Patil Pallavi M et al: Development & Validation of UV Derivative Spectrophotometric Methods

Journal of Pharmaceutical and Scientific Innovation

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Research Article

DEVELOPMENT AND VALIDATION OF UV DERIVATIVE SPECTROPHOTOMETRIC

METHODS FOR THE DETERMINATION OF GLIMEPIRIDE, METFORMINE HCL AND
PIOGLITAZONE HCL IN BULK AND MARKETED FORMULATION
Patil Pallavi M*, Rathod Sonali D, Chaudhari Praveen.D.
1
Department of Pharmaceutical Chemistry. Modern College Of Pharmacy, Pune University, Pune, Maharashtra, India,
*Email: [email protected]
Received on: 22/04/12 Revised on: 19/05/12 Accepted on: 16/06/12

ABSTRACT
UV Derivative Spectrophotometric methods for the simultaneous determination of Glimepiride (GLM), Metformin HCL (MFN) and Pioglitazone HCL (PLZ)
in tablets were developed in the present work. The various parameters, such as linearity, precision, accuracy, specificity, robustness, limit of detection and
limit of quantitation were studied according to (ICH) International Conference on Harmonization guidelines.The first derivative UV spectrophotometric
method was performed at 227nm, 233nm and 265.5nm for GLM, MET and PIO respectively in 0.1N NaOH solution and distilled water (50:50). The proposed
methods are highly sensitive, precise and accurate and therefore can be used for its intended purpose.
KEYWORDS: Anti-diabetic drugs; Glimepiride, Metformin HCL and Pioglitazone HCL, Validation.

INTRODUCTION resistance by enhancing insulin action on peripheral tissues

UV Spectrophotometry is applicable for colorless compounds whereas glimepiride is a sulfonylurea group oral anti-diabetic
which is having double or triple bonds in structure. drug with prolonged effect and more over it maintains a more
Absorption of sample increases with the increase in sample physiological regulation of insulin secretion than
concentration. By preparing different concentrations in beer’s glibenclamide during physical exercise, suggesting that there
law range and constructing the calibration curve sample can may be less risk of hypoglycaemia with glimepiride, and act
be estimated quantitatively. by increasing the secretion of insulin by the functioning β-
Diabetes is one of the costliest health problems in the world. cells of the pancreas11. Fig 1 shows the structure of [A]
Globally, diabetes is likely to be the fourth leading cause of Metformine, [B] Pioglitazone and [C] Glimepiride
death1. Approximately 90% of people with diabetes have type
2 diabetes. It usually begins as insulin resistance, a disorder
in which the cells do not use insulin properly. As the need for
insulin rises; the pancreas gradually loses its ability to
produce insulin. Type II diabetes is associated with older age,
obesity, family history of gestational diabetes, impaired
glucose metabolism, physical inactivity and race/ ethnicity2.
If the glycemic target level is not achieved with one oral
agent alone, combination oral and/or insulin therapy is
recommended3, 4. Combination oral therapy becomes an Fig 1: [A] Metformin, [B] Pioglitazone and [C] Glimepiride
obvious choice when glycemic control is not achieved with
conventional monotherapy5. The advantages of oral dose This combination can be achieved by taking each of the drugs
combinations as compared to their components which are separately or alternatively fixed formulations have been
taken alone are lower cost and better patient compliance6. developed. A combination tablet formulation is beneficial in
Combination therapy has been shown to achieve greater terms of its convenience and patient compliance. The review
blood glucose lowering than monotherapybecause different of literature reveals that there were analytical methods of all
classes have different and complimentary mechanisms of the three drugs individually in pharmaceutical dosage forms
action. Therefore, it is more logical to add another drug than and even in biological samples12-20 and a few methods
replace the existing drug. The rapid introduction of reported for combination of either of the two drugs. 20-22
combination therapy with two or three complementary oral MATERIAL AND METHODS
anti diabetics help in targeting the dual effect and also Apparatus
reduced adverse effects8. A double beam UV/Visible spectrophotometer, Shimadzu
Chemically, metformin is 1,1-dimethyl biguanide UV- 1700 Pharmaspec, was employed with a pair of 1 cm
hydrochloride, pioglitazone is (± )-5-[p- [2-(5-ethyl-2- quartz cells for all analytical work.
pyridyl)-ethoxy] benzyl]-2,4-thiazolidinedione whereas Reagents and chemicals
glimepiride is 1-(4-(2-(3-ethyl-4-methyl-2-oxo-2,5-dihydro- Glimepiride was obtained from Zim Lab. Nagpur, Metformin
1H-pyrrole-1-carboxamido)ethyl)phenylsulfonyl)-3-(4-meth and Pioglitazone were obtained from Gen Pharmaceuticals
ylcyclohexyl)urea10 (structures shown in figure 1a, 1b and Ltd. Pune, Maharashtra, India as gift sample and were used as
1c). Metformin improves hepatic and peripheral tissue working standards. Sodium hydroxide of analytical grade and
sensitivity to insulin without the problem of serious lactic double distilled water were used throughout the analysis.
acidosis, pioglitazone has been shown to effect abnormal
glucose and lipid metabolism associated with insulin

