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1 Spectrophotometric Method for Estimation of Sitagliptin Phosphate in Bulk... I J P F A International Science Press Spectrophotometric Method for Estimation of Sitagliptin Phosphate in Bulk and Tablet Dosage Form Maste Meenaxi M.*, Bhat A. R., Mohite Mukesh and Patil Deepak Abstract: Two simple, precise and economical UV methods have been developed for estimation of Sitagliptin Phosphate in bulk formulation. Sitagliptin Phosphate has the absorbance maxima in zero order spectra is 267nm. First order derivative for the analysis of Sitagliptin Phosphate at 275nm. Drug followed Beer-Lambert s law in the concentration range of 20-70µg/ml for both the methods. Results of analysis were validated statistically and were found to be satisfactory. Thus proposed methods can be successfully applied for estimation of Sitagliptin phosphate in routine analytical work. Keywords: Sitagliptin Phosphate, Zero order spectra, First order derivative, UV Spectrophotometer. INTRODUCTION Sitagliptin Phosphate is described chemically 1,2,4- triazolo[4,3-a]pyrazine, 7-[(3R)-3-amino-1-oxo-4- (2,4,5-trifluorophenyl)butyl]-5,6,7,8-tetrahydro-3- (trifluoromethyl), phosphate(herman,2005; Januvia side effects, 2007; USFDA, 2006; Herman, 2006). The structure is given in Figure 1.It is a new class of oral anti hyperglycemic (anti-diabetic) drug of dipeptidyl peptidase-4 (DPP-4) inhibitor class. This enzymeinhibiting drug is used either alone or in combination with other oral antihyperglycemic agents such as Metformin (Puranik, 2005) for treatment of diabetes mellitus type 2. The benefit of this medicine is its lower side effects (e.g. less hypoglycemic, less weight gain) in the control of blood glucose values. An UVspectrophotometric method for the quantitation of Sitagliptin phosphate at 430 nm which based on condensation of primary amino group of Sitagliptin phosphate with acetyl acetone & formaldehyde producing yellow colored product has been developed (Sekaran, 2010). The drug is not official in any Pharmacopoeia. Literature survey reveals that only LC-MS methods were reported for the determination of sitagliptin phosphate in plasma and urine of humans, rats and dogs (Stella, 2007; Zeng, 2008; Nirogi, 2008; Beconi, 2007). So far, no derivation methods are available for the determination of this drug, spectrophotometer continues to be very popular, because of their simplicity, specificity and low cost. This study presents new spectrophotometric method for the determination of sitagliptin phosphate in bulk and Pharmaceutical formulations. EXPERIMENTAL Materials Sitagliptin phosphate was obtained as gift sample from Matrix Ltd. All other chemicals used were of analytical grade. A shimadzu UV-1700 UV/VIS Spectrophotometer was used with 1cm matched quartz cells and double distilled water used throughout the experiment. Methods Accurately about 10 mg of Sitagliptin Phosphate was weighed and transferred to 100 ml volumetric flask; 40 ml of methanol was added to dissolve the drug completely with vigorous shaking. Then the volume was made up with the distilled water up to the mark to give the drug stock solution of concentration 100µg/ml. Aliquots of standard stock solution were pipetted out and suitably diluted with distilled water to get the final concentration of standard solution. Zero order derivative method at n = 0 (Method A) showed a sharp peak at 267nm (figure 2). The absorbance difference at n = 6 (da/dλ) is calculated by the inbuilt software of the instrument which was directly proportional to the concentration of the standard solution. The standard drug solution was diluted so as to get the final concentration in the range * Department of Pharmaceutical Chemistry KLEU s College of Pharmacy, Belgaum , Karnataka, Vol. 2, No. 2, September

2 Maste Meenaxi M., Bhat A. R., Mohite Mukesh and Patil Deepak Figure 1: Structure of Sitagliptin Phosphate Graph 1: Standard calibration curve of zero order derivative spectrum Figure 2: Zero order Spectrum µg/ml and scanned in the zero order derivative spectra (Table 1). The calibration curve of (da/dë) against concentration of the drug showed linearity (Graph 1). Table 1 Standard Calibration for Sitagliptin Phosphate Sr. No. Conc. µg/ml Absorbance Method A Method B Graph 2: Standard calibration curve of first order derivative spectrum Similarly, for First order derivative, same method was employed at n = 1 (Method B) showed a sharp peak at 275 nm (Figure 3). The standard drug solution was diluted so as to get the final concentration in the range of 20-70µg/ml and scanned in first order derivative spectra (Table 1). The calibration curve of da/dλ against concentration of the drug showed linearity (Graph 2). For estimation of Sitagliptin Phosphate in tablet formulation by the two methods, for the analysis STP, ten tablets (Januvia 100 mg) were weighed and ground into a fine powder. An accurately weighed Figure 3: First Order Spectrum portion of the powder equivalent to 10 mg of powder of Sitagliptin Phosphate weighed and transferred to 100 ml volumetric flask and dissolve in 40 ml of methanol. It was kept for ultra sonification for 45 min, 48 International Journal of Pharmaceutical Formulation and Analysis

