Dispersive liquid-liquid microextraction for the determination of vitamins D and K in foods by liquid chromatography with diode-array and atmospheric pressure chemical ionization-mass spectrometry detection.
A simple and rapid method was developed using reversed-phase liquid chromatography (LC) with both diode array (DAD) and atmospheric pressure chemical ionization mass spectrometric (APCI-MS) detection, for the simultaneous analysis of the vitamins ergocalciferol (D2), cholecalciferol (D3), phylloquinone (K1), menaquinone-4 (K2) and a synthetic form of vitamin K, menadione (K3). The Taguchi experimental method, an orthogonal array design (OAD), was used to optimize an efficient and clean preconcentration step based on dispersive liquid-liquid microextraction (DLLME). A factorial design was applied with six factors and three levels for each factor, namely, carbon tetrachloride volume, methanol volume, aqueous sample volume, pH of sample, sodium chloride concentration and time of the centrifugation step. The DLLME optimized procedure consisted of rapidly injecting 3 mL of acetonitrile (disperser solvent) containing 150 µL carbon tetrachloride (extraction solvent) into the aqueous sample, thereby forming a cloudy solution. Phase separation was performed by centrifugation, and the sedimented phase was evaporated with nitrogen, reconstituted with 50 µL of acetonitrile, and injected. The LC analyses were carried out using a mobile phase composed of acetonitrile, 2-propanol and water, under gradient elution. Quantification was carried out by the standard additions method. The APCI-MS spectra, in combination with UV spectra, permitted the correct identification of compounds in the food samples. The method was validated according to international guidelines and using a certified reference material. The validated method was applied for the analysis of vitamins D and K in infant foods and several green vegetables. There was little variability in the forms of vitamin K present in vegetables, with the most abundant vitamer in all the samples being phylloquinone, while menadione could not be detected. Conversely, cholecalciferol, which is present in food of animal origin, was the main form in infant foods, while ergocalciferol was not detected.
Department of Analytical Chemistry, Faculty of Chemistry, Regional Campus of International Excellence "Campus Mare Nostrum", University of Murcia, E-30071 Murcia, Spain.
Pilar
P
Viñas
María
M
Bravo-Bravo
Ignacio
I
López-García
Manuel
M
Hernández-Córdoba
D019840:2-Propanol; D000097:Acetonitriles; D002251:Carbon Tetrachloride; D002762:Cholecalciferol / Q000032:analysis*; D056148:Chromatography, Reverse-Phase; D004872:Ergocalciferols / Q000032:analysis*; D005163:Factor Analysis, Statistical; D005504:Food Analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D041943:Infant Formula / Q000737:chemistry*; D059627:Liquid Phase Microextraction / Q000379:methods; D000432:Methanol; D012015:Reference Standards; D012965:Sodium Chloride; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors; D014675:Vegetables / Q000737:chemistry*; D010837:Vitamin K 1 / Q000032:analysis*; D024482:Vitamin K 2 / Q000032:analysis*; D024483:Vitamin K 3 / Q000032:analysis*; D014867:Water
D000097:Acetonitriles; D004872:Ergocalciferols; D014867:Water; D024482:Vitamin K 2; D002762:Cholecalciferol; D012965:Sodium Chloride; D024483:Vitamin K 3; D010837:Vitamin K 1; D002251:Carbon Tetrachloride; D019840:2-Propanol; D000432:Methanol; C032159:acetonitrile
Dispersive liquid–liquid microextraction; Foods; LC–APCI-MS; Vitamin D (ergocalciferol (D(2)), Cholecalciferol (D(3))); Vitamin K (phylloquinone (K(1)), Menaquinone 4 (K(2)), Menadione (K(3)))