363 | P a g e International Standard Serial Number (ISSN .... RPA1415146015.pdf365 | P a g e...

363 | P a g e International Standard Serial Number (ISSN): 2319-8141

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International Journal of Universal Pharmacy and Bio Sciences 3(6): November-December 2014

INTERNATIONAL JOURNAL OF UNIVERSAL

PHARMACY AND BIO SCIENCES IMPACT FACTOR 2.093***

ICV 5.13*** Pharmaceutical Sciences RESEARCH ARTICLE……!!!

SIMULTANEOUS ESTIMATION OF ALOGLIPTIN AND PIOGLIAZONE

IN BULK AND COMBINED TABLET DOSAGE

FORM BY UV-SPECTROPHOTOMETRIC METHODS

Anusha M*, Manzoor Ahmed, Satishkumar Shetty A, Vijaya Krishna.C. Aradhya.

Department of Pharmaceutical Analysis, National College of Pharmacy,

Shivamogga 577 201, Karnataka, India.

KEYWORDS:

Alogliptin, Pioglitazone,

second order derivative

method, Area under

curve method.

For Correspondence:

Anusha M*

Address: Department of

Pharmaceutical Analysis,

National College of

Pharmacy, Shivamogga

577 201, Karnataka,

India.

Email:

anusha.udupa.92@gmail.

com

ABSTRACT

Two simple, accurate, rapid and precise UV Spectrophotometric

methods have been developed for simultaneous estimation of

Alogliptin (ALO) and Pioglitazone (PIO) in bulk and combined

tablet dosage form. The methods employed were (A) Second order

derivative and (B) Area under curve method. Method-A involves

measurement of second order derivative spectrum absorbance at

276nm (λmax of ALO) and 226nm (λmax of PIO). Method-B involves

measurement of peak area in the wavelength range 271-281 nm and

221-231 nm for Alogliptin and Pioglitazone respectively. In both the

methods linearity was found in the concentration range of 2.5-15

µg/ml and 3-18µg/ml respectively. Both the methods were found to

be rapid, specific, precise and accurate. Hence these methods can be

applied for routine analysis of Alogliptin and Pioglitazone in

combined dosage form without any interference by the excipients.

The above methods are validated according to ICH guidelines.

mailto:[email protected]

mailto:[email protected]



1.INTRODUCTION:

Chemically, Alogliptin is prepared as a benzoate salt, which is identified as 2-({6-[(3R)-3-

aminopiperidin-1-yl]-3-methyl-2, 4-dioxo-3, 4-dihydropyrimidin- 1(2H)-yl} methyl)

benzonitrilemonobenzoate (figure 1). Alogliptin inhibits dipeptidyl peptidase 4 (DPP-4), which

normally degrades the incretins glucose-dependent insulinotropic polypeptide (GIP) and

glucagon like peptide 1 (GLP-1). The inhibition of DPP-4 increases the amount of active plasma

incretins which helps with glycemic control1.

NN

N

CNO

O

NH2

CH3

HOOC.

NCH3

ONH

S

O

O

. HCl

Fig. 1 Chemical structure of Alogliptin Fig. 2 Chemical structure of Pioglitazone

Pioglitazone is an oral anti-hyperglycemic agent that acts primarily by decreasing insulin

resistance. Chemically, pioglitazone is prepared as hydrochloride salt, which is identified as (±)-

5-[[4-[2-(5-ethyl-2-pyridinyl)ethoxy] phenyl] methyl]-2, 4-thiazolidinedionemonohydrochloride.

Pioglitazone is a drug belongs to the drug class of thiazolidinedione, which is used to decrease

insulin resistance. It is an anti-diabetic agent to manage NIDDM (non- anti-hyperglycemic

insulin-dependent diabetes mellitus, sugar diabetes) called type 2 diabetes. Pioglitazone acts as

an agonist at peroxisome proliferator activated receptors (PPAR) in target tissues for insulin

action such as adipose tissue, skeletal muscle, and liver 2,3

.

Survey of literature revealed that few analytical methods has been developed for the

determination of ALO and PIO individually 4, 5

and in combination with other drugs 6-11

. Hence

an attempt has been made to develop a simple, accurate, rapid and precise UV

spectrophotometric methods for simultaneous estimation of ALO and PIO in combined dosage

form with validation as per recommendation of ICH guidelines.

2. EXPERIMENTAL

2.1 Instrument and materials:-

For both the methods Shimadzu 1800 UV-Visible Spectrophotometer was used with 1 cm

match quartz cell of 10 mm optical path length spectral band width of 1 ± 0.2 nm and

wavelength accuracy of ± 0.3 nm . The tablet formulation of ALO and PIO (Label claim:



Alogliptin 25 mg and Pioglitazone 30 mg), Oseni tablets was purchased from local market. The

pure API samples of Alogliptin and Pioglitazone were obtained as free gift samples Swapnroop

drug & pharmaceuticals (Aurangabad, Maharashtra) and Ontop pharmaceuticals pvt ltd

(Bangalore) respectively. AR grade methanol was used as solvent throughout the

experiment.

