DEVELOPMENT AND VALIDATION OF RP HPLC METHOD FOR THE ESTIMATION OF SITAGLIPTIN PHOSPHATE IN BULK AND TABLET DOSAGE FORM

DEVELOPMENT AND VALIDATION OF RP -HPLC METHOD

FOR THE ESTIMATION OF SITAGLIPTIN PHOSPHATE IN

BULK AND TABLET DOSAGE FORM

Ne ha Sunil Dangi*, S. R. Bavas kar and Dr. S. D. Barhate

Departme nt of Qualit y Assura nce, Shree Sureshda da Jain Instit ute of

Pharmac e ut ic a l Educat io n and Researc h, Jamne r - 424206.

ABSTRACT

A simple new, precise, economic reverse phase high performance

chromatographic method has been developed and validated for the

estimation of Sitagliptin phosphate in bulk and tablet dosage form. The

estimation is done by using C18 Primesil (250mm x 4.6mm;5µm)

column and mobile phase is Methanol: Water (50:50 % v/v), pH is

maintained at 3 by using Orthrophosphoric Acid. The chromatographic

separation was performed with the UV detector set at 264 nm and flow

rate is maintained at 0.7ml/min. The method was validated and found

to be linear in the range of 20 - 100 µg/ml and correlation coefficient

was found to be 0.999. The assay of Sitagliptin was found to be

99.15%. The results of the study showed that the proposed RP-HPLC

method is simple, accurate and economical which is useful for the

routine determination of Sitagliptin phosphate in bulk and its pharmaceutical dosage form.

KEYWORDS: Sitagliptin Phosphate, RP-HPLC, Method development, Validation.

INTRODUCTION

Fig. 1 Structure of Sitagliptin

Volume 5, Issue 9, 773-781. Research Article ISSN 2277– 7105

Article Received on 02 July 2016,

Revised on 23 July 2016, Accepted on 12 Aug 2016

DOI: 10.20959/wjpr20169-6878

*Corresponding Author

Ne ha S uni l Dang i

Department of Quality

Assurance, Shree

Sureshdada Jain Institute

of Pharmaceutical

Education and Research,

Sitagliptin Phosphate chemically

7-[(3R)-3-amino-1-oxo-4-(2,4,5-triflurophenyl]-5,6,7,8-tetra hydro-3-(trifluromethyl)-1,2,4-triazole [4,3] pyrazoline phosphate(1:1) monohydrate. It

is a novel hypoglycemic drug that belongs to dipeptidyl-peptidase 4 inhibitor class which

stimulates glucose-dependent insulin release.[1,2] Recently the combination of two drugs has been recommended in the treatment of diabetes mellitus to improve glycemic control.

Sitagliptin generally used in combination e.g. Sitagliptin-Metformin, Sitagliptin-Simvastatin.

Sitagliptin increases the level of incretin level (GLP-1, GIP) which inhibits the release of

glucagon by pancreas, and decreases blood glucose, but more significantly increases Insulin

secretion.[3]

Literature survey reveals that various bio-analytical RP-HPLC methods are there[1,2] as well as few spectrophotometric methods are available for estimation of Sitagliptin in bulk and

pharmaceutical dosage form.[3-4] Two RP-HPLC methods are also there, but these methods are not economical.[8-9] Hence there is need to develop an RP-HPLC method for the estimation of Sitagliptin Phosphate in the Tablet formulations. The aim of present analytical

research is to develop simple, precise, accurate, rapid and economical RP-HPLC method for

the estimation of Sitagliptin in tablet formulation.

MATERIALS AND METHODS Materials

Sitagliptin was purchased from Swapnroop drug and pharmaceuticals. Commercial Tablet of

Sitagliptin phosphate named ISTAVEL was purchased from local market. All other

chemicals and reagents used are HPLC grade manufactured by Merck Ltd. India.