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 Patil Pallavi M et al: Development & Validation of UV Derivative Spectrophotometric Methods

Commercial formulation Preparation of sample solutions

A commercial pharmaceutical preparation, Gluconorm PG- Sample solution containing both the drugs was prepared by
2tablet (2 mg Glimepiride, 15 mg Pioglitazone and 500mg dissolving 10 mg of each drug in 100mL volumetric flask
Metformin) was procured from the local market. using 50 ml of 0.1N Sodium hydroxide to give stock
Preparation of standard solution solutions then both sonicate for 20 min and further with
Standard stock solution of GLM, MFN and PLZ was distilled water and made 100 μg/mL stock solution, working
prepared by dissolving 10 mg of each drug separately in standard solution of 10 μg/mL concentration was prepared by
100mL volumetric flask using 0.1N sodium hydroxide as appropriate dilution. Seven standard dilutions of
solvent up to 50 ml and volume make up with distilled water concentrations of 5, 10, 15,20,25,30,35,40,45 and 50μg/mL
and both sample sonicate upto 20 min. Stock solutions of 100 was prepared from working standard solution. The
μg/mL were obtained in this manner. From these stock absorbance of this sample solution was measured at 227nm,
solutions, working standard solutions of concentration were 233nm and 265.5nm and their concentrations were
prepared by appropriate dilutions. Working standard determined using proposed analyticalmethods.
solutions were scanned in the entire UV range to determine Simultaneous equations method and preparation of
the λmax. The λmax of GLM, MFN and PLZ were found to solutions
be 227 nm,233nm and 265.5 nm respectively. Method was based on simultaneous equation method of
Calibration curve Vierodt. The method is applicable in the case of sample
Standard dilutions of each drug were prepared separately containing two drugs, each of which absorbs at the λ max of
having concentrations of 2-20 μg/mL for GLM and MFN and the other (Beckett et al, 1997). Three equations were
concentration of 5-50μg/mL for PLZ.The absorbances of developed using absorptivity coefficient values as an X
these standard solutions were measured at 227nm,233nm and componant. The content in the mixture was determined by
265.5nm and calibration curve was plotted. The absorptivity using the following three component equations/ Cramer’s
coefficients of the three drugs were determined using rule:
calibration curve graph shown below; X COMPONANT =A1 (β2γ3 - β3γ2) -A2 (β1γ3- β3γ1) + A3
GLIMEPIRIDE (β1γ2- β2γ1)/α1 (β2γ3 - β3γ2) - α 2 (β1γ3- β3γ1) + α 3
ABS (β1γ2-β2γ1)
Similarly, y and z component can be estimated.
0.8 Triple combination equations were constructed based upon
the fact that the absorbance of the mixture of GLM, MFN and
0.6 y = 0.0452x - 0.0156
PLZ at 227nm, 233nm and 265.5nm is the sum of the
R² = 0.9955
0.4 ABS absorbances at respective wavelengths and the spectra shown
0.2 in fig no 1. From the absorbance value obtained of all the
three λ max, absorptivity were calculated and shown in table
0 1.
-0.2 0 5 10 15

PIOGLITAZONE HCL

Fig: 1Spectra of Mixture i.e; GLM, MFN and PLZ in 25:

50: 50 ratios
METFORMINE HCL Quantitative equations method
ABS Method was based on Quantitative equation method. Primary
stock solution was prepared by using 0.1N NaOH. From this
1.5 different dilutions were prepared to determine λmax and
y = 0.0798x - 0.025 beer’s law range. Calibration curve was prepared by using
1 different concentrations of standard solution. GLM, MFN and
R² = 0.999
ABS PLZ in dosage form were estimated by calibration curve25, 26.
0.5
Developed method was validated as per ICH27, 28 guidelines
0 with the help of several parameters like accuracy, precision,
0 5 10 15 LOD, LOQ, and stability.29, 30
-0.5

