Bioanalytical method development validation and in vtro stability studies of ddp 4 inhibitors by different analytical tools

Abstract

quotObjective: To develop and validate a rapid, simple, accurate, precise Liquid Chromatography Mass Spectroscopic [LC-MS] methods for Gemigliptin Tartarate Sesquihydrate and Teneligliptin Hydrobromide Hydrate newlinePART-I newlineExperimental Work: A simple, rapid and sensitive liquid chromatographic-tandem mass spectrometric method equipped with Turbo Ion Spray (TIS) source, operating in the Positive ion and Multiple Reaction Monitoring (MRM) mode was devised to quantify Gemigliptin Tartarate Sesquihydrate in human plasma. A superior procedure of protein precipitation (compared to Solid Phase extraction (SPE) and liquid-liquid Extraction) was followed to extract analyte and ISTD from human plasma. LC-MS method was carried out by using Phenomenex Gemini C18 (3 µm, 30.0 × 2.0 mm) column and 0.1% Formic acid in 10 mM Ammonium acetate in water: Acetonitrile (10:90 % v/v) as mobile phase at 0.5 ml/min flow rate. The retention times of Gemigliptin and Sitagliptin (Internal Standard) were 3.29 min and 4.22 min and the total run time was 5.5 min. Monitoring of the fragmentation of m/z 490.160 and#8594; 338.100 and 408.142 and#8594; 235.000 performed during MS/MS detection of Gemigliptin and Internal Standard (IS) on the mass spectrometer. After optimization, the method was validated according to USFDA and EMEA guidelines for Bioanalysis. newline newlineResults and Discussion: Sample preparation was performed using Protein Precipitation Method and this method yielded extremely clean extracts with very good recovery of 84.84 % and 89.36 % for drug and ISTD respectively. The response of the LC-MS/MS method for analyte was linear over the dynamic range of 1 2000 ng/ml with correlation coefficient r2 and#8805; 0.998. The Accuracy was well within the accepted limit of ±20% at lower limit of quantification (LLOQ) and ±15% at all the other concentrations in the linear range. This method was fully validated for the Accuracy, Precision and Stability studies. newlinePART-II newlineExperimental Work: A simple, rapid and sensitive liquid chromatographic-tandem mass spectrometric method equipped with