DEVELOPMENT AND VALIDATION OF ANALYTICAL METHODS FOR THE SIMULTANEOUS ESTIMATION OF SITAGLIPTIN PHOSPHATE AND METFORMIN HYDROCHLORIDE IN PHARMACEUTICAL DOSAGE FORM

ABOUT AUTHORS:
R.B.Desireddy, *Sure.Lakshmi Sindhuri, A. Charitha, G.Naga sowjanya
Nalanda institute of pharmaceutical sciences,
kantepudi, Sattenapalli.
*suresindhuri@gmail.com

ABSTRACT:
The scope and need of the present work is to develop and validate an analytical method for the analysis of an anti-diabetic drug. Literature survey reveals various UV, HPLC and LC/MS methods are available for individual estimation of the Metformin Hydrochloride and Sitagliptin Phosphate and in combination with other drugs. But, none of the reported analytical methods describe a stability indicating RP-HPLC method for the simultaneous analysis of Metformin and Sitagliptin in tablet dosage form. Hence the present works aims at developing a simple, sensitive, precise, economic and validated second order derivative UV spectroscopic method & stability indicating RP-HPLC method for the simultaneous estimation of Sitagliptin and Metformin in pharmaceutical dosage form. Incomparision with direct UV method, the second order derivative UV spectroscopic method eliminates the interference from UV-absorbing excipients. This method was found to be fast and can be used for formulation screening. HPLC is an integral analytical tool in assessing drug product stability.  HPLC methods should be able to separate, detect and quantify the various drug-related degradants that can form on storage or manufacturing, plus detect and quantify any drug-related impurities that may be introduced during synthesis. Stability studies were carried out under various stress conditions like acid hydrolysis, base hydrolysis, water hydrolysis, oxidation, photolytic degradation and under UV-light. Both the drugs Sitagliptin and metformin were degraded more in the alkaline condition.

REFERENCE ID: PHARMATUTOR-ART-1912

INTRODUCTION
Sitagliptin Phosphate and Metformin Hydrochloride are available in combined dosage forms as film coated tablets (JANUMET). Each tablet contains 50mg of Sitagliptin Phosphate and 500 mg of Metformin Hydrochloride. It is used for the treatment of Diabetes Mellitus. Sitagliptin which belongs to class triazolopyrazines(phenylethylamines) works competitively to inhibit the enzyme dipeptidyl peptidase 4 (DPP-4)[1-2]. This enzyme breaks down the incretins GLP-1(Glucagon like particle-1) and they have the ability to potentiate the secretion of insulin and suppress the release of glucagon from pancreas.

Metformin is an oral anti-diabetic first line drug for treating type II diabetes. It is a biguanide drug activates AMP-activated Protein kinase (AMPK), a liver enzyme that plays an important role in Insulin signaling,[3-5] whole body energy balance and the metabolism of glucose and fats. It improves hyperglycemia by suppressing “hepatic gluconeogenesis”.

The combination of Metformin and a dipeptidyl peptidase 4 inhibitor (Sitagliptin) has been shown to be safe, effective and well-tolerated treatment for type II diabetes. When both these drugs are combinedly administered the mean HbA1C levels are reduced by 0.65-1.1% from a baseline of 7.8-8.4%.

According to an FDA guidance document, a stability-indicating method is “a validated[16] quantitative analytical procedure that can detect the changes with time in the pertinent properties of the drug substance and drug product. A stability-indicating method[17] accurately measures the active ingredients, without interference from degradation products, process impurities, excipients or other potential impurities. High performance liquid chromatography (HPLC) is an integral analytical tool in assessing drug product stability. HPLC methods should be able to separate, detect and quantify the various drug-related degradants that can form on storage or manufacturing, plus detect and quantify any drug-related impurities that may be introduced during synthesis.

Reagents and materials
All the chemicals and solvents used are of analytical grade which were purchased from Qualigens, Merk & S.D Fine chemicals, Mumbai. Both the drug samples of Sitagliptin Phosphate and Metformin Hydrochloride were received as a gift samples. Commercially available JANUMET tablets were purchased from local market. Each tablet consists of 500mg of Metformin Hydrochloride (MET) and 50mg of Sitagliptin phosphate(STG).

Instrumentation:
Schimadzu Digital Electronic Balance - BL 220H, Labindia SAB 5000 pH meter, Value Vaccum pump, Schimadzu UV-1800, UV/Vis-Spectrophotometer, Schimadzu HPLC, Phenomenex column, UV detector, Ultrasonic cleaner, Life care equipments pvt.ltd.

Procedure:
U.V Spectrophotometric method for simultaneous estimation of Sitagliptin phosphate & Metformin hydrochloride by second order derivative spectroscopy:

In derivative spectral method [6-8], firstly UV spectrum of drug would be recorded at 200-400 nm and processed to get derivative spectrum. At the zero crossing point of one drug, the second drug would be measured which gives a reasonable means of estimating drug without interference of additives or impurities and thereby improves the sensitivity of the method. Thus a derivative spectrum shows better resolution of overlapping bands than the fundamental spectrum and may permit the accurate determination of the lmax of the individual bands.

SOLVENT SELECTION:
The UV spectra of Sitagliptin Phosphate (STG) and Metformin Hydrochloride(MET) were recorded individually in various solvents like water, methanol, acetonitrile, ethanol etc. All the spectra were processed to obtain derivative spectra. The derivative spectra of STG and MET in solvents like methanol, acetonitrile showed no favorable zero crossing points i.e., at any particular wavelength where one drug has zero crossing point, the other drug also have similar zero crossing point hence no spectral isolation was found. When dissolved in water, the derivative spectra of both drugs, STG and MET showed zero crossing point in 1st and 2nd derivative spectra (Fig: 1, 2). The zero crossing points and their absorbance are shown in table 1.

Though both first and second derivative spectra showed zero crossing points and their absorbances were considerably better, 2nd derivative spectra was selected because the spectral characteristics were good in this particular derivative spectra and it obeys Beer Lambert's Law. The zero crossing points of Sitagliptin Phosphate in water were found to be 249, 252 and 257nm and 272, 278 and 280 for Metformin Hydrochloride.

Out of this 249nm and 278nm have been selected for the present study based on its linearity data. At 249nm Sitagliptin Phosphate showed zero absorbance but Metformin Hydrochloride had considerable absorbance. Similarly at 278nm, Metformin Hydrochloride showed zero absorbance but Sitagliptin Phosphate had considerable absorbance.

Table:1. Zero crossing points of Sitagliptin (STG) & Metformin (MET)

Type of Derivative

Drug

Zero crossing point (nm)

Ist

STG

241nm

MET

217nm

2nd

STG

249nm

MET

278nm


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