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* Corresponding author: M.Venkatesh
E-mail address: venkateshpharma@yahoo.com
IJPAR |Volume 2 | Issue 2 | Apr - Jun- 2013 ISSN: 2320-2831
Available Online at: www.ijpar.com
[Research article]
Analytical method Development and Validation for the estimation of
Pioglitazone hydrochloride in Bulk and Tablet dosage form
by UV_Spectroscopy
*M.Venkatesh, B.Narender, D.Anusha, G.Srikanthbabu, J.Mounika, S.Laxman.
Moonray institute of Pharmaceutical sciences, Raikal(V), Mahabubnagar (D),
Andhrapradesh, India – 509 216
ABSTRACT
This paper describes the analytical method suitable for validation of Pioglitazone hydrochloride by UV
Spectrophotometric method. The method utilized UV spectroscopy and the solvent system was consists of 6 N
Glacial acetic acid at wave length 270 nm. Validation experiments were performed to demonstrate Specificity,
Precision, Linearity, Accuracy, ruggedness. The method was linear over the concentration range of 10-50 µg/ml.
The Proposed method was simple, sensitive & reliable with good Precise, Accurate, and Reproducible and rapid
for the determination of Pioglitazone. The commercial formulations are estimated without interference. Hence
this method can be used for routine determination of Pioglitazone hydrochloride in bulk and their
pharmaceutical dosage forms.
Key words: UV-Spectrophotometry, Pioglitazone hydrochloride, Pharmaceutical dosage form.
INTRODUCTION
Pioglitazone hydrochloride is an oral anti-
hyperglycemic agent which is used in the treatment
of type 2diabetes mellitus. This type of diabetes
mellitus is also known as non-insulin-dependent
diabetes mellitus (NIDDM). This syndrome is
characterized by hyper glycemia resulting from
abnormalities in insulin secretion, action or both
Systematic (IUPAC) Name: (±)-5-{[4-[2-(5-ethyl-
2-pyridinyl) ethoxy] phenyl] methyl}-2,4-
thiazolidinedione mono hydrochloride(1,2)
. The aim
of the present study was to develop and validate a
simple, UV Spectroscopic method for the
determination of Pioglitazone hydrochloride in
bulk and tablets. The developed method was
validated using ICH guidelines for validation
(ICH,1995) (2)
Materials and Methods
Instrument
Absorption spectral measurements were carried out
with a UV – Visible spectrophotometer (Analytical
technologies model spectro 2060 plus version 5)
was employed with spectral band width of 5 nm
and wavelength accuracy of 0.3nm (with automatic
wavelength correction with a pair of 5 cm matched
quartz cells).
Chemicals
Pioglitazone hydrochloride pure drug was supplied
by Hetero Labs, India as gift sample and used as
such. Spectroscopy graded water and analytical
reagent grade glacial acetic acid were used.](https://image.slidesharecdn.com/03-191213062502/85/Analytical-method-Development-and-Validation-for-the-estimation-of-Pioglitazone-hydrochloride-in-Bulk-and-Tablet-dosage-form-by-UV_Spectroscopy-1-320.jpg)
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M.Venkatesh et al / Int. J. of Pharmacy and Analytical Research Vol-2(2) 2013 [55-58]
www.ijpar.com
Preparation of standard stock solution
Standard solution of pioglitazone hydrochloride
was prepared by dissolving 10 mg of Pioglitazone
hydrochloride in 10ml of mobile phase (6N GAA)
to get concentration 1000µg/ml. Different aliquots
of above solution in the range 0.1 to 0.5 ml were
transferred into series of 10ml volumetric flask and
volume made up to the mark with water to obtain
the concentrations 10 to 50µg/ml. scanning ranges
was finalized for study and solutions were scanned
on spectrophotometer in the UV range of 200-
400nm.
