Human adipose tissue pretreatment method development for tocopherols’ and tocotrienols’ targeted metabolomics in breast cancer disease

The “vitamin E” family consists primarily of four naturally occurring tocopherols (alpha-, beta-, gammaand delta-) and four unsaturated corresponding tocotrienols, which differ in their structure by the composition of methyl groups attached to the chromanol ring. Apart from their potent antioxidant activity, it is hypothesized that some of these compounds might play protective role against cancer diseases, for instance breast cancer and prostate cancer. In the field of cancer prevention, developing metabolomic approach is one to be considered in potential biomarkers’ research. For this reason, targeted metabolomic analysis is required for the evaluation of tocochromanols’ utility in breast cancer prevention. The aim of this study was development of a selective and rapid method for isolation of tocopherols and tocotrienols from human adipose tissue. After mechanical extraction with isopropanol/ethanol mixture (5:1) with addition of BHT and in the presence of internal standard (deuterated alpha-tocopherol), samples were centrifuged in 4°C and the supernatant was collected. Then samples were diluted with water and solid-phase extraction was performed. For this purpose, polymeric SPE tubes were used (Bond Elut Plexa, 30 mg, 1 ml, Agilent Technologies). After samples application, SPE bed was purified with 80% methanol solution and target analytes were eluted with isopropanol. Finally, extracts were evaporated to dryness and residues were dissolved in 100 μl of 0.04% BHT in ethanol. Tocopherols’ and tocotrienols’ contents in human adipose tissue were determined with the use of LC-APCI-MS/MS. The chromatographic conditions were as follows: 3.0×100 mm Cosmosil 2.5π-NAP column (Nacalai Tesque, Japan) with the mobile phase methanol/water (9:1, v/v) in an isocratic flow. Mass spectrometry experiments were performed with multiple reaction monitoring (MRM) in the positive polarization mode. The developed pretreatment method is efficient for tocochromanols’ determination in human adipose tissue.