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 Patil Pallavi M et al: Development & Validation of UV Derivative Spectrophotometric Methods

Estimation in the marked formulation successfully applied to the determination of GLM, MFN and
Twenty tablets were weighed and crushed to a fine powder. PLZ in the commercially available tablets dosage form.
An accurately weighed powder sample equivalent to 10mg of The recovery studies were carried out at different
GLM, MFN and PLZ was transferred to a 10mL volumetric concentrations by spiking a known concentration of standard
flask, dissolved in 5mL 0.1N NaOH, shaken for 10 min and drug to the reanalyzed sample and contents were reanalyzed
the volume was made up to the mark with 0.1N NaOH. The by proposed methods. The results of marketed formulation
solution was then filtered through Whatman filter paper no. analysis and Recovery studies are depicted in Table2. The
41. The solution was further diluted to get different method was validated statistically for range, linearity,
concentrations in the range of 5-50μg/mL of both the drugs. precision, accuracy, repeatability, LOD, and LOQ Table 3-5.
The analysis procedure was repeated three times with the Accuracy was ascertained on the basis of Recovery studies.
formulation. The result of analysis of the formulation is Precision was calculated as inter and intraday Variation for
shown in Table 1. both the drugs table 6-8. The percentage recoveries for of
Method validation GLM, MFN and PLZ were found to be 99.77%±1.5409,
The method validation parameters like linearity, precision, 100.29%±1.7891 and 99.99±0.7662 for this method
accuracy, repeatability, limit of detection and limit of respectively. The relative standard deviation was found to be
quantitation were checked as per ICH guidelines. within the limit, indicating good accuracy, precision, and
Linearity and range repeatability of the proposed method.
The linearity for GLM, MFN and PLZ were determined at CONCLUSION
some concentration levels for GLM and MFN from 2-20 The proposed method based on the UV is suitable for
μ/mL and for PLZ ranging from 5-50μ/mL using working determination of GLM, MFN and PLZ in the commercial
standards. tablets. The methods are simple, reliable, fast and
Precision and Accuracy reproducible. The spectrophotometric method requires only
Journal of Applied Pharmaceutical Science 01 (01); 2011: wavelength scan and automatic calculation of the first
46-49 the precision of the method was evaluated by interday derivative value. Furthermore, the proposed methods are
and intraday variation studies. In intraday studies, working inexpensive and low polluting, because small volumes are
solutions of standard and sample were analyses thrice in a required for preparation of samples.
day and percentage relative standard deviation (% RSD) was ACKNOWLEDGEMENT
calculated. In the interday variation studies, working solution I sincerely thanks to Zim Laboratory, Nagpur, Maharashtra
of standard and sample were analysed on three consecutive and Gen Pharmaceuticals, Pune, Maharashtra for providing
days and percentage relative standard deviation (% RSD) was me the gift sample of GLM, MFN and PLZ and I thank my
calculated. The data is shown in table 1-6. lab technicians for their contribution.
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Table 1: The absorptivity values of GLM, MFN and PLZ in the proposed method
Absorptivity value 227nm 233nm 265.5 nm
Ax1 0.0639 - -
Ax2 - 0.0599 -
Ax3 - - 0.0233
Ay1 0.0526 - -
Ay2 - 0.0523 -
Ay3 - - 0.000484
Az1 0.03834 - -
Az2 - 0.0286 -
Az3 - - 0.01547
Whereas; Ax1, Ax2 and Ax3 = are the absorptivity value of GLM at the respective wavelength.
Ay1, Ay2 and Ay3 = are the absorptivity value of MFN at the respective wavelength.
Az1, Az2 and Az3 = are the absorptivity value of PLZ at the respective wavelength.
1mg / ml solution was used as primary stock solution. The working solution of 0.1 mg / ml prepared by transferring 5ml from respective stock solution to a 50
ml volumetric flask and completing to volume with the distilled water. The drug proporation for Q-method was 25µg/ml GLM, 50 µg/ml MFN and 50 µg/ml
PLZ ( means the proportion is 25:50:50)