2.2 Preparation of standard stock solution:-

100 mg each of Alogliptin and Pioglitazone were weighed separately and transferred in two

different 100 ml volumetric flasks. Both the drugs were dissolved in 50 ml of methanol by

ultrasonication and then volume was made up to the mark with methanol to obtain the

concentration of 1000 µg/ml of each component (stock A and A' solution). From the above stock

A and A' solution 10 ml of aliquot was pipetted out into a 100 ml volumetric flask and the

volume was made up to the mark with methanol to obtain the final concentration of 100 µg/ml of

each component (stock B and B' solution).

2.3 Preparation of sample stock solution using formulation:-

Twenty tablets of Alogliptin and Pioglitazone (Oseni) in combination were weighed and their

average weight was determined. The tablets were crushed to fine powder and from the triturate,

tablet powder equivalent to 25 mg of Alogliptin was weighed which also contains 30 mg of

Pioglitazone and transferred to 100 ml volumetric flask and dissolved in 50 ml methanol and the

content was kept in ultrasonicator for 15 min. The solution was filtered through Whatmann filter

paper No.41, finally the volume was made up to the mark with methanol, which gave a

concentration of 250µg/ml of Alogliptin and 300µg/ml of Pioglitazone and this solution was

used as stock ‘A’ solution.

From the above stock ‘A’ solution, 5 ml of the aliquot was pipetted out and was transferred to a

50 ml volumetric flask. The volume was made upto 50 ml with methanol to obtain final

concentration of 25 µg/ml Alogliptin and 30 µg/ml Pioglitazone (stock B).

2.4 Methods:

a) Second order derivative method:-

The standard solutions of both the drugs were scanned in the spectrum mode from 400

to 200 nm. These spectrums were converted to second order derivative spectra by using

derivative mode in UV probe software 2.43.



The second order derivative spectra o f Alogliptin and Pioglitazone shows maximum

absorbance at 276 n m ( λ1) a nd 226 n m ( λ2) respectively and the same wavelengths have

been selected for the second order derivative spectroscopy.

b) Area under curve method:-

It involves the calculation of integrated value of absorbance with respect to the wavelength

between two selected wavelengths λ1 and λ2. Area calculation processing item calculates

the area bound by the curve and the horizontal axis. The horizontal axis is selected by

entering the wavelength range over which the area has to be calculated.

This wavelength range is selected on the basis of repeated observations so as to get the

linearity between area under curve and concentration. For the selection of analytical

wavelength suitable dilutions of Alogliptin (2.5-15μg/ml) and Pioglitazone (3-18μg/ml) of

the standard stock solutions (10μg/ml) of both the drugs were prepared separately and

scanned in the range of 400-200 nm. Maximum absorbance was observed at 276 nm and

226 nm for Alogliptin and Pioglitazone respectively.

The wavelength ranges selected for the estimation of Alogliptin and Pioglitazone are 271-

281 nm (λ1 and λ2) and 221-231 nm (λ3 and λ4) respectively. Aliquots were prepared for

the sample solution in the concentration range of 2.5-15μg/ml and 3-18μg/ml for ALO and PIO

and their area under curve was measured at above selected wavelengths.

2.5 VALIDATION OF THE METHODS:-

All the methods were validated according to ICH guidelines by carrying out analysis of six

replicate samples of tablet. Recovery studies were carried out at three different levels i.e.,

80%, 100% and 120% by adding the pure drug to previously analyzed tablet powder

sample. From the amount of t h e drug found, percentage recovery was calculated.

3. RESULT AND DISCUSSION:-

The estimation of Alogliptin and Pioglitazone in bulk and tablet formulation was found to be

accurate and reproducible with a linearity range of 2.5-15µg/ml and 3-18µg/ml respectively

for both the methods and the correlation coefficient was found to be 0.9999 and 0.9999

for the methods A and B respectively. The optical characteristics such as linearity range,

molar absorptivity, percentage relative standard deviation of recovery studies and

precision in each method were calculated and the results were reported in Table 1 and

Table 2 for method A and B respectively.



Also the regression characteristics like slope (m), intercept (c) and correlation coefficient

(r2) were calculated and are presented in Table 1 and Table 2 for method A and B

respectively. The accuracy was found by recovery studies at three different levels i.e. 80%,

100% and 120%. The values of standard deviation were satisfactory and the recovery studies

were close to 100%. The % RSD value was less than 2, an indicative of the accuracy of the

methods. The results for formulation were reported in Table 3.