Preliminary solubility studies of drugs
A small quantity of standard drug was dissolved
in different solvents like distilled water, methanol,
ethanol, acetonitrile, isopropyl alcohol, and in
various buffer solutions. By the solubility studies
we determined that the drug was dissolved in 6N
glacial acetic acid, hence this solvent system was
used in the present study.
Determination of λ max
From the stock solutions, a working standard was
prepared. The absorption spectrum for pioglitazone
hydrochloride, the absorption spectrum was
recorded using 10µg/ml solution and the maximum
absorption was found to be 270nm. The Calibration
curves were prepared for Pioglitazone the
concentration range of 10-50 µg/ml at selected
wave lengths by diluting aliquot portions of stock
solution of each drug. The plots of Beer’s law limit
are shown in Fig.1.
Fig.1 Calibration curve of Pioglitazone hydrochloride
Preparation of Sample solution
Sample label claim 30 mg. The average weight was
determined with 20 tablets, which were grounded
in a mortar until fine powder. Accurately weighed
amount of powder equivalent to 10 mg of
Pioglitazone hydrochloride was quantitatively
transferred to a 10 ml calibrated volumetric flask
with the mobile phase (6N GAA). The volume was
made up to mark, shake for 15 min and filter the
sample solution using What man filter paper No-1.
From above solution 1ml was transferred to 10 ml
calibrated volumetric flask and made up to mark
with the aid of 6 N GAA to obtain the
concentration 100 µg /ml. From above solution 0.3
ml was transferred to 10 ml calibrated volumetric
flask and made up to mark with the aid of 6N GAA
to obtain the concentration 30 µg/ml. Then the
solution was scanned from 200-400 nm.
METHOD VALIDATION
The method was validated with reference to
linearity, accuracy, precision, and specificity,
ruggedness. (5)
Linearity
Linearity was performed by taking aliquots of
0.1, 0.2, 0.3, 0.4 and 0.5 ml from stock solution
(1mg/ml) in 10ml volumetric flasks and diluted up
to the mark with the (6N GAA ) such that the final
concentration of Pioglitazone in the range of 10
to50 µg/ml. Under the experimental conditions
described the graphs obtained by plotting
concentration (µg/ml) vs absorbance. The
observations and calibration curve is shown in
Table 1 and Fig.1](https://image.slidesharecdn.com/03-191213062502/85/Analytical-method-Development-and-Validation-for-the-estimation-of-Pioglitazone-hydrochloride-in-Bulk-and-Tablet-dosage-form-by-UV_Spectroscopy-2-320.jpg)
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M.Venkatesh et al / Int. J. of Pharmacy and Analytical Research Vol-2(2) 2013 [55-58]
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TABLE: 1 OPTICAL CHARACTERISTIC AND LINEARITY DATA
Parameters Observations
λmax for Pioglitazone hydrochloride
Beer’s law limits
Correlation coefficient
Regression equation (Y=mx+c)
Intercept(a)
Slope(b)
Molar absorptivity
Sandell sensitivity
270 nm
10-50 µg/ ml
0.999
Y= -0.009+0.025x
-0.0094
0.02528
9888.67L/Mole/Cm
0.039735 µg/ ml
Accuracy
The accuracy was assessed by determining the
%RSD values for 24 µg/ml, 30µg/ml, 36 µg/ml,
(n=10) i.e at 80%, 100%, 120% level the resulting
solutions were then reanalyzed by proposed
method. The results are shown in table 2.
TABLE: 2 ACCURACY STUDIES
Level of accuracy %RSD
80% 0.66
100% 0.59
120% 0.66
Precision
Precision of the methods was studied as intra-day,
inter day and repeatability. Intra-day, study was
performed by analyzing, the three different
concentration of the drug (80%, 100%, 120%) in
the same day. Inter-day precision was performed
by analyzing three different concentration of the
drug (80%, 100%, and 120%) for three days in a
week (4).
Repeatability was performed by analyzing
the three different concentration of drug (80%,
100%, and 120%) for 10 times at each
concentration and the results are shown in table
3&4.
