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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Anees et al.

World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 2.786

Volume 4, Issue 04, 1329-1339.

Research Article

ISSN 2278 – 4357

SIMULTANEOUS ESTIMATION OF ALOGLIPTIN AND METFORMIN FROM ITS TABLET DOSAGE FORM BY AREA UNDER CURVE AND MULTICOMPONENT UV SPECTROPHOTOMETRIC METHOD *Ana Nikalje, Mirza Shahed Baig, Mohammed Imran Anees, Afaq Qureshi. Dept. of Pharmaceutical Chemistry, Y.B. Chavan College of Pharmacy. Dr. Rafiq Zakaria Campus, Rauza Bagh, Aurangabad-431001, Maharashtra, India. Article Received on 10 Feb 2015,

ABSTRACT

Revised on 04 March 2015, Accepted on 24 March 2015

estimation of Alogliptin (ALO) and Metformin (MET) in Tablet

Two simple, accurate and precise UV methods were developed for the

dosage form. Both the drugs are used as Antidiabetic Drug. Method A *Correspondence for

is Area under Curve Spectrophotometry and in this the wavelength

Author

range selected for Quantitation are 215 – 240nm for Alogliptin and 265

Mohammed Imran

- 293nm for Metformin. Method B is Multicomponent mode

Anees

wavelength selected for Quantitation method were MET and ALO

Dept. of Pharmaceutical

therefore for 284nm (λ max of MET) and 274 (λ max of ALO) for the

Chemistry, Y.B. Chavan College of Pharmacy. Dr.

analysis. In both the methods linearity for detector response was

Rafiq Zakaria Campus,

observed in the concentration range of 10-50 microgram/ml for MET

Rauza Bagh, Aurangabad-

and ALO respectively. The results of tablet analysis for Area under

431001, Maharashtra,

Curve was found to be 99.90 ± 0.151 for ALO and 99.62 ± 0.220 for

India.

MET and results obtained for Multicomponant was 99.73 ± 0.306 for

[email protected] [email protected]

ALO and 99.30 ±

0.224 for MET. The proposed methods were

successfully applied for the Simultaneous determination of both the drugs in bulk as well as commercial tablet preparation. The results of the analysis have been validated statistically and by recovery studies which were according to ICH guidelines. KEYWORDS: Alogliptin, Metformin, UV-Spectrophotometry, Area under Curve, Multicomponent mode method.

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INTRODUCTION Alogliptin is white crystalline powder and is chemically 2-({6-[(3R)-3-aminopiperidin-1-yl]3- methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl}methyl)benzonitrile. Alogliptin is an orally administered anti-diabetic drug in the DPP-4 inhibitor. Like other medications for the treatment of Type 2 diabetes, Alogliptin does not decrease the risk of heart attack and stroke. Like other members of the gliptin class, it causes little or no weight gain, exhibits relatively little risk of causing hypoglycemia, and exhibits relatively modest glucose-lowering activity. Alogliptin and other gliptins are commonly used in combination with Metformin in patients whose diabetes cannot adequately be controlled with Metformin alone. Alogliptin is a dipeptidyl peptidase-4 inhibitor (DPP-4i) that is designed to slow the inactivation of incretin hormones GLP-1 (glucagon-like peptide-1) and GIP (glucose-dependent insulinotropic peptide).

Fig 1: Alogliptin Metformin is white crystalline powder and is chemically N,N-Dimethylimidodicarbonimidic diamide drug that decreases glucose production in the liver, increases insulin sensitivity and enhances peripheral glucose uptake. It does not stimulate secretion of endogenous insulin.

