INTRODUCTION:
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.
MATERIALS AND METHODS:
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.
EXPERIMENTAL:
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.
Assay:
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.
Robustness:
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.
RESULTS AND DISCUSSION:
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.