Hitesh Shah, Shivan, Puthli
Jul 3, 2013
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Abstract
Introduction: Chemically, Darifenacin hydrobromide is (S)-2-{1-[2-(2,3-dihydrobenzofuran-5yl)ethyl]-3-pyrrolidinyl}2, 2diphenylacetamide hydrobromide. The empirical formula of Darifenacin hydrobromide is C28H30N2O2.HBr. Darifenacin is a muscarinic antagonist indicated for the treatment of overactive bladder with symptoms of urge urinary incontinence, urgency and frequency. Darifenacin has greater affinity for the M3 receptor. M3 receptors are involved in contraction of human bladder and gastrointestinal smooth muscle, saliva production, and iris sphincter function. A novel high performance liquid chromatography using positive ion APCI ionization Tandem Mass Spectrometry method was developed and validated for quantification of Darifenacin in Human plasma. Method: The analyte in the plasma was extracted using Solid Phase Extraction method. The analyte was separated using an binary mobile phase on a reserve phase column to meet the demands of the clinical laboratory for speed of analysis and chromatographic resolution. Detection was carried out by MS/MS in the multiple reaction monitoring mode using the respective (M+H)+ ions Q1 m/z 427.3 Q3 m/z 14.6 and Q1 m/z 431.3 Q3 m/z 151.1 for analyte and internal standard. The developed method was validated. Results: The assay exhibited a linear dynamic range of 0.102-15.066ng/ml for Darifenacin in human plasma. The lower of quantification was 0.102 ng/ml with relative standard deviation of less than 13.03 %. No interference by endogenous substances or matrix effect was observed. The intra-day and inter-day accuracy and precision (% CV) values were in the range of 91.76-107.72% (2.69-8.96%) and 91.18-105.57% (3.84-9.01%) respectively. The spiked plasma samples were found to be stable even after 3 freezethaw cycles -700C & -200C. The processed plasma samples were found to be stable in autosampler for 30 hrs at 10°C. A short run time 3.0 min for each sample made it a high throughput method for estimation of clinical samples. The developed and validated method for estimation of Troxipide in human plasma was effectively used to determine plasma concentration profiles in human subjects.