Table 2: Determination of Accuracy by percentage recovery method for GLM, MFN and PLZ
Ingredients Tablet amount Amount added Level of Amount Percentage Average % recovery
(µg/ml) (µg/ml) addition recovered recovery
(µg/ml)
GLM 25(µg/ml) 2(µg/ml) 80% 4.5(µg/ml) 99.97% 99.77%±1.5409
25(µg/ml) 2.5(µg/ml) 100% 5 (µg/ml) 99.08%
25(µg/ml) 3(µg/ml) 120% 5.5(µg/ml) 100.25%
MFN 50(µg/ml) 2(µg/ml) 80% 4.5(µg/ml) 99.98% 100.29%±1.7891
50(µg/ml) 2.5(µg/ml) 100% 5(µg/ml) 99.99%
50(µg/ml) 3(µg/ml) 120% 5.5(µg/ml) 100.90%
PLZ 50(µg/ml) 2(µg/ml) 80% 4.5(µg/ml) 99.77% 99.99±0.7662
50(µg/ml) 2.5(µg/ml) 100% 5(µg/ml) 99.98%
50(µg/ml) 3(µg/ml) 120% 5.5(µg/ml) 100.24%

Table 3: Validation parameters for GLM

Sr. No Parameters Results
1 Absorption (nm) 227nm
2 Linearity range (µg/ml) 2-20 µg/ml
3 Standard regression equation y=0.045x- 0.015
4 Correlation coefficient (r2) r2=0.995
5 A (1%, 1cm) 61636
6 Accuracy (% recovery± SD) 99.77%±0.1518
7 Precision (% CV) 100.6 %, 101.3%
8 Specificity A 25 µg/ml solution of candidate drug in solvent (0.1 N NaOH and
distilled water mixture in the ratio of 50:50 respectively ) at UV
detection ʎ of 227 nm will show an absorbance value of 1.5409
9 LOD 0.02261
10 LOQ 0.07565

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 Patil Pallavi M et al: Development & Validation of UV Derivative Spectrophotometric Methods

Table 4: Validation parameters for MFN

Sr.No Parameters Results
1 Absorption (nm) 233nm
2 Linearity range (µg/ml) 2-20 µg/ml
3 Standard regression Y = 0.079
4 equation r2 = 0.999
5 Correlation coefficient (r2) 35781
6 A (1%, 1cm) 100.29%±0.2567
7 Accuracy (% recovery± 100.7 %,101.3%
8 SD) A 25 µg/ml solution of candidate drug in solvent (0.1 N NaOH and
Precision (% CV) distilled water mixture in the ratio of 50:50 respectively ) at UV
Specificity detection ʎ of 233 nm will show an absorbance value of 1.7891
LOD
LOQ
9 0.067
10 0.2055

Table 5: Validation parameters for PLZ

Sr.No Parameters Results
1 Absorption (nm) 265.5nm
2 Linearity range (µg/ml) 5-25 µg/ml
3 Standard regression y=0.016x
4 equation r2=0.995
5 Correlation coefficient (r2) 15324
6 A (1%, 1cm) 99.99±0.1315
7 Accuracy (% recovery± SD) 101.5 %,100.9%
8 Precision (% CV) A 50 µg/ml solution of candidate drug in solvent (0.1 N NaOH and
Specificity distilled water mixture in the ratio of 50:50 respectively ) at UV
detection ʎ of 265.5 nm will show an absorbance value of 0.7662
9 LOD 0.0077
10 LOQ 0.0235

Table 6: Precision data for the developed method Assays of GLM as % of labeled amount
Sample number Analyst –I Analyst –II
(Intra-day precision) (Inter-day precision)
1 101.0 101.2
2 100.6 101.6
3 99.9 101.9
4 100.3 101.5
5 100.1 101.4
6 100.8 101.1
Average S.D. 100.6 101.3
0.423 0.327

Table 7: Precision data for the developed method Assays of MFN as % of labeled amount
Sample number Analyst –I Analyst –II
(Intra- day precision) (Inter- day precision)
1 100.4 101.2
2 100.6 101.5
3 100.8 101.1
4 100.8 101.5
5 100.4 101.0
6 100.7 101.1
Average S.D. 100.7 101.3
0.444 0.343

Table 8: Precision data for the developed method Assays of PLZ as % of labelled amount
Sample number Analyst –I Analyst –II
(Intra- day precision) (Inter- day precision)
1 101.6 101.2
2 101.9 100.1
3 101.5 100.8
4 101.4 101.0
5 101.1 100.6
6 100.3 99.9
Average S.D. 101.5 100.9
0.324 0.493

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