γ tocopherol

CO₂ and N₂ were obtained from Gaschema (Jonava, Lithuania); α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, β-carotene, and hexane were obtained from Sigma-Aldrich (Steinheim, Germany).

Albumax I and RPMI-1640 were purchased from Fisher Scientific^® (Leicestershire, UK). All solvents used were HPLC-grade or higher quality and purchased from Sigma-Aldrich (St. Louis, Missouri USA). Radiolabeled isoprenic precursors [1-(n)-³H] geranylgeranyl pyrophosphate triammonium salt {[1-(n)-³H]-GGPP; 14 Ci/mmol}, [1-(n)-³H] phytol (20 Ci/mmol), and {[1-(n)-³H] phytyl-PP ([³H]-phytyl-PP; 20 Ci/mmol) were obtained from Amersham-Pharmacia Biotech (Buckinghamshire, UK). Adenosyl-L-methionine, S-[methyl-³H] ([³H]-SAM, 82 Ci/mmol) and [1-(n)-³H] FPP triammonium salt ([³H]-FPP 23 Ci/mmol) were purchased from Perkin Elmer^® (Waltham, Massachusetts, EUA). PK, α-tocopherol, γ-tocopherol, phytol, phytyl-PP, GGPP, UQ-8, UQ-9 and MK-4 pure standards were purchased from Sigma-Aldrich. Saponin, hypoxanthine, gentamycin sulfate, D-sorbitol, glucose, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and another reagent not cited here were also purchased from Sigma-Aldrich.

Methanol (MeOH), ethanol (EtOH), and acetonitrile (ACN), HPLC grade, and diethyl ether and petroleum ether, analytical grade, were purchased from Fischer Scientific (Geel, Belgium). 2-propanol and acetone, HPLC grade, were purchased from Honeywell (Offenbach, Germany). All the standard compounds (lutein, chlorophyll A 87%, β-carotene, α-tocopherol, γ-tocopherol, δ-tocopherol, and squalene) were purchased from Sigma Aldrich (Stenheim, Germany). Stock solutions containing 1000 mg/L of each analyte were prepared in methanol for lutein, in ethanol for β-carotene and tocopherols, in diethyl ether for squalene, and in acetone for chlorophyll, and stored at −20 °C during the study. Intermediate standard solutions of 100–500 mg/L of each compound were prepared in 2-propanol.
Mixed working standard solutions were prepared from the intermediate solutions, in order to construct the calibration curves of the analytes used for quantification. For the optimization of the method, solid phase extraction (SPE) Strata Silica 1 g/6 mL cartridges (55 μm, 70 Å) were purchased from Phenomenex (California, USA). All samples were filtered using Chromafil Regenerated Cellulose (RC) syringe filters, 0.22 μm, purchased from Macherey-Nagel (Düren, Germany) prior to injection.