The spectra of Alogliptin, Pioglitazone and formulation are reported by Second order

derivative method (Fig. 3, 4 and 5), calibration curve was plotted (Fig. 6, 7, 8 and 9).

Fig: 3: Second order derivative spectra of Alogliptin at 276 nm.

Fig: 4: Second order derivative spectra of Pioglitazone at 226 nm.



Fig: 5: Second order derivative spectra of Formulation at 276 and 226 nm.

Fig: 6: Calibration curve for ALO at 276 nm by Second Order Derivative Method.



Fig: 7: Calibration curve for PIO at 226 nm by

Second Order Derivative Method.

Fig: 8: Calibration curve for Formulation at 276 nm by

Second Order Derivative Method.



Fig: 9: Calibration curve for Formulation at 226 nm by

Second Order Derivative Method

The absorption spectra of Algliptin, Pioglitazone and formulation by Area under Curve

method are reported (Fig. 10, 11 and 12) and calibration curve was plotted (Fig. 13, 14, 15

and 16)

Fig. 10: Spectra showing AUC for ALO in the wavelength

range of 271 (λ1) to 281 (λ2) nm and 221(λ3) nm to 231 (λ4) nm



Fig. 11: Spectra showing AUC for PIO in the wavelength

range of 271 (λ1) to 281 (λ2) nm and 221 (λ3) to 231 (λ4)

Fig: 12: Spectra showing AUC for Formulation in the wavelength range of

271 (λ1) to 281 (λ2) nm and 221 (λ3) to 231 (λ4) nm in the Tablet dosage form.



Fig: 13: Calibration curve of Alogliptin at 271 – 281 nm by

Area under Curve Method.

Fig: 14: Calibration curve of Pioglitazone at 221-231nm by




Fig: 15: Calibration curve of Formulation at 271- 281 nm by Area under Curve Method.

Fig: 16: Calibration curve of Formulation at 221 - 231 nm by




Table 1: Optical characteristics and other parameters for Method A

Parameters ALO PIO

Linear range (µg/ml) 2.5-15 3-18

λmax / wavelength range (nm) 276 226

Coefficient of correlation 0.9999 0.9999

Slope*(m) -0.009x 0.013 x

Intercept*(c) -0.003 -0.001

Accuracy

(%RSD)

80% 1.452 0.6504

100% 0.5852 0.8282

120% 0.7072 0.5320

Precision

(%RSD)

Intra-day 0.5741 0.48360

Inter-day 0.09504 0.04163

Limit of Detection (µg/ml) 0.1782 0.293

Limit of Quantification (µg/ml) 0.54 0.88

*y = mx + c; when x is the concentration in µg/ml and y is absorbance unit.

Table 2: Optical characteristics and other parameters for Method B

*y = mx + c; when x is the concentration in µg/ml and y is absorbance unit.

Parameters ALO PIO

Linear range (µg/ml) 2.5-15 3-18

λmax / wavelength range (nm) 271-281 221-231

Coefficient of correlation 0.9999 0.9999

Slope*(m) 0.016x 0.024x

Intercept*(c) 0.002 0.008

Accuracy

(%RSD)

80% 1.4254 0.6504

100% 0.5852 0.6150

120% 0.7072 0.5005

Precision

(%RSD)

Intra-day 0.5741 0.3322

Inter-day 0.5452 0.8374

Limit of Detection (µg/ml) 0.156 0.124

Limit of Quantification (µg/ml) 0.475 0.376



Table 3: Results of formulation

Method Brand

name

Label

claim of

ALO(mg)

Label

claim of

PIO(mg)

Amount

found for

ALO(mg)

Amount

found for

PIO(mg)

%Recovery

±SD** for

CIL

%Recovery

±SD** for

OLM

A Oseni 25 30 24.50 29.86 99.72±0.3123 99.64±0.8282

B 25 30 25.07 30.12 99.72±0.3123 99.69±

0.62749

** Average of six determinations.

4. CONCLUSION:-

The proposed two spectrophotometric methods were found to be simple, accurate and

precise and inexpensive and can be used for routine analysis of Alogliptin and Pioglitazone

in bulk and its formulation.

5. ACKNOWLEDGEMENT:-

The authors express their gratitude to the National Education Society, National College of

Pharmacy, Shimoga for providing all the facilities and Swapnroop drug & pharmaceuticals

(Aurangabad, Maharashtra) and Ontop pharmaceuticals pvt ltd, Bangalore for providing me the

gift samples of Alogliptin and Pioglitazone. The authors are also thankful to Vision Group of

Science and Technology, Government of Karnataka for their grants to our college.

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