Supercritical Fluid Chromatography with Photodiode Array Detection in the Determination of Fat-Soluble Vitamins in Hemp Seed Oil and Waste Fish Oil
In the presented study for the first time a new, optimized, fast SFC (supercritical fluid chromatography) method was applied to separate in one run fat-soluble vitamins from waste fish oil, including cis-and trans-retinyl palmitate, cisand trans-retinyl acetate, retinol, α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, ergocalciferol (D 2 ), cholecalciferol (D 3 ), cisand trans-phylloquinone (K 1 ) and menaquinone-4 (K 2 -MK4). Vitamins were baseline separated on an Acquity UPC 2 (ultra
... quity UPC 2 (ultra performance convergence chromatography) HSS C18 SB (highly strength chemically modified silica) column within 13 min. The influence of the stationary phase, such as Torus 1-AA (1-aminoanthracene), Torus Diol (high density diol), Torus DEA (diethylamine), BEH (silica with no bonding), BEH-2EP (2-ethylpirydine), CSH Fluoro-Phenyl (silica with fluoro-phenyl groups), column temperature, flow rate and back pressure on the separation of the compounds was described. The application of the modified saponification procedure allowed us to increase concentration in the sample prepared for the analysis of γ-tocopherol from less than 1% (wt %) to 14% for the first time. In addition, α-tocopherol, γ-tocopherol, δ-tocopherol and retinol were identified in waste fish oil. Vitamin purification and analysis in waste fish oil are reported for the first time here. Due to the short time and effectiveness of the proposed method, it can be easily applied in industrial processes. The vitamin content in fish oil may vary depending on the species as well as the part of the fish. According to Stancheva , the fish oil constituted retinol and α-tocopherol in the amount of 9-142 µg and 308-1113 µg per 100 g of the raw tissue, respectively, in four different fish species. However, the literature lacks information on the content of fat-soluble vitamins in waste fish oil. The analysis of vitamins is a challenging task in many ways. Firstly, the vitamins are sensitive to heat, light, oxygen and other factors causing their isomerization. Moreover, unstable polyene chains in the vitamin structures increase their degradation    . On the other hand, fat-soluble and water-soluble vitamins constitute a group of compounds with different chemical structures and characteristics, which may be used while performing separation  . Another aspect is that SFC methods are performed at subcritical conditions, but also with the addition of co-solvent and low temperatures. Often, the mobile phase is intentionally kept at subcritical conditions, in which the properties of the mobile phase are continuous and satisfactory separation is obtained [12, 13] . Various separation techniques have been applied in the analysis of vitamins in foods, vegetables, and biological samples. It was proved by a number of researchers that SFC is as effective as other analytical techniques in vitamin separation. In analytical studies by Galuba , the content of tocopherols (α-, γ-, δ-) analyzed by the HPLC (high performance liquid chromatography) and SFC (supercritical fluid chromatography) method was comparable. An increased number of applications in the pharmaceutical industry concerns the separation of isomers by SFC  . In previous studies, supercritical fluid chromatography equipped with different detectors was applied to separate tocopherols and tocotrienols    , vitamin A and carotenoids [9,10,19,20], vitamin D [21,22] and vitamin K [15, 23] , as well as the mixture of fat-soluble vitamins [24, 25] and water-soluble vitamins  . Thus far, there has been a lack of an efficient and fast methods for the one-step separation of a higher number of vitamins. The aim of this study is to propose a new efficient method for the separation of fourteen fat-soluble vitamins with the use of supercritical fluid chromatography. The optimization includes the influence of different parameters, such as back pressure, temperature, flow rate and stationary phase on the separation of analytes. Moreover, the group of mentioned vitamins are to be purified and quantitated in hemp seed oil, and in the case of waste fish oil are to be identified and purified for the first time. Materials and Methods Standards and Materials Authentic analytical standards, retinol, retinyl palmitate (cis-and trans-isomer), retinyl acetate (cisand trans-isomer), α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, cis-phylloquinone (cis-K 1 ), trans-phylloquinone (trans-K 1 ), menaquinone-4 (K 2 -MK4), cholecalciferol (D 3 ) and ergocalciferol (D 2 ) were purchased from Sigma-Aldrich (Poznań, Poland). Vitamin D compounds and β-tocopherol were received from Sigma-Aldrich as standard solutions, 1 mg/mL in ethanol and 50 mg/mL in hexane, respectively. According to standard certificates, retinyl acetate as well as retinyl palmitate and phylloquinone (K 1 ) contain 5% of cis-isomer. As for the materials for separation and analyses, commercially-available, cold-pressed hemp seed oil was used as well as the waste fish oil delivered by the A-SENSE company (Puławy, Poland). Each standard solution was injected alone to measure the corresponding retention time and UV spectrum (Sigma-Aldrich, Poznań, Poland).