Instrument and chromatographic conditions

High performance liquid chromatography YounglineAcme9000 having autochro-3000

software containing primesilC18 column (250mm X 4.6mm, 5µm) was used for the study.

UV-Spectrophotometer is used as a detector. The mobile phase used was Methanol: Water

(0.05% Orthophosphoric acid) in the ratio of 50:50 % v/v maintained at pH 3 with a flow

rate 0.7ml/min. Mobile phase and sample solutions were filtered through a 0.45 μm

membrane filter and degassed in Sonicator. The effluent was detected at 264 nm. The

Preparation of mobile phase

The mobile phase was prepared by Mixing Methanol (50ml) and Water (50ml) and adjusted

to the pH 3 with Ortho Phosphoric Acid. The prepared mobile phase was degassed by

ultrasonication for 20 min so as to avoid the disturbances caused by dissolved gases. This

degassed mobile phase was filtered through 0.45 g membrane nylon filter to remove smaller

particles that may be present in mobile phase.

Preparation of solutions Standard stock solution

10 mg of Sitagliptin pure drug was dissolved in 10 ml volumetric flask by sufficient quantity

of Methanol: water (50:50). Volume was made up to 10ml, this will produce 1000μg/ml

standard stock solution of Sitagliptin.

Working standard solution

1mL of standard stock solution was pipetted into 10mL volumetric flask and diluted up to the

mark with diluent and filtered through 0.45μ Millipore Nylon filter to obtain concentration of

10μg/ml.

EXPERIMENTAL

Study of spectra and selection of wavelength

UV Detector was selected and solution 10 µg/mL of Sitagliptin was scanned in the range of

400 – 200nm. A fix concentration of analyte were analysed at different wavelengths. As per

response of analyte, 264nm wavelength was selected. The UV Spectra of Sitagliptin is as

shown in Fig. 2.

Analysis of marketed formulation

For analysis of the tablet dosage form, 20 tablets were weighed individually and their average

weight was determined which is 4.22gm, after that they were crushed to fine powder. 42.2

gm sample of Sitagliptin accurately weighed and transferred to 10 ml volumetric flask which

is equivalent to 10mg of pure drug. Powder was dissolved with some amount of mobile phase

and sonicate to dissolve it completely and to remove dissolved gases. Then it is filtered

through 0.45 µg nylon membrane filters. Then volume was made up to mark with mobile

phase. It will make the solution of 1000 µg/ml. From the above solutions 0.60 ml from Stock

was taken and diluted to 10 ml with mobile phase to get a solution containing 60µg/ml. The

amount of Sitagliptin was calculated by extrapolating the value of area from the calibration

curve. Shown in Fig. 3.

Fig. 3 Chromatogram of Sitagliptin

Table 1 Analysis of marketed formulation Sr.

no. Concentration Area

Amt. found

%Label Claim 1 60 1774.32 59.40 99.00 2 60 1779.25 59.57 99.28 - Mean 1776.78 59.49 99.15 - SD 3.4860 0.1202 0.1979

- %RSD 0.20 0.20 0.20

Method validation

The method was validated in terms of the following parameters; linearity, accuracy,

precision, repeatability, robustness, LOD, LOQ and system suitability parameters as per the

1. Linearity

From Sitagliptin standard stock solution, different working standard solution (20, 40, 60, 80,

100μg/ml) were prepared in mobile phase. Chromatogram was recorded. The plot of linearity

plotted graphically as a function of analyte concentration is as shown in fig. 4 and table 2 and

3.