Fig 2: Metformin Both Alogliptin and Metformin are official in IP and are marketed as combined tablet dosage formulation in the Ratio is 100:100 mg ALO: MET and were named as Kazano as brand name. Literature survey revealed that a number of methods have been reported for estimation of both the drugs individually and in combination of other drugs, but not a single method is

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reported for the estimation of both the drugs simultaneously. Present work describes two precise, accurate, and reproducible methods for simultaneous estimation of ALO and MET in tablet formulation. MATERIAL AND METHODS Instruments: UV-VISIBLE SPECTROPHOTOMETER (DOUBLE BEAM) Make: Jasco Model: UV V-630 Spectrophotometer Spectral Bandwidth: 2nm MATERIALS Standard gift sample of Alogliptin (ALO) and Metformin (MET) were provided by Wockhardt Pharmaceutical Ltd and Swapnroop Drugs and Pharmaceutical Ltd and Combined dose Tablet Kazano were purchased from local market for analysis. Solvent used: Methanol AR grade used as solvent. Stock solution: Stock solution of both the drugs 100mcg/ml is prepared by dissolving 10mg each drug in 100ml volumetric flask and the final volume is make up by Methanol AR grade.

Fig 3: Calibration Curve of ALO

Fig 4: Calibration Curve of MET www.wjpps.com

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Table 1: Calibration Curve Table of ALO Concentration (mcg/ml) 10 20 30 40 50 Regression equation R2

Absorbance 0.053 0.1038 0.1536 0.2022 0.2597 Y= 0.005x + 0.002 0.999

Table 2: Calibration Curve Table of MET Concentration (mcg/ml) 10 20 30 40 50 Regression equation R2

Absorbance 0.22 0.458 0.678 0.901 1.12 Y= 0.011x + 0.002 0.999

Method A – Area under Curve (AUC) Method A stock solution of ALO and MET (100µg/ml) was prepared, by accurately weighing 10 mg of each drug and dissolving in separate 100 ml volumetric flasks. They were dissolved first in 25 ml of methanol sonicated and then the volume was making up to the mark to get 100µg/ml. For each drug, appropriate liquid were pipette out from the standard stock solution into a series of 10mL volumetric flasks, to get a set of dilutions for each drug respectively is scanned in the spectrum mode from 400 nm to 200 nm. The absorption spectra thus obtained is selected for analysis, from the overlain spectra of both the drugs (fig.5), wavelength selected for Quantitation are 215 – 240nm for Alogliptin and 265 - 293nm for Metformin. The calibration curves for ALO and MET was plotted in the concentration range of 10-50 mcg/ml exhibiting the Beer’s and Lamberts range (table 1, 2). The concentration of individual drug present in the mixture was determined by using the simultaneous AUC equation calculations.

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FIGURE NO. 5 – Overlain Spectra of ALO & MET showing AUC (b)Limit of detection (LOD) and Limit of Quantitation (LOQ) The LOD and LOQ of MET and ALO by the proposed methods were determined using calibration standards. The value of LOD for MET was 0.36, and for ALO 3.96 and value of LOQ for MET was 1.11and for ALO was 1.57respectively for area under curve method respectively. (c) Specificity Comparison of area under curve method the spectrum of MET and ALO in bulk drug with drug formulation (KAZANO) solutions shows that the wavelengths of maximum and minimum absorbance do not change. According to the results obtained, the area under curve Spectrophotometric methods are able to access the MET and ALO in the presence of excipients and hence, methods can be considered specific. (d) Accuracy The accuracy was determined by standard addition method. Three different levels (80%, 100% and 120%) of standards were spiked to commercial capsule in triplicate. The mean of percentage recoveries and the %RSD was calculated. The mean % recoveries of MET were found to be 99.90%, for area under curve. ALO were found to be 99.62%, for area under curve method respectively.

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Table 3: Accuracy by recovery values of ALO and MET in pharmaceutical preparation Amount present (mg/cap)

Level of % Recovery

Amount of standard added (mg)

Total amount recovered (mg)

%Recovery

ALO

MET

ALO

MET

ALO

MET

ALO

MET

80

10

20

8

16

17.8

35.55

98.88

98.74

100

10

20

10

20

19.84

39.77

99.10

99.44

120

10

20

12

24

22.2

44.66

100.9

101.5

SD: Standard deviation, R.S.D: Relative standard derivation (n*=3) Table 4: Statistical Data for Recovery Studies Component