TABLE: 3 RESULTS FROM INTERDAY PRECISION
INTERDAY PRECISION
DAY-1 DAY-2 DAY-3
24
µg/ml
30
µg/ml
36
µg/ml
24
µg/ml
30
µg/ml
36
µg/ml
24
µg/ml
30
µg/ml
36
µg/ml
%RSD 0.27 0.35 0.55 0.34 0.37 0.38 0.34 0.31 0.25
TABLE:4 RESULTS FROM INTRADAY PRECISION & REPEATABILITY
INTRA DAY PRECISION REPEATABILITY
10.30AM 3.30PM
Concentration(µg/ml)concentration(µg/ml) concentration(µg/ml)
24 30 36 24 30 36 24 30 36
% RSD 0.44 0.54 0.20 0.41 0.25 0.25 0.36 0.38 0.30
RUGGEDNESS OF TEST METHOD
ANALYST TO ANALYST
Analyst to Analyst variability study was conducted
with different analysts under similar conditions at
different concentration levels (24 µg/ml, 30 µg/ml,
36 µg/ml). Triplicate samples were prepared and
each was analyzed as per test method and the
results are shown in table 5.](https://image.slidesharecdn.com/03-191213062502/85/Analytical-method-Development-and-Validation-for-the-estimation-of-Pioglitazone-hydrochloride-in-Bulk-and-Tablet-dosage-form-by-UV_Spectroscopy-3-320.jpg)
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M.Venkatesh et al / Int. J. of Pharmacy and Analytical Research Vol-2(2) 2013 [55-58]
www.ijpar.com
TABLE 5: RESULTS FROM RUGGEDNESS
RUGGEDNESS
Analyst-1 Analyst-2
24 µg/ml 30 µg/ml 36 µg/ml 24 µg/ml 30µg/ml 36 µg/ml
%RSD 0.56 0.66 0.16 0.28 0.35 0.28
RESULTS AND DISCUSSION
Development and validation of spectro photometric
method for the estimation pioglitazone could be
used as a valuable analytical tool in routine
analysis, to check the batch to batch variations.
After the drug is approved, pharmaceutical
validation and development of finger printing are
necessary to ensure that the drug product will
meet/set pharmaceutical standards for identity,
strength, quality, purity, safety and efficacy.
The wavelength 270nm (λmax for Pioglitazone)
was selected for analysis of the drugs in 6N glacial
acetic acid and linearity was observed in the range
10-50 µg/ml (r =0.999) for the amount of drugs
estimated by the proposed methods was in good
agreement with the label claim. The proposed
methods were validated with reference to linearity,
accuracy, precision, and specificity. The accuracy
of the methods was assessed by recovery studies at
three different concentration levels. Molar
absorptivity (e), low values of Sandell sensitivity
indicated the high sensitivity of the proposed
method.
The method was found to be precise as indicated by
the repeatability, intra-day, inter-day analysis,
showing %RSD less than 2. The results did not
show any statistical difference between analysts
suggesting that the method which is developed is
rugged. The results of precision shown in table 3.
All statistical data proves validity of the methods
and can be used for routine analysis of
pharmaceutical formulation.
TABLE 4: ANALYSIS DATA OF TABLET FORMULATION
Drug Label claim (mg/tab) Assay(% of label claim) ± %RSD
Piosis 30 99.1±0.51
ACKNOWLEDGEMENT
The authors are grateful to the Management &
Principal of Moonray institute of pharmaceutical
sciences, Raikal(v), Mahabubnagar (Dist), Andhra
pradesh, India – 509216, for their continuous
support and encouragement and for providing the
necessary facilities.
REFERENCES
[1] Indian pharmacopoeia. Controller of Publications, Delhi, volume-III, 2010, pp.1916, 1917
[2] International conference on Harmonization,”Q2A: Text on validation of Analytical Procedures,”
Federal register 60(40), 11260-11262(1995).