Fig. 4 Calibration curve of Sitagliptin

Table 2 Data of Linearity

Sr. no. Concentration μg/ml Area

1 20 500.98

2 40 1034.08

3 60 1583.81

4 80 2164.20

5 100 2771.20

Table 3 Statistical data of linearity

Regression Equation Data Y = mx+c

Slope (m) 28.353

Intercept© 90.314

Correlation coefficient 0.999

2. Accuracy

The accuracy study was performed at three different levels (80%, 100% and 120% of the test

concentration). The mean % recoveries were found to be between 98–102% as required by

ICH guidelines. The results of the recovery studies and its statistical validation data are given

Table 4 Recovery study of Sitagliptin Level of Recovery %

80% Sitagliptin

100% Sitagliptin

120% Sitagliptin

Amount Present(mcg) 20 20 20

Amount of Std Added (mcg) 16 20 24 Amount Recovered (mcg) 16.20 19.77 24.08 Mean % Recovery 100.94 98.83 100.32

Standard Deviation 0.90 0.95 1.27

%RSD 0.89 0.96 1.26

*Mean of each three readings

3. Precision

Precision of the analytical method is expressed as the series of the measurement. It was

ascertained by replicate estimation of the drug by the proposed method as shown in Table No.

5.

Table 5 Intra-day precision

Conc. Mean Area Amt Found % Amt Found SD RSD 40 1044.85 40.03 100.27 3.10 0.30

60 1589.11 59.23 98.73 1.97 0.12

80 2163.30 79.49 99.36 5.65 0.26

*Mean of each three readings

Table: 6 Inter-day precision

Sr No. Conc Mean Amt Found % Amt Fnd SD RSD 1 40 1042.85 40.11 100.27 5.93 0.57 2 60 1589.11 59.43 99.06 7.93 0.50 3 80 2161.30 79.42 99.27 7.06 0.33 *Mean of each three readings

4. Repeatability

Table 7 Repeatability study of Sitagliptin

Conc. µg/ml Peak Area Amount Found Percent Amount Found

40 1010.35 38.82 97.05

40 1032.13 39.59 98.98

40 1033.12 39.62 99.32

- Mean 39.34 98.45

- SD 0.45 1.22

5. Robustness

The changes were did in flow rate (± 0.1 ml/min), pH of mobile phase composition and

wavelength (± 1 nm). %RSD for peak area was calculated which should be less than 2%. As

shown in Table No. 8.

Table: 8 Result of Robustness study of Sitagliptin

6. LOD and LOQ

The Limit of detection and Limit of Quantitation were separately determined which is based

on the standard deviation of response of the calibration curve. LOD and LOQ is as follow

 LOD of Sitagliptin = 0.8299  LOQ of Sitagliptin = 2.51

7. System suitability

System suitability parameters were studied as shown in Table No. 9.

Table: 9 System Suitability studies result

System Suitability Parameters Proposed Method

Retention Time 4.8890

Area 1025.2073

Theoretical Plate Number 14882.1

Tailing Factor 1.2354

RESULT AND DISCUSSION

The proposed method was developed and validated as per the ICH guidelines. Linearity was

observed over a concentration range of 20 to 100μg/ml. The accuracy of method was

determined by calculating mean percentage recovery at 80,100 and 120 % level and % RSD

was found to be less than 2. In this method precision was studied as repeatability and inter

and intra-day variations for both drugs and both were found in limit. System suitability

parameters were satisfactory and the theoretical plates were obtained above 2000. Tailing

Parameter Concentration

(Mcg)

Area

factor was found below 2. %RSD also found below 2%. The assay of Sitagliptin was found to

be 99.15% and the low % RSD value confirms the robustness of the method.

CONCLUSION

The proposed RP-HPLC method was found to be simple, accurate, precise, linear, robust and

economic for quantitative estimation of Sitagliptin phosphate in bulk and its formulation. The

proposed RP-HPLC method was cost effective and less time consuming. The values for

system suitability parameters showed feasibility of this method for routine pharmaceutical

application. Hence, the present RP-HPLC method is suitable for routine assay of Sitagliptin

phosphate in bulk and tablet dosage form in the quality control laboratories.

ACKNOWLEDGEMENT

The authors are thankful to S.S.J.I.P.E.R, Jamner, Maharashtra, India, for providing the

necessary facilities and help in my dissertation work.

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