Mean

Standard Deviation

Coefficient of Variation

ALO

99.62 %

0.01

0.0439182

MET

99.90 %

0.04

0.0555012

(e)Precision The repeatability of the method was determined by assaying five standard solutions of MET and ALO at the concentration 20μg/ml, and 20μg/ml respectively. The reproducibility of proposed method was determined by performing tablet assay at different time intervals (2 hour interval) on same day (Intra-day precision) and on three different days (Inter-day precision). Results of Intra-day and Inter-day precision is expressed in %RSD. Table 5: Precision study by % RSD Method

Intraday precision ALO (10µg/ml) MET(10µg/ml) % % SD SD RSD RSD

Interday precision ALO (10µg/ml) MET (10µg/m) % % SD SD RSD RSD

Area Under 0.002 1.823 0.005 0.539 0.0021 1.926 Curve Method SD: Standard deviation, R.S.D: Relative standard derivation (n*=3)

0.0094

1.018

METHOD B – Multicomponant Method In this method, the stock solution of both the drugs 100mcg/ml is prepared by dissolving 10mg each drug in 100ml volumetric flask and the volume is makeup by Methanol (A.R. grade), by appropriate dilution of standard stock solutions of both the drugs to 10mcg/ml

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dilution respectively is scanned in the spectrum mode from 400 nm to 200 nm. The absorption spectra thus obtained is selected for analysis, from the overlain spectra of both the drugs (fig.6), wavelength selected for wavelength selected for Quantitation are 284nm for Metformin and 274nm for Alogliptin respectively which are the λmax of both the drugs. The calibration curves for TEN and LAM was plotted in the concentration range of 10-50 mcg/ml exhibiting the Beer’s and Lamberts range (table 1, 2). The concentration of individual drug present in the mixture was determined by using the simultaneous Multicomponant equation calculations. .

FIGURE NO 6. : Spectra showing Multicomponant mode Method validation for Multicomponent mode method spectroscopy of ALO and MET Data of Bulk Drug Analysis Table 6: Data of Bulk Drug Analysis Sr. No.

Amount present in mcg/ml

Amount found in mcg/ml

Amount found in %

ALO

MET

ALO

MET

ALO

MET

1.

10

20

9.91

19.72

99.10

98.60

2.

10

20

9.97

19.87

99.70

99.35

3.

10

20

9.99

19.82

99.90

99.10

4.

10

20

9.89

19.78

98.90

99. 90

5.

10

20

9.93

19.78

99.30

99.95

n=5

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Table 7: Statistical Data for Recovery Studies Mean

Standard Deviation


ALO

99.38%

0.038714

0.0038601

MET

99.39 %

0.0166325

0.0016521

Component

a) Specificity Comparison of Multicomponent mode method the spectrum of ALO and MET in bulk drug with drug formulation (KAZANO) solutions shows that the wavelengths of maximum and minimum absorbance do not change. According to the results obtained, the Multicomponent mode Spectrophotometric methods are able to access the ALO and MET in the presence of excipients and hence, methods can be considered specific. (b) Accuracy The accuracy was determined by standard addition method. Three different levels (80%, 100% and 120%) of standards were spiked to commercial tablets in triplicate. The mean of percentage recoveries and the %RSD was calculated. The mean % recoveries of MET were found to be 99.66%, for area under curve ALO were found to be 99.54%, for area under curve method respectively. Table 8: Accuracy by recovery values of MET and ALO in Pharmaceutical Preparation Amount present (mg/tab)

Level of % Recovery

Amount of standard added (mg)

Total amount recovered (mg)

%Recovery

ALO

MET

ALO

MET

ALO

MET

ALO

MET

80

10

20

8

16

17.85

35.90

99.16

99.74

100

10

20

10

20

19.91

39.85

99.55

99.65

120

10

20

12

24

23.95

43.78

99.79

99.52

X: %Mean, SD: Standard deviation, R.S.D: Relative standard derivation (n*=3) Table 9: Statistical Data for Recovery Studies Mean

Standard Deviation


ALO

99.50%

0.06657

0.06646

MET

99.63 %

0.01285

0.01244

Component

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Table 10: Validation Parameters of Alogliptin and Metformin PARAMETERS Linearity range µg/ml Slope Regression coefficient Limit of detection (µg/ml) Limit of quantitation (µg/ml) Accuracy (% Recovery) 80 100 120 Precision (%RSD) Intraday precision Interday precision Specificity