[3] Dr.Chowdary, K.P.R.; Devalo Rao, G.; Himabindu, G.Validation of analytical methods, The Eastern
Pharmacist. 1999, 497, 39-41.
[4] Pragati Shakya et al (2006) has performed the determination of pioglitazone hydrochloride in bulk and
pharmaceutical formulations by UV Spectro photometric method. IJPSR, 2010; Vol. 1 (11): 153-157
ISSN: 0975-8232.
*******************************](https://image.slidesharecdn.com/03-191213062502/85/Analytical-method-Development-and-Validation-for-the-estimation-of-Pioglitazone-hydrochloride-in-Bulk-and-Tablet-dosage-form-by-UV_Spectroscopy-4-320.jpg)
Analytical method Development and Validation for the estimation of Pioglitazone hydrochloride in Bulk and Tablet dosage form by UV_Spectroscopy
- 1. 55 * Corresponding author: M.Venkatesh E-mail address: venkateshpharma@yahoo.com IJPAR |Volume 2 | Issue 2 | Apr - Jun- 2013 ISSN: 2320-2831 Available Online at: www.ijpar.com [Research article] Analytical method Development and Validation for the estimation of Pioglitazone hydrochloride in Bulk and Tablet dosage form by UV_Spectroscopy *M.Venkatesh, B.Narender, D.Anusha, G.Srikanthbabu, J.Mounika, S.Laxman. Moonray institute of Pharmaceutical sciences, Raikal(V), Mahabubnagar (D), Andhrapradesh, India – 509 216 ABSTRACT This paper describes the analytical method suitable for validation of Pioglitazone hydrochloride by UV Spectrophotometric method. The method utilized UV spectroscopy and the solvent system was consists of 6 N Glacial acetic acid at wave length 270 nm. Validation experiments were performed to demonstrate Specificity, Precision, Linearity, Accuracy, ruggedness. The method was linear over the concentration range of 10-50 µg/ml. The Proposed method was simple, sensitive & reliable with good Precise, Accurate, and Reproducible and rapid for the determination of Pioglitazone. The commercial formulations are estimated without interference. Hence this method can be used for routine determination of Pioglitazone hydrochloride in bulk and their pharmaceutical dosage forms. Key words: UV-Spectrophotometry, Pioglitazone hydrochloride, Pharmaceutical dosage form. INTRODUCTION Pioglitazone hydrochloride is an oral anti- hyperglycemic agent which is used in the treatment of type 2diabetes mellitus. This type of diabetes mellitus is also known as non-insulin-dependent diabetes mellitus (NIDDM). This syndrome is characterized by hyper glycemia resulting from abnormalities in insulin secretion, action or both Systematic (IUPAC) Name: (±)-5-{[4-[2-(5-ethyl- 2-pyridinyl) ethoxy] phenyl] methyl}-2,4- thiazolidinedione mono hydrochloride(1,2) . The aim of the present study was to develop and validate a simple, UV Spectroscopic method for the determination of Pioglitazone hydrochloride in bulk and tablets. The developed method was validated using ICH guidelines for validation (ICH,1995) (2) Materials and Methods Instrument Absorption spectral measurements were carried out with a UV – Visible spectrophotometer (Analytical technologies model spectro 2060 plus version 5) was employed with spectral band width of 5 nm and wavelength accuracy of 0.3nm (with automatic wavelength correction with a pair of 5 cm matched quartz cells). Chemicals Pioglitazone hydrochloride pure drug was supplied by Hetero Labs, India as gift sample and used as such. Spectroscopy graded water and analytical reagent grade glacial acetic acid were used.
