Salting-out Assisted Liquid-Liquid Extraction for Analysis of Caffeine and Nicotinic Acid in Coffee by HPLC-UV/Vis Detector

A salting-out assisted liquid-liquid extraction method followed by high performance liquid chromatography with ultraviolet-visible detector (SALLE-HPLC-UV/Vis) has been proposed for determination of caffeine and nicotinic acid in raw and roasted coffee samples. Various parameters affecting chromatographic separations including mobile phase composition, injection volume, flow rate and column temperature were studied. Experimental parameters affecting the extraction efficiency of SALLE method such as type and volume of the organic solvent, type and amount of salt, pH of the sample, and concentration of the ion-pairing reagent were also studied and thereby optimum conditions were established. The calibration curves which were constructed at five different concentration levels using the optimum conditions exhibited good linearity, with the coefficient of determination (R-2) 0.999 and 0.995 for caffeine and nicotinic acid, respectively. The limits of detection (LOD) and quantification (LOQ) which were determined at 3 and 10 times signal-to-noise ratio were 0.05 and 0.13 mg/L for nicotinic acid and 0.20 and 0.63 mg/L for caffeine, respectively. Intra- and inter-day precision studies were demonstrated satisfactory precision with RSD values below 10. Relative recoveries were also studied, and their results were ranging from 82-122% for both raw and roasted coffee samples. The findings demonstrated that the proposed method could be used as an effective and best alternative for the determination of caffeine and nicotinic acid in raw and roasted coffee samples.