 2.160 100%
5 405948 70592 6739 1.2
Benfotiamine 100%
6 2.186 408732 70526 5837 1.2

Mean 406142.3

Std. Dev. 1287.197

% RSD 0.316933

Table 14: Results of Intermediate precision for Metformin

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P. Aravinda Reddy. et al. J Pharm Res, 2016; 05 (12): 268-281

Area %Assay
Height (µV) USP Plate Resolution
S.No Peak Name Rt (µV*sec) USP Tailing
count

1 Metformin 98%
3.623 1608292 235473 5372 1.1 10.1

2 Metformin 98.2%
3.611 1609283 235938 5927 1.1 10.1

3 Metformin 10.1 98.7%

3.696 1617836 235738 6129 1.1
Metformin 3.696 10.1 99.7%
4 1619743 235963 5284 1.1
Metformin 3.696 10.1 98.5%
5 1614262 231938 5284 1.1
Metformin 10.1 98.2%
6 3.642 1608471 235948 6347 1.1

Mean 1611315

Std. Dev. 6077.093

% RSD 0.377151

Day 2:

Table 15: Results of Intermediate precision Day 2 for Benfotiamine

Area Height %Assay

S.No Peak Name RT (µV*sec) (µV) USP Plate count USP Tailing

1 Benfotiamine 100%
2.198 405423 70572 5672 1.2

2 Benfotiamine 100%
2.196 405927 70516 5639 1.2

3 Benfotiamine 100%
2.178 405029 70572 6183 1.2
Benfotiamine 2.142 100%
4 405432 70372 5923 1.2
Benfotiamine 2.177 100%
5 405062 70592 6739 1.2
Benfotiamine 101%
6 2.177 408417 70526 5837 1.2

Mean 405881.7

Std. Dev. 1283.857

% RSD 0.316313

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P. Aravinda Reddy. et al. J Pharm Res, 2016; 05 (12): 268-281

Table 16: Results of Intermediate precision Day 2 for Metformin

Area Height %Assay

S.No Peak Name RT (µV*sec) (µV) USP Plate count USP Tailing Resolution

1 Metformin 100%
3.611 1638732 244384 5363 1.1 10.1

2 Metformin 100%
3.623 1637438 235827 6282 1.1 10.1

3 Metformin 10.1 100%

3.684 1638474 236382 5938 1.1
Metformin 3.697 10.1 99.7%
4 1634273 239183 6194 1.1
Metformin 3.684 10.1 99.8%
5 1636372 231931 5402 1.1
Metformin 10.1 100%
6 3.684 1639283 234356 5837 1.1

Mean 1637429

Std. Dev. 1860.366

% RSD 0.113615

ACCURACY:

Table 17: The accuracy results for Benfotiamine

%Concentration Amount
Amount Found
Added
Area % Recovery Mean Recovery
(at specification
(ppm)
Level) (ppm)

50% 201472.3 15 14.8 98.6

100% 406193 30 30.1 100.3 99.7%

150% 607144 45 45.1 100.2

Table 18: The accuracy results for Metformin

%Concentration Amount
Amount Found
Added
Area % Recovery Mean Recovery
(at specification
(ppm)
Level) (ppm)

50% 826527.7 30 14.8 101.6

100% 1622241 60 30.1 99 99.6%

150% 2422702 90 45.1 98.2

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P. Aravinda Reddy. et al. J Pharm Res, 2016; 05 (12): 268-281

The results obtained for recovery at 50%, 100%, 150% are QUANTITATION LIMIT
within the limits. Hence method is accurate.
The quantitation limit of an individual analytical procedure
LIMIT OF DETECTION is the lowest amount of analyte in a sample which can be
quantitatively determined.
The detection limit of an individual analytical procedure is
the lowest amount of analyte in a sample which can be detected LOQ=10×σ/S
but not necessarily quantitated as an exact value.
Where
LOD= 3.3 × σ / s
σ = Standard deviation of the response
Where
S = Slope of the calibration curve
σ = Standard deviation of the response
BENFOTIAMINE
S = Slope of the calibration curve
Result: =10×4269.822/13396
BENFOTIAMINE
=3.1µg/ml
Result: = 3.3×4269.822/13396
METFORMIN
= 1.05µg/ml
Result: =10×57796.93/27563
METFORMIN
=20.9µg/ml
Result: =3.3×57796.93/27563

= 6.9µg/ml

Robustness
Table 19: Results for Robustness Benfotiamine

Parameter used for sample Theoretical

Peak Area Retention Time Tailing factor
analysis plates

Actual Flow rate of 1.0 mL/min 406433 2.121 4009 1.2

Less Flow rate of 0.9 mL/min 398841 2.210 3800.8 0.9

More Flow rate of 1.1 mL/min 389947 2.184 4800.8

Less organic phase 413898 2.200 4890.8 0.9

More Organic phase 389578 2.172 4190.8 0.7

Table 19: Results for Robustness Metformin

Theoretical
Parameter used for sample analysis Peak Area Retention Time Tailing factor
plates

