Development and Validation of Method to Quantify Piperine in Black Pepper Seed

Abstract

This study describes the development and validation of a rapid, highly sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the precise isolation and quantification of piperine from black pepper (Piper nigrum L.) seeds. The extraction process was optimized by subjecting 10g of finely ground black pepper seed powder to exhaustive solid-liquid extraction using 150mL of 95% ethanol in a Soxhlet apparatus for 3 hours. The resulting crude extract was filtered, concentrated on a water bath at 80°C, and treated with 10 mL of a 10% potassium hydroxide KOH solution under continuous stirring to precipitate and remove interfering resinous components. The remaining alcoholic extract was kept overnight to yield highpurity piperine crystals via selective filtration. Chromatographic separation of the isolated analyte was achieved on an analytical C18 column maintained at 40°C using an isocratic mobile phase of acetonitrile and water (50:50) supplemented with 0.1% formic acid. Delivered at a flow rate of 0.7mL/min with a 10μL injection volume, the total analytical runtime was optimized to just 7 minutes. Mass spectrometric detection was operated in positive electrospray ionization mode (ESI+), targeting the protonated molecular precursor ion of piperine ([M+H]+ at m/z 286.1). Under an optimized collision energy of 25V, collision-induced dissociation consistently generated two highly specific diagnostic daughter product ions at m/z 201.0 and m/z 134.9 for definitive quantitative identification via Multiple Reaction Monitoring. The correlation coefficient R2 was obtained 0.999.