Determination of benfothiamine in nutraceuticals using dispersive liquid-liquid microextraction coupled to liquid chromatography

This paper describes a dispersive liquid-liquid microextraction (DLLME) procedure coupled to liquid chromatography (LC) with fluorimetric detection for the determination of benfothiamine (BT). First, the sample was subjected to an enzymatic hydrolysis step using alkaline phosphatase. Then, a derivatization by chemical oxidation with ferricyanide at pH 13 to form fluorescent thiochrome was carried out. For DLLME, 0.5 mL of acetonitrile (disperser solvent) containing 90 mu L of 1,1,2,2-tetrachloroethane (extraction solvent) was rapidly injected into 10 mL of a sample solution containing the derivatized thiochrome and 24% (w/v) sodium chloride, thereby forming a cloudy solution. Phase separation was carried out by centrifugation, and a volume of 20 mu L of the sedimented phase was submitted to LC. The mobile phase was a mixture of a 90% (v/v) 10 mM KH2PO4 (pH 7) solution and 10% (v/v) acetonitrile at 1 mL min(-1). An amide-based stationary phase involving a ligand with amide groups and trimethylsilyl endcapping was used. The calibration graph was obtained using aqueous standards (correlation coefficient 0.9998). The limit of detection was 0.8 ng mL(-1). The selectivity of the method was judged from the absence of interfering peaks at the BT elution time for blank chromatograms. A relative standard deviation of 2.8% was obtained from a series of ten consecutive DLLME-LC analyses of an aqueous standard solution containing 10 ng mL(-1) BT. The method was applied to the determination of benfothiamine in different nutraceuticals. The main advantage of the method is that, while direct injection of thiochrome cannot be carried out due to the high alkalinity of the extract, the use of DLLME provides a non-corrosive extract of BT and permits a lower detection limit as the preconcentration factor was close to 250. This is the first time that BT has been determined using a clean, environmentally friendly procedure.