- 2. 56 M.Venkatesh et al / Int. J. of Pharmacy and Analytical Research Vol-2(2) 2013 [55-58] www.ijpar.com Preparation of standard stock solution Standard solution of pioglitazone hydrochloride was prepared by dissolving 10 mg of Pioglitazone hydrochloride in 10ml of mobile phase (6N GAA) to get concentration 1000µg/ml. Different aliquots of above solution in the range 0.1 to 0.5 ml were transferred into series of 10ml volumetric flask and volume made up to the mark with water to obtain the concentrations 10 to 50µg/ml. scanning ranges was finalized for study and solutions were scanned on spectrophotometer in the UV range of 200- 400nm. Preliminary solubility studies of drugs A small quantity of standard drug was dissolved in different solvents like distilled water, methanol, ethanol, acetonitrile, isopropyl alcohol, and in various buffer solutions. By the solubility studies we determined that the drug was dissolved in 6N glacial acetic acid, hence this solvent system was used in the present study. Determination of λ max From the stock solutions, a working standard was prepared. The absorption spectrum for pioglitazone hydrochloride, the absorption spectrum was recorded using 10µg/ml solution and the maximum absorption was found to be 270nm. The Calibration curves were prepared for Pioglitazone the concentration range of 10-50 µg/ml at selected wave lengths by diluting aliquot portions of stock solution of each drug. The plots of Beer’s law limit are shown in Fig.1. Fig.1 Calibration curve of Pioglitazone hydrochloride Preparation of Sample solution Sample label claim 30 mg. The average weight was determined with 20 tablets, which were grounded in a mortar until fine powder. Accurately weighed amount of powder equivalent to 10 mg of Pioglitazone hydrochloride was quantitatively transferred to a 10 ml calibrated volumetric flask with the mobile phase (6N GAA). The volume was made up to mark, shake for 15 min and filter the sample solution using What man filter paper No-1. From above solution 1ml was transferred to 10 ml calibrated volumetric flask and made up to mark with the aid of 6 N GAA to obtain the concentration 100 µg /ml. From above solution 0.3 ml was transferred to 10 ml calibrated volumetric flask and made up to mark with the aid of 6N GAA to obtain the concentration 30 µg/ml. Then the solution was scanned from 200-400 nm. METHOD VALIDATION The method was validated with reference to linearity, accuracy, precision, and specificity, ruggedness. (5) Linearity Linearity was performed by taking aliquots of 0.1, 0.2, 0.3, 0.4 and 0.5 ml from stock solution (1mg/ml) in 10ml volumetric flasks and diluted up to the mark with the (6N GAA ) such that the final concentration of Pioglitazone in the range of 10 to50 µg/ml. Under the experimental conditions described the graphs obtained by plotting concentration (µg/ml) vs absorbance. The observations and calibration curve is shown in Table 1 and Fig.1
- 3. 57 M.Venkatesh et al / Int. J. of Pharmacy and Analytical Research Vol-2(2) 2013 [55-58] www.ijpar.com TABLE: 1 OPTICAL CHARACTERISTIC AND LINEARITY DATA Parameters Observations λmax for Pioglitazone hydrochloride Beer’s law limits Correlation coefficient Regression equation (Y=mx+c) Intercept(a) Slope(b) Molar absorptivity Sandell sensitivity 270 nm 10-50 µg/ ml 0.999 Y= -0.009+0.025x -0.0094 0.02528 9888.67L/Mole/Cm 0.039735 µg/ ml Accuracy The accuracy was assessed by determining the %RSD values for 24 µg/ml, 30µg/ml, 36 µg/ml, (n=10) i.e at 80%, 100%, 120% level the resulting solutions were then reanalyzed by proposed method. The results are shown in table 2. TABLE: 2 ACCURACY STUDIES Level of accuracy %RSD 80% 0.66 100% 0.59 120% 0.66 Precision Precision of the methods was studied as intra-day, inter day and repeatability. Intra-day, study was performed by analyzing, the three different concentration of the drug (80%, 100%, 120%) in the same day. Inter-day precision was performed by analyzing three different concentration of the drug (80%, 100%, and 120%) for three days in a week (4). Repeatability was performed by analyzing the three different concentration of drug (80%, 100%, and 120%) for 10 times at each concentration and the results are shown in table 3&4. TABLE: 3 RESULTS FROM INTERDAY PRECISION INTERDAY PRECISION DAY-1 DAY-2 DAY-3 24 µg/ml 30 µg/ml 36 µg/ml 24 µg/ml 30 µg/ml 36 µg/ml 24 µg/ml 30 µg/ml 36 µg/ml %RSD 0.27 0.35 0.55 0.34 0.37 0.38 0.34 0.31 0.25 TABLE:4 RESULTS FROM INTRADAY PRECISION & REPEATABILITY INTRA DAY PRECISION REPEATABILITY 10.30AM 3.30PM Concentration(µg/ml)concentration(µg/ml) concentration(µg/ml) 24 30 36 24 30 36 24 30 36 % RSD 0.44 0.54 0.20 0.41 0.25 0.25 0.36 0.38 0.30 RUGGEDNESS OF TEST METHOD ANALYST TO ANALYST Analyst to Analyst variability study was conducted with different analysts under similar conditions at different concentration levels (24 µg/ml, 30 µg/ml, 36 µg/ml). Triplicate samples were prepared and each was analyzed as per test method and the results are shown in table 5.
