Lichrospher si60 column

Sample preparation for the analysis of tocopherols in extra virgin olive oil was performed prior to injection to the LiChrospher Si60 column (inner diameter 25 cm, length 4.6 mm and particle size 5 µm) (Merck, Darmstadt, Germany), fitted in an Agilent 1200 high performance liquid chromatography (HPLC) system equipped with a fluorescent detector (Agilent Technologies, Santa Clara, CA, USA). The sample preparation method is herein: 0.15 g of olive oil was diluted in 10 mL of n-hexane followed by centrifugation with a rotor speed of 25,000× g for 10 min in a laboratory cold centrifuge (HERMLE Labortechnik, Wehingen, Germany). The supernatant was transferred to a chromatographic vial and 20 µL of the sample was injected into the HPLC system. Chromatographic separation was performed by the isocratic mixture of isopropanol:hexane 0.5:99.5 (v/v) mobile phase, operated with a 0.7 mL·min⁻¹ flow rate. The fluorescence detector was set to excitation and emission wavelengths of 296 nm and 330 nm, respectively. Peaks were identified on the basis of the retention times of the standards α-, β-, γ- and δ-tocopherol separately and their concentrations were calculated using respective external calibration curves [23 (link)].

Tocopherol and tocotrienol contents
were determined according to the method published in ref (48 (link)). 400 μL of n-heptane was added to 2 mL Eppendorf tubes containing a
frozen leaf disk and glass beads (kept on a precooled aluminum box),
before grinding the leaf material in a Geno Grinder (SPEX CertiPrep)
for 3 min at 1700 strokes min^–1. Eppendorf tubes
containing the extracts were kept for one night in a −20 °C
freezer. The following day, samples were vortexed and centrifuged
for 10 min at 16,000 g (Biofuge Fresco), before 100 μL of the
extract was added to a microvial for HPLC analysis. Chromatographic
analysis of tocopherols was done by injecting 20 μL of extract
into a Shimadzu HPLC system equipped with an RF-10A XL fluorescence
detector, 10-series (Shimadzu Corporation, Kyoto, Japan). Tocopherol
separation was obtained using a LiChrospher Si 60 column (5 μm/250
x 4 mm, Merck, Darmstadt, Germany) and an isocratic system with a
flow of 1 mLmin^–1 of the eluent [n-heptane and isopropanol (99/1, v/v)]. Four different kinds of tocopherols
and tocotrienols (α-, β-, γ-, and δ-tocopherol,
respectively) were detected by their fluorescence at 328 nm after
excitation at 290 nm. Peaks were identified based on their retention
time in comparison with standards (Merck KGaA, Darmstadt, Germany).
Tocopherol standards were measured at the beginning, after every five
samples, and at the end of the analysis.

The content of tocols was determined according to the procedure described by Mikołajczak et al. [40 (link)]. The oil sample was diluted in n-hexane and centrifuged on an Eppendorf Centrifuge 5417R type (Eppendorf AG, Hamburg, Germany) for 10 min (16,000 rpm). The resultant solution was analyzed using an HPLC Agilent Technologies 1200 chromatograph (Santa Clara, CA, USA) equipped with a fluorescence detector of the same company and a LiChrospher Si 60 column (250 mm × 4 mm × 5 μm, Merck, Darmstadt, Germany). A 0.7% solution of iso-propanol in n-hexane was used as the mobile phase at a flow rate of 1 mL/min. The fluorescence detector was set at excitation and emission wavelengths of 296 nm and 330 nm, respectively. The content of tocols was determined from calibration curves prepared for tocopherol standards. The repeatability for determining tocopherol contents was 2.5% (coefficient of variation). The LOQs were 0.45, 0.4, and 0.2 μg/g of the sample for α-, γ-, and δ-tocopherol, respectively. The linearity of the calibration curves was confirmed in the range of 0.02–16 mg/L.