ALO 10-50 0.005 0.999

MET 10-50 0.009 0.999

3.96

0.36

1.57

1.11

99. 57

99. 90

1.823 1.926 Specific

0.539 1.018 Specific

RESULTS AND DISCUSSION The methods discussed in the present work provide a convenient and accurate way for simultaneous analysis of Alogliptin and Metformin. In AUC method wavelength selected for Quantitation are 215 – 240nm for Alogliptin and 265 - 293nm for Metformin. In both the methods linearity for detector response was observed in the concentration range of 10-50 mcg/ml for Alogliptin and Metformin, both. Percent label claim for tablet analysis by both the methods was found in the range of 99.62 ± 0.01 for ALO and 99.90 ± 0.04 for MET. Standard deviation and coefficient of variance for three determination of tablet sample, by both the methods was found to be less than + 2.0 indicating precision of both the methods. Percent recovery was found in the range of 99.98 for ALO and 99.36 for MET, values of standard deviation and coefficient of variation was satisfactorily low indicating the accuracy of both the methods. In Method B which was Multicomponent mode wavelength selected for Quantitation method of ALO and MET were 284nm (λ max of MET) and 274 (λ max of ALO). In tablet analysis by both the methods was found in the range of 99.50 ± 0.006 for ALO and 99.63 ± 0.003 for MET. Standard deviation and coefficient of variance for three determination of tablet sample, by both the methods was found to be less than + 2.0 indicating precision of both the methods. Accuracy of both the methods was ascertained by recovery studies and the results are expressed as % recovery. Percent recovery was found in the range of 99.50 for ALO and 99.63 for MET, values of standard deviation and coefficient of variation was satisfactorily

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low indicating the accuracy of both the methods. LOD of ALO was 3.9 and 0.36 for MET and LOQ was 1.57 and 1.11 for ALO and MET respectively. The result of analysis shows that the developed methods are accurate, precise, reproducible and economical and can be employed for routine quality control analysis of Alogliptin and Metformin in combined dose tablet formulation. ACKNOWLEDGEMENTS The authors are very much thankful to the Chairman, Mrs. Fatma Rafiq Zakaria, Maulana Azad Educational Trust, Dr. Rafiq Zakaria Campus for providing necessary facilities for the project work. The authors are also thankful to Wockhardt Pvt. Ltd, for providing gift samples of Alogliptin and Metformin. REFERENCES 1. Instruction Manual User’s System Guide UV-630 Jasco Spectrophotometer (P/N: 03027002D). JASCO Corporation, Analytical instrument division, Tokyo, Japan. 2. Indian Pharmacopoeia. Govt. of India, health ministry & family welfare, published by IP commission Ghaziabad., 2007; 2: 673, 1276-77. 3. B. K. Sharma. Instrumental methods of chemical analysis. 2000, Introduction to analytical chemistry. 19th edition. Goel publishing house, 2000; 200-203: 1-4. 4. U.S. Pharmacopoeia. United States Pharmacopoeial Convention, Inc., 1994; 1982-84. 5. International Conference on Harmonization. Draft Guideline on Validation of Analytical Procedures Definitions and Terminology. Federal Register., 1995; 60: 112-160. 6. Mohammed Imran Anees, et al: Determination of Cefixime and Moxifloxacin in Pharmaceutical dosage form by UV Spectrophotometric method. WJPPS, 2015; 2278– 4357: Volume 4, Issue 03: 1172-1179. 7. Mohammed Imran Anees, et al: Determination of Gemifloxacin and Ambroxol in Pharmaceutical Dosage form by UV Spectrophotometric method.JMCDD, 2015; 23479027: 402-408. 8. Mohammed Imran Anees, et al: Determination of Fluoxetine and Zonisamide by UV Spectrophotometric method. JMCDD, 2015; 2347-9027: 438-444. 9. Beckett AH, Stenlake JB. Practical Pharmeceutical chemistry. CBS Publishers, New Delhi. 4th edition, 275-337. 10. Skoog DA, Holler J, Crouch R.Instrumental analysis.Cengage learning India (P) Ltd, New Delhi., 2007; 378-437.

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