- 4. 58 M.Venkatesh et al / Int. J. of Pharmacy and Analytical Research Vol-2(2) 2013 [55-58] www.ijpar.com TABLE 5: RESULTS FROM RUGGEDNESS RUGGEDNESS Analyst-1 Analyst-2 24 µg/ml 30 µg/ml 36 µg/ml 24 µg/ml 30µg/ml 36 µg/ml %RSD 0.56 0.66 0.16 0.28 0.35 0.28 RESULTS AND DISCUSSION Development and validation of spectro photometric method for the estimation pioglitazone could be used as a valuable analytical tool in routine analysis, to check the batch to batch variations. After the drug is approved, pharmaceutical validation and development of finger printing are necessary to ensure that the drug product will meet/set pharmaceutical standards for identity, strength, quality, purity, safety and efficacy. The wavelength 270nm (λmax for Pioglitazone) was selected for analysis of the drugs in 6N glacial acetic acid and linearity was observed in the range 10-50 µg/ml (r =0.999) for the amount of drugs estimated by the proposed methods was in good agreement with the label claim. The proposed methods were validated with reference to linearity, accuracy, precision, and specificity. The accuracy of the methods was assessed by recovery studies at three different concentration levels. Molar absorptivity (e), low values of Sandell sensitivity indicated the high sensitivity of the proposed method. The method was found to be precise as indicated by the repeatability, intra-day, inter-day analysis, showing %RSD less than 2. The results did not show any statistical difference between analysts suggesting that the method which is developed is rugged. The results of precision shown in table 3. All statistical data proves validity of the methods and can be used for routine analysis of pharmaceutical formulation. TABLE 4: ANALYSIS DATA OF TABLET FORMULATION Drug Label claim (mg/tab) Assay(% of label claim) ± %RSD Piosis 30 99.1±0.51 ACKNOWLEDGEMENT The authors are grateful to the Management & Principal of Moonray institute of pharmaceutical sciences, Raikal(v), Mahabubnagar (Dist), Andhra pradesh, India – 509216, for their continuous support and encouragement and for providing the necessary facilities. REFERENCES [1] Indian pharmacopoeia. Controller of Publications, Delhi, volume-III, 2010, pp.1916, 1917 [2] International conference on Harmonization,”Q2A: Text on validation of Analytical Procedures,” Federal register 60(40), 11260-11262(1995). [3] Dr.Chowdary, K.P.R.; Devalo Rao, G.; Himabindu, G.Validation of analytical methods, The Eastern Pharmacist. 1999, 497, 39-41. [4] Pragati Shakya et al (2006) has performed the determination of pioglitazone hydrochloride in bulk and pharmaceutical formulations by UV Spectro photometric method. IJPSR, 2010; Vol. 1 (11): 153-157 ISSN: 0975-8232. *******************************