Abstract

DEVELOPMENT AND VALIDATION OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY MASS SPECTROMETRIC METHOD FOR ESTIMATION OF DEXMETHYLPHENIDATE FROM HUMAN PLASMA USING LIQUID-LIQUID EXTRACTION

Malini Sharma* and N.B. Sridharamurthy

ABSTRACT

A novel, simple, specific, sensitive and reproducible liquid chromatography mass Spectrometric (LC/MS-MS) assay method has been developed and validated for estimation of Dexmethylphenidate in human plasma biological matrix. The LC/MS-MS method includes use of D Threo-Methylphenidate-D10 as an internal standard (IS). Samples were extracted using liquid-liquid extraction methodology. The detection was performed using API 3200 (AB Sciex) and PC based data system with Analyst 1.4.2 software. Chromatographic separation was achieved on ZORBAX× SB -C8, 100×4.6mm, 3.5μm column using isocratic mobile phase composition with (5 mM Ammonium acetate Solution with 0.1 % Formic Acid: Acetonitrile: 7:3 V/V) at a flow rate of 0.8 mL/min at 40ºC column oven temperature, without splitter over a total run time of 3 min. Method validation was performed as per USFDA guidelines and the results met the pre-defined acceptance criteria. The calibration curve was linear over a concentration range of 0.205 to 80.195 ng/mL for Dexmethylphenidate (using D Threo-Methylphenidate-D10 as internal standard) with correlation value r2 ≥ 0.995. Interbatch quality control samples accuracy for Dexmethylphenidate ranged from 98.47 to 103.94% with inter-batch precision values of 0.98% to 4.78% during the course of validation, demonstrating acceptable assay accuracy and precision. Further experiments like; Matrix Effect, Bench-Top, Freeze-Thaw, Dry extract, Wet extract, Effect of Haemolysis, anticoagulant, Lipemic plasma, etc were conducted to assess the suitability of proposed method for intended usage. Method was found to be suitable for quantitation of Dexmethylphenidate clinical study samples.

Keywords: Dexmethylphenidate; LCMS-MS; Human plasma, Method validation.