Development of HPLC Method for Determination of Sitagliptin in Human Plasma using Fluorescence Detector by Experimental Design Approach

Abstract

This study describes a rapid and sensitive HPLC method using fluorescence detection, which enables the determination of Sitagliptin mono phosphate (SIT) and Itopride Hydrochloride (ITP) as internal standard with good accuracy and precision to allow its detection in human plasma. SIT and ITP were extracted by liquid liquid extraction (LLE) using ethyl acetate. All the factors were studied to optimize the chromatographic conditions using full factorial design and central composite design for screening and optimization design respectively. Chromatographic separation was achieved using C18 column (250 mm, 4.6 mm,5 μm), and mobile phase composed of 0.01M phosphate buffer (pH 4.5) and acetonitrile in a ratio 73:27 pumped at flow rate 1 mL/min and detected at 267 nm and 310 nm for excitation and emission. The method was validated over a concentration range 0.1-3 μg/mL for SIT using FDA guidelines. The intraday and inter-day precision did not exceed 5% from the nominal concentration and accuracy was within 90-105% at all quality control levels. The validated method was applied successfully to human plasma samples.