Validated UV Spectrophotometric Method for Estimation of Sitagliptin Phosphate in Tablet Dosage Form.


Sachin Patil1*, B. Ramesh1, A.R. Hareesh2 and Kiran Patil3

1Sri Adichunchanagiri College of Pharmacy, B. G. Nagara-571448, Tal: Nagamangala Dist: (Mandya), Karnataka, India;2Government College of Pharmacy, Vidyanagar, Karad, Dist-Satara, Maharashtra-415 124, India.

*Corresponding Author E-mail:



Sitagliptin phosphate is a new class of oral antihyperglycemic agents and it is potent and selective inhibitor of dipeptidyl peptidase-IV (DPP-IV) for the treatment of Type 2 diabetes. No method is available for routine analysis of sitagliptine phosphate. Here we have developed simple, accurate and rapid UV spectrophotometric method for estimation of Sitagliptin phosphate from tablet formulation. The drug obeyed the Beer’s law and showed good correlation. It showed absorption maxima at 272.5 nm in methanol. The linearity was observed between 35 – 85 mcg/ml. The results of analysis were validated by recovery studies. The recovery was more than 99%. The method was found to be simple, rapid, accurate, precise and reproducible


KEYWORDS: Sitagliptin phosphate, UV–Vis Spectrophotometry, Recovery



Sitagliptin [(2R)-1-(2,4,5-trifluorophenyl)-4-oxo-4-[3-(trifluoromethyl)-5,6 dihydro [1,2,4]triazolo [4,3-a]pyrazin- 7(8H)-yl] butan-2-amine] (Fig. 1) is an orally active, potent and selective inhibitor of dipeptidyl peptidase-IV (DPP-IV) for the treatment of Type 2 diabetes). Sitagliptin phosphate is a new class of oral antihyperglycemic agents, which enhance the body's own ability to lower blood glucose when it is, elevated2. Sitagliptin phosphate prolongs the activity of proteins that enhance the release of insulin after blood sugar rises.


Sitagliptin phosphate is a selective inhibitor of the enzyme dipeptidyl peptidase-4 (DPP-4), which metabolizes the naturally occurring incretin hormones glucagon-like peptide-1(GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) resulting in enhanced glucose-dependent insulin secretion from the pancreas and decreased hepatic glucose production. Since GLP-1 enhances insulin secretion in the presence of raised blood glucose levels, inhibiting DPP-IV activity will increase and prolong the action of GLP-1 by reducing its rate of inactivation in plasma3,4. Sitagliptin phosphate reduces hemoglobin A1c (HbA1c), fasting and postprandial glucose by glucose-dependent stimulation of insulin secretion and inhibition of glucagon secretion5. GLP-1 has other widespread effects including delaying gastric emptying, significantly reducing glucagons levels and possible central effects on the appetite3.


Literature survey revealed that the analytical techniques published for Sitagliptin phosphate, have mainly involved clinical pharmacokinetic studies, a method for the determination of sitagliptin in human plasma has been developed and validated using HTLC/LC–MS/MS6, determination of sitagliptin in human urine and hemodialysate using turbulent flow online extraction and tandem mass spectrometry7.


The objective of the present work was to develop simple, rapid, accurate, precise and reproducible UV spectrophotometric method for the estimation of Sitagliptin phosphate in pharmaceutical dosage forms. The method was further validated for the parameters like precision, accuracy, sensitivity, and linearity. The limit of detection (LOD) and limit of quantification (LOQ) were also determined. The results of analysis were validated statistically and by recovery studies.



Instrument Used:

A Shimadzu UV/VIS 1700 (E), 230VCE spectrophotometer with Spectral bandwidth of 1 nm.


Reagents and Solutions:

Sitagliptin phosphate was obtained as a gift sample from Rao’s Pharma Ltd., Hyderabad, India. Sitagliptin phosphate tablets were procured from local pharmacy. Methanol used was of analytical grade. Glass double distilled water was used throughout the experiment.


Fig. 1: Chemical structure of Sitagliptin Phosphate.



Determination of λmax:

Weighed amount of Sitagliptin phosphate was dissolved in Methanol to obtain a 1000mcg/ml solution. This solution was subjected to scanning between 200 – 400 nm and absorption maximum was determined. The effect of dilution on absorption maxima was studied by diluting the above solution to 75mcg/ml and scanned from 200 – 400nm.


Standard Stock Solution:

An accurately weighed 100 mg of Sitagliptin phosphate was dissolved in 10 ml methanol in a 100 ml volumetric flask and the volume was adjusted up to the mark with methanol to obtain a 1 mg/ml. 10 ml of this solution in 100 ml volumetric flask and volume adjust to the mark with methanol gives stock solution with concentration of 100µg/ml.