Actual Flow rate of 1.0 mL/min 1592811 3.643 7849 1.1

Less Flow rate of 0.9 mL/min 1613422 4.498 3312.2 0.9

More Flow rate of 1.1 mL/min 1619138 3.505 4312.2 0.8

Less organic phase 1616104 4.504 4392.2 0.9

More organic phase 1623185 3.512 4292.2 0.9

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P. Aravinda Reddy. et al. J Pharm Res, 2016; 05 (12): 268-281

SUMMARY

Table 20: Summary of validation data for Benfotiamine:

S.No Parameter Observation Acceptance criteria

System suitability
Theoretical plates 4009 Not less than 2000
1
Tailing 1.2 Not more than 2
%RSD 0.9 Not more than 2.0%
Specificity
2
%Assay 99% 98-102%
3 Method Precision (%RSD) 0.7 Not more than 2.0%
Linearity 10-50 µg/ml
4 Slope 13396
Correlation coefficient(r2) 0.99 ≤0.99
Accuracy
5
Mean % recovery 99.7 98 - 102%
Robustness All the system
a) Flow rate variation suitability
6
b) Organic phase parameters are
variation within the limits.

Table 21: Summary of validation data for Metformin:

S.No Parameter Observation Acceptance criteria

System suitability
Theoretical plates 7849 Not less than 2000
1
Tailing 1.1 Not more than 2
%RSD 0.1 Not more than 2.0%

Specificity
2
%Assay 99% 98-102%

3 Method Precision (%RSD) 0.7 Not more than 2.0%

Linearity 20-100 µg/ml

4 Slope 27563
Correlation coefficient(r2) 0.99 ≤0.99

Accuracy
5
Mean % recovery 99.6 98 - 102%

All the system

Robustness
suitability
6 a) Flow rate variation
parameters are
b) Organic phase variation
within the limits.

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P. Aravinda Reddy. et al. J Pharm Res, 2016; 05 (12): 268-281

CONCLUSION 9. Detectors used in HPLC (online

)URL:http://wiki.answers.com/Q/What_detectors_are_used_in_
In the present investigation, a simple, sensitive, precise and HPLC
accurate RP-HPLC method was developed for the quantitative
estimation of Benfotiamine and Metformin in bulk drug and 10. Detectors (online)
pharmaceutical dosage forms. ,URL:http://hplc.chem.shu.edu/NEW/HPLC_Book/Detectors/det
_uvda.html
This method was simple, since diluted samples are directly used
without any preliminary chemical derivatisation or purification 11. Detectors (online)
steps. ,URL:http://www.dionex.com/enus/webdocs/64842-31644-
02_PDA-100.pdf
Benfotiamine and Metformin are freely soluble in ethanol,
methanol and sparingly soluble in water. 12. Detectors
(online),URL:http://www.ncbi.nlm.nih.gov/pubmed/8867705
Methanol: Triethylamine Buffer was chosen as the mobile phase.
The solvent system used in this method was economical. 13. Detectors
(online),URL:http://www.chem.agilent.com/Library/application
The %RSD values were within 2 and the method was found to be s/59643559.pdf
precise.
14. Detectors
The results expressed in Tables for RP-HPLC method was (online),URL:http://hplc.chem.shu.edu/new/hplcbook/detector
promising. The RP-HPLC method is more sensitive, accurate and
precise compared to the Spectrophotometric methods. 15. Draft ICH Guidelines on Validation of Analytical Procedures
Definitions and terminology. Federal Register, vol 60. IFPMA,
This method can be used for the routine determination of Switzerland, (1995), PP 1126.
Benfotiamine and Metformin in bulk drug and in Pharmaceutical
dosage forms. 16. Code Q2B, Validation of Analytical Procedures; Methodology.
ICH Harmonized Tripartite Guidelines, Geneva, Switzerland,
ACKNOWLEDGEMENT (1996), PP 1- 8.

Thе Authors arе thankful to the Management and Principal, 17. Introduction to analytical method validation (online),
Department of Pharmacy, Vaageswari College of Pharmacy, available from: URL:
Karimnagar, for extending support to carry out the research http://www.standardbase.hu/tech/HPLC%20validation%20PE.
work. Finally, the authors express their gratitude to the Sura pdf.
Labs, Dilsukhnagar, Hyderabad, for providing research
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P. Aravinda Reddy. et al. J Pharm Res, 2016; 05 (12): 268-281

How to cite this article:

P. Aravinda Reddy, et al., DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF
BENFOTIAMINE AND METFORMIN IN COMBINED DOSAGE FORM. J Pharm Res, 2016; 05 (12): 268-281. DOI:

Conflict of interest: The authors have declared that no conflict of interest exists.
Source of support: Nil

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