Simultaneous Determination of Nine Water and Fat Soluble Vitamins After SPE Separation and RP-HPLC Analysis in Pharmaceutical Preparations and Biological Fluids

Abstract

An automated reversed phase high performance liquid-chromatography (RP-HPLC) method is described, for the simultaneous analysis of water soluble [ascorbic acid (C), nicotinic acid, nicotinamide, folic acid, cyanocobalamine (B12), and riboflavin (B2)] and fat soluble (retinol, α-tocopherol, α-tocopherol acetate) vitamins. The compounds are separated after solid-phase extraction (SPE) on C18 cartridges, where water soluble vitamins pass unretained, while fat soluble vitamins are retained on the sorbent. After isolation of the two fractions, water soluble vitamins are separated on a Lichrosorb RP-18 250x4.0 mm, 5 μm analytical column, using a gradient elution system consisting of CH3OH-0.05 M CH3COONH4 (5:95 v/v) changing to (30:70 v/v) over a period of 20 min at a flow rate 1 mL/min. Fat soluble vitamins are separated on a Spherisorb RP-18 220x4.6 mm, 5 μm analytical column with an isocratic mobile phase consisting of CH3OH-CH3CN (30:70 v/v) at a flow rate of 1.5 mL/min. A UV-vis detector operated at 270 nm and 290 nm for water soluble and fat soluble vitamins, respectively, is used for detection and quantitation of the analytes. Xanthine is used as internal standard at a concentration of 4.2 ng/L for water soluble vitamins, while anthraquinone is used as internal standard at a concentration of 3.5 ng/mL for fat soluble vitamins. Linearity is observed up to 10 ng/mL for ascorbic acid, folic acid, and riboflavin, up to 15 ng/mL for nicotinic acid, nicotinamide, and cyanocobalamine, while up to 20 ng/mL for all fat soluble vitamins. Limits of detection ranged from 2.5 - 5.0 ng for water soluble vitamins and 2.0 - 5.0 ng for fat soluble vitamins. Method's validation is achieved in terms of day-to-day and within-day reproducibility studies. Long term stability study is conducted during routine operation of the system over a period of ten consecutive days. The developed method is applied to the analysis of pharmaceutical preparations: (tablets, injection solutions) and biological fluids: (blood serum, urine). SPE technique is used for the isolation of the vitamins from the matrix of human biological fluids: blood serum (40 μL) and urine (100 μL). High extraction recoveries are achieved using Merck Lichrolut RP-18 cartridges.