STABILITY-INDICATING RP- HPLC METHOD FOR ANALYSIS OF SITAGLIPTIN IN THE BULK DRUG AND IT’S PHARMACEUTICAL DOSAGE FORM
V.DEEPTHI *, POORNIMA.Y, DR.G.DEVALA RAO, T.SANDEEP REDDY
Department of Pharmaceutical Analysis,
K.V.S.R.Siddharthacollege of pharmaceutical sciences,
A novel stability-indicating RP-HPLC method has been develop and validated for quantitative analysis of Sitagliptin in the bulk drug and in its pharmaceutical dosage forms using Hypersil–BDS- C18 column (250x4.6mmi.d, 5µ particle size) with 10mM Phosphate buffer (PH-3.5): ACN 60:40%v/v as isocratic mobile phase enabled separation of the drug from its degradation products. UV detection was performed at 260 nm. The method was validated for linearity, accuracy (recovery), precision, sensitivity, ruggedness and robustness. The linearity of the method was excellent over the range 10–60μg/ml (correlation coefficient 0.999). The limits of detection and quantification were 0.21 and 0.640μg/ml, respectively. Recovery of Sitagliptinfrom the pharmaceutical dosage form ranged from 99.99 to 100.05%.
Sitagliptin was subjected to stress conditions (Hydrolysis (acid, base), oxidation,thermal and photo degradation) and the stressed samples were analysed by use of the method. Degradation was observed in acid, base, and 30% H2O2. The drug was stable under the other stress conditions investigated. The degradation products were well resolved from main peak. The forced degradation studies prove the stability indicating power of the method.
Reference Id: PHARMATUTOR-ART-1529
Sitagliptin1,2 is a new oral hypoglycemic (anti-diabetic drug) of the new dipeptidyl peptidase-4 (DPP-4) inhibitor class of drugs. This enzyme-inhibiting drug is to be used either alone or in combination with metformin or a thiazolidinedione for control of type 2 diabetes mellitus. The drug works to competitively inhibit a protein/enzyme, dipeptidyl peptidase 4 (DPP-4), that results in an increased amount of active incretins (GLP-1 and GIP), reduced amount of release of glucagon (diminishes its release) and increased release of insulin.
Fig. 1: structural formula of Sitagliptin
Stability testing forms an important part of the process of drug product development. The purpose of stability testing is to provide evidence on how the quality of a drug substance or drug product varies with time under a variety of environmental conditions, for example temperature, humidity, and light, which enables storage conditions, retest periods, and shelf life to be recommended.The two main aspects of study of the stability of a drug product that play an important role in shelf life determinations are assay of the active drug and the degradation products generated during stability studies. Assay of a drug product in a stability test sample must be performed with stability?indicating method, as recommended by the International Conference on Harmonization (ICH).
A literature survey revealed that very few methods3,4,5,6,7,8 were developed and none of the reported procedures enables analysis of the Sitagliptin in pharmaceutical dosage forms in the presence of their degradation products. This manuscript describes the development and validation, in accordance with ICH guidelines,ofarapid,economical, precise, and accurate stability?indicatingisocratic RP?HPLC method for analysis of Sitagliptininthepresence of its degradation products.
MATERIALS AND METHODS
Chemicals and solutions
An analytically pure sample of Sitagliptin (purity 99.7%) wasprocured as gift sample from Granules pharma (Hyderabad, India) and Tablet formulation [JANUVIA (Brand name), Granules pharma, Hyderabad, India] was procured from a localpharmacy with labelled amount 50mg. Acetonitrile and Methanol (HPLC grade) were obtained from Merck Fine Chemicals(Mumbai, India), Potassium Di-hydrogen phosphate (AR grade) from Loba chemicals ( Mumbai, India), sodium-hydroxide (NaOH), hydrochloric acid (HCl), and hydrogen peroxide(H202) were from Qualigens Fine Chemicals (Glaxo, Mumbai, India).The 0.45?μm Nylon pump filter was obtained from AdvancedMicrodevices (AmbalaCantt., India). Doubledistilled water was usedthroughout the experiment. Other chemicals used were of analyticalor HPLC grade.
