Cyclohexane

Fat was extracted with a ternary mixture of cyclohexane, 2-propanol and aqueous NaCl solution (0.9%, w/v), enabling a fast and clean separation of the lipid phase (Smedes, 1999 , Cruz et al., 2013 , Santos et al., 2015 ). In brief, 500 mg of homogenized sample were extracted with 1.6 mL of 2-propanol and 2 mL of cyclohexane (analytical grade from Carl Roth GmBH, Germany) after addition of an internal standard for total fat estimation (glyceryl triundecanoate, Sigma–Aldrich, Spain). After vortex mixing and overnight maceration at 4 °C, aqueous NaCl was added (1%; 2.8 mL), thoroughly mixed and centrifuged at 5000 rpm for 5 min, and the upper phase was transferred to derivatization vials. After repeating extraction with further 2 mL of cyclohexane, the combined supernatants were evaporated under a stream of nitrogen at 60 °C.

Cyclohexane (tech. 99.5%) and ammonia water (p.a., 25 wt % NH₃) were purchased from Roth. Oleic acid (OA, 90%), erbium chloride hexahydrate (ErCl₃·6H₂O, 99.9%), ytterbium chloride hexahydrate (YbCl₃·6H₂O, 99.9%) and yttrium chloride hexahydrate (YCl₃·6H₂O, 99.9%) were received from ABCR. Sodium hydroxide (NaOH, 99%) was obtained from Grüssing, Ethanol (EtOH, 100%) from Berkel AHK and hydrofluoric acid (HF, 30%) from Riedel de Haën. Polyoxyethylene (5) nonylphenyl ether (Igepal^® CO-520), ammonium fluoride (NH₄F, 99.8%), 1-octadecene (tech. 95%), sodium oleate (82%), tetraethyl orthosilicate (TEOS, 98%) as well as yttrium-, ytterbium- and erbium standards for inductively coupled plasma optical emission spectroscopy (ICP-OES) measurements (TraceCERT^®, c = 1000 mg/mL) were purchased from Sigma Aldrich. All chemicals were used without further purification.

Concentrations of pyruvate were quantified using high-performance liquid chromatography (HPLC; Shimadzu Prominence, Kyoto, Japan) equipped with an Aminex HPX 87H column (Bio Rad, Vienna, Austria) and an SPD-M10A VP photo-diode array detector (flow rate 0.6 mL min⁻¹; oven at 40 °C, 5 mM H₂SO₄ as eluent).
For n-hexadecane quantification, deuterated n-hexadecane (D34, Sigma-Aldrich, St. Louis, MO, USA) was added to the samples as internal standard (20 mg L⁻¹) before extracting the entire vial using 9 mL cyclohexane (purity 99.9%, Carl ROTH, Karlsruhe, Germany). Vials were shaken at 300 rpm for half an hour, phases were allowed to separate overnight, and subsamples of the cyclohexane were used to quantify the remaining n-hexadecane concentrations via gas chromatography (GC; Agilent 6890N GC) coupled with mass spectrometry (MS; Agilent 5973 MS). For separation, a J + W Scientific DB-5MS (30 m length, 0.25 mm ID, 0.25 µm film thickness) capillary column was used. The device was operated in a pulsed splitless mode with a Helium flow of 0.8 mL/min. Oven temperature was initiated at 65 °C (4 min), then ramped at 10 °C/min to 220 °C, further ramped at 20 °C/min to 310 °C, and held at this temperature for 5 min.