3 Spectrophotometric Method for Estimation of Sitagliptin Phosphate in Bulk... finally the volume was made up to the mark with distilled water, this was then filtered through Whatman filter paper to get tablet stock solution of concentration to 100 µg/ml. Various dilutions of the tablet solution were prepared and analyzed for six times and concentration was calculated by using calibration curve for two methods. Both the methods were validated according to ICH guidelines (1996) by carrying out analysis of six replicate samples of the tablets (Table 2 & 3), Assay (Table 4) and recovery studies were carried out at three different levels i.e. 80%, 100% and 120% by adding the pure drug (8, 10 and 12mg respectively) to previously analyzed tablet powdered sample. From the found drug amount, percentage recovery was calculated (Table 5). Table 2 Optical Characteristics and Parameters Parameters Method A Method B λ max (nm)/wavelength range (nm) Beer, s-lamberts range (µg/ml) Coefficient of Correlation (r 2 ) Regression Equation y = mx+c a. Slope (m) b. Intercept (c) LOD LOQ Table 3 Statistical Validation Parameter Mean* S.D* C.O.V.* S.E.* Method A Method B Method A Method B Method A Method B Method A Method B r Slope X X X Intercept X10-05 Table 4 Assay of the Tablet Sr. No. Tablet Conc. (µg/ml) Amount present (mg/tab) Amount found (mg /Tab)* % of Drug Found Method A Method B Method A Method B Method A Method B 1 T *Average of six readings Table 5 Recovery Studies Sr. Tablet Level of % Mean* S.D.* C.O.V.* S.E.* No. Sample recovery (%) A B A B A B A B 1 T When *n=3 at each level of recovery RESULTS AND DISCUSSION Sitagliptin phosphate is not official in any of the Pharmacopoeias and only listed in The Merck Index, Martindale and The Complete Drug Reference. There is no official analytical method reported for the estimation of Sitagliptin Phosphate. Literature survey reveals that the developed UV spectroscopic method for the estimation of Sitagliptin Phosphate is not reported anywhere. Hence the objective of the work was to develop simple, precise, accurate, sensitive, rapid and economical UV-Visible Spectrophotometric methods for the estimation of Sitagliptin Phosphate in bulk and pharmaceutical formulation. The methods were based on the development of calibration curve for the standard drug and the analysis of the formulation was done using the calibration curve equation in the quantitation mode of the Spectrophotometer (Method A). The standard drug solution were scanned in the spectrum mode and the first order derivative spectra with (n=1) Vol. 2, No. 2, September

4 Maste Meenaxi M., Bhat A. R., Mohite Mukesh and Patil Deepak processed showed zero crossing at the absorbance maxima of the normal spectra, the derivative da/dλ is plotted against the concentration of the standard solution (Method B). Both methods A & B for the estimation of Sitagliptin phosphate in tablet form were found to be simple, precise, accurate, rapid & reproducible. Beer- Lambert s law was obeyed in the concentration range of 20-70µg/ml in both methods. The validated parameters confirmed to be reproducible, accurate and satisfactory. Hence the two methods can be employed for routine analysis of the drugs in quality control, R&D laboratories. CONCLUSION The present method was intended to study the zero & first order kinetics by UV Spectrophotometric method. The method developed is simple, precise, accurate, sensitive, rapid & economical for the estimation of Sitgliptin Phosphate in bulk & Pharmaceutical formulation. References [1] Herman, G.; et al. Pharmacokinetics and Pharmacodynamics of Sitagliptin, an Inhibitor of Dipeptidyl Peptidase IV, in Healthy Subjects: Results from Two Randomized, Double Blind, Placebo Controlled Studies with Single Oral Doses. Clinical Pharmacology and Therapeutics 2005, 78: [2] Januvia Side Effects & Drug Interactions. Rx List.com, [3] U. S. Food and Drug Administration. FDA Approves New Treatment for Diabetes [4] Herman, G.; et al. Pharmacokinetics and Pharmacodynamic Effects of the oral DPP 4 Inhibitor Sitagliptin in Middle Aged Obese Subjects. Journal of Clinical Pharmacology 2006, 46: [5] Puranik, M.; et al. Simultaneous Estimation of Metformin HCl and Rosaglitazone Maleate in Solid Dosage form by Ultra-violet Spectrophotometry. Indian Drugs 2005, 42(7), 428. [6] Sekaran, B. C.; et al. Development and Validation of Spectrophotometric Method for the Determination of dpp4 inhibitor, Sitagliptin, in its Pharmaceutical Dosage forms, International Journal Pharmacy and Pharmaceutical Science 2010, 2, [7] Stella, V. H.; et al. Metabolism and Excretion of Dipeptidase 4 inhibitor (14C) Sitagliptin in Humans. Drug Metabolism and Disposition. 2007, 35, [8] Zeng, W.; et al. Determination of Sitagliptin in Human Urine and Hemodialysate using Turbulent Flow Online Extraction and Tendem Mass Spectrophotometry. Journal Pharmaceutical and Biomedical Analysis 2008, 46, [9] Nirogi, R.; Sensitive Liquid Chromatography Tandem Mass Spectrometry Method for the Quantification of Sitagliptin, a DPP 4 inhibitor, in Human Plasma using Liquid-liquid Extraction. Biomedical Chromatography 2008, 22, [10] Beconi, M. G.; et al. Disposition of the Dipeptidyl Peptidase 4 inhibitor Sitagliptin in Rats and Dogs. Drug Metabolism Disposition 2007, 35, [11] Text Validation of Analytical Procedures Q2B in; ICH, Harmonised Tripartite Guidelines, International Journal of Pharmaceutical Formulation and Analysis

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