Preparation of standard solutions
Accurately weigh and transfer 10 mg of SitagliptinWorkingstandard into a 100 ml volumetric flask, add about 50 ml of Methanol (Diluent)andsonicate to dissolve it completely and make volume up to themark with the same solvent (100μg/ml, Stock solution). Furtherpipette 1 ml of the above stock solution into a 10ml volumetric flaskand dilute up to the mark with Mobile phase (diluent), it was 10μg/ml. Standardcalibration solutions (10–60μg/ ml) for assessment of linearitywere prepared from this stock solution by dilution with suitablediluent.
Preparation of sample solutions
The commercially available Tablet contains Sitagliptin (100mg). 20 tablets were weighed individually and finely powdered. A powder blend equivalent to 10mg of SIT was transferred to a 10mL volumetric flask containing 10 ml of theMethanol(diluent) and sonicated for 5min. and then final solution was made with diluent to get thesolution of 100μg/ml From this solution, 1 ml was taken in 100ml standard volumetric flask and diluted to 100 ml withMobile phase(diluent)togive a solution of 10μg/ml From this stock solution, variousdilutions of solution were prepared and analysed.
The liquid chromatographic system consisted of followingcomponents: Shimadzu HPLC model containing LC?20AT (VP series)pump, variable wavelength programmable UV/ VIS detector SPD?20A (VP series) and Hamilton syringe (705NR,50μl).Chromatographic analysis was performed using Empower -2 software on a Hypersil–BDS-C18 column with 250 x 4.6 mmi.d, 5μm particle size.The optimized mobile phase was consisted of 10mM Phosphate buffer (PH-3.5): ACN with 60:40%v/v .The flow rate was 1.2ml / min. Detection wavelength 260 nm was selected by scanning drug over a wide range of wavelength 200 nm to 400 nm in spectrophotometer. The 20μl sample was injected and the total run time was 6 min. Chromatogram showed peak of Sitagliptinat retention time of 2.157 ± 0.02 min.
Forced degradation study
To study the effect of acid, approximately 100 mg Sitagliptinwas accurately weighed and dissolved in 25 ml of 1M hydrochloric acid (HCl) and refluxed for 70°C for approximately 2 hr in water bath. The solution was then left to reach ambient temperature, and neutralized to pH 3.5 by addition of 1M sodium hydroxide( NaOH) then diluted to 50 ml with diluent (Mobile Phase) from this solution target concentration was prepared and injected.
To study the effect of alkali, approximately 100 mg Sitagliptin was accurately weighed and dissolved in 25 ml of 1M sodium hydroxide(NaOH) and refluxed for 70°C for approximately 2 hr in water bath. The solution was then left to reach ambient temperature, and neutralized to pH 7 by addition of 1M hydrochloricacid (HCl) then diluted to 50 ml with diluent (Mobile Phase) from this solution target concentration was prepared and injected.
To study the effect of oxidising conditions, approximately 50 mgSitagliptin was accurately weighed and dissolved in 25 ml of30% H2O2 and refluxed for 40°C for approximately 2 hr in waterbath. The solution was then left to reach ambient temperature, anddiluted to 50 ml with diluent (Mobile Phase) from this solutiontarget concentration was prepared and injected.
To study the effect of photolytic effect, approximately 50mgSitagliptin was accurately weighed and exposed to UV light for 24 hours. Then the drug substance was made to 100ug/ml with diluent (mobile phase) and refluxed for 70°C for approximately 2 hrinwater bath. The solution was then left to reach ambient temperature, from this solutiontarget concentration was prepared and injected.
To study the effect of temperature, approximately 50 mgSitagliptin was stored at 80°C for 48 hr, then dissolved in fewml of diluent and sonicate for 5min then diluted to 50 ml withdiluent (Mobile Phase) from this solution target concentration wasprepared and injected.
The method was validated according to International Conference on Harmonization 9,10guidelines for validation of analyticalprocedures.
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