Linearity and Calibration:

Aliquots of 3.5 to 8.5 ml portions of standard solution were transferred to a series of 10 ml volumetric flasks and volume in each flask were adjusted to 10 ml with methanol to obtain a concentration of range of 35-85 µg/ml. A calibration curve for Sitagliptin phosphate was obtained by measuring the absorbance at the λmax of 272.5 nm. Statistical parameters like the slope, intercept, coefficient of correlation, standard deviation, Relative standard deviation, and error were determined.

Relative standard deviation of was observed for analysis of 9 replicate samples, indicating precision and reproducibility. Limit of detection (LOD) and limit of quantification (LOQ) were calculated by Eqs. (1) LOD =  and (2) LOQ =, respectively, where δ is the standard deviation of blank and s is slope of calibration 8.



Twenty tablets each containing of 100 mg of sitagliptin phosphate was weighed and powdered. Powder equivalent to 100mg of sitagliptin phosphate was transferred into 100 ml volumetric flask dissolved in 10 ml Methanol solution. Volume was then made up to 100 ml with methanol solution to get a solution of strength 1mg/ml. The solution was then filtered through Whattman filter paper No.40 (0.45 micron). Then 10 ml of above filtered solution was further diluted to 100 ml with Methanol to produce a solution of strength 100 mg/ml.  Aliquots of the sample were removed and diluted to 10 ml with methanol to obtain strengths as 35mcg/ml, 45mcg/ml, 55mcg/ml 65mcg/ml and 75mcg/ml and determined the respective absorbance at 272.5 nm against the methanol as blank.


Recovery studies:

Recovery studies were performed to judge the accuracy of the method. Recovery studies were carried out by adding a known quantity of pure drug to the preanalyzed formulation and the proposed method was followed. From the amount of drug found, percentage recovery was calculated.



The evaluation of robustness was performed for system suitability to ensure the validity of analytical procedure. This was done by varying the instrument, analyst, and time of study.  The analysis was performed on Shimadzu UV Visible spectrophotometer, model- 1700 (Japan). Interday and intraday analysis was performed by changing the analyst.



The UV scan of standard solution between 200 – 400 nm showed the absorption maxima at 272.5nm, shown in fig. 2.


Fig. 2: lmax of Sitagliptin Phosphate in Methanol.


The Beer’s law was verified from the calibration curve by plotting a graph of concentration vs. absorbance. The plot is shown in fig. 3.


Fig. 3: Calibration curve of Sitagliptin phosphate in Methanol at 272.5 nm.



Sr. No.




λmax (nm)



Beer’s range (µg/ml)



Molar absorptivity  (l/mol/cm)



Sandell’s sensitivity (mg/cm2/0.001AU)



Correlation coefficient (r2)



Regression equation

y = .006 x - 0.006


Intercept (a)



Slope (b)



Limit of detection (LOD µg/ml)



Limit of quantification (LOQ µg/ml)






Label Claim mg

% Estimated


C.O.V. (%)


% Recovery








Table Legends: TABLE 2, S.D.: Standard Deviation; S.E.: Standard Error; C.O.V.: Coefficient of Variation




Regression analysis showed very good correlation. The calibration plot revealed zero intercept which is clear by the regression analysis equation Y = mX + C. (Where Y is absorbance, m is the slope and X is the concentration of sitagliptin phosphate in mcg/ml) as obtained by the least square method. The results thus obtained are depicted in Table No. I. The results of analysis for assay and recovery studies were studied and are shown in Table No. II.


No significant variations were observed on interday and intraday analysis. Also no significant variations were observed on changing the instrument make and model.



The spectrum of Sitagliptin phosphate in methanol showed the absorption maxima at 272.5 nm. No effect of dilution was observed on the maxima, which confirmed the maxima at 273nm.


The statistical analysis of data obtained for the calibration curve of Sitagliptin phosphate in pure solution indicated a high level of precision for the proposed method, as evidenced by low value of coefficient of variation. The coefficient of correlation was highly significant. The linearity range was observed between 35 – 85 mcg/ml. The plot clearly showed a straight line passing through origin (y=0.006x-0.006). The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.373849 µg/ml and 1.132782 µg/ml respectively.


The assay method was validated by low values of % RSD and standard error, indicating accuracy and precision of the methods. The Excellent percentage recovery value indicates that there is no interference from the excipients present in formulation, further proves the accuracy of the method.

Robustness of the method was studied by varying the instrument, time of study and analyst. Reproducibility of the results confirmed the robustness of the method.



From the results and discussion the method described in this paper for the determination of Sitagliptin phosphate from tablet formulation is simple, rapid, accurate and precise and reproducible. This method can be successfully applied routine estimation of Sitagliptin phosphate in bulk and pharmaceutical dosage form.



We are grateful to Rao’s Pharma Ltd., Hyderabad, India, for providing gift sample of Sitagliptin phosphate drug for research work. We are thankful to Principal, Shri Adichunchunagiri College of Pharmacy, B. G. Nagara for providing laboratory facility and constant encouragement.



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Received on 14.01.2010       Modified on 20.02.2010

Accepted on 12.03.2010      © RJPT All right reserved

Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 798-800