One μL was injected by a split injector (50:1) at an inlet temperature of 250 °C. The oven temperature was programmed as follows: started at 80 °C, heating rate 5 °C/min up to 175 °C, followed by another gradient of 3 °C/min to 230 °C, and hold at this temperature for 5 min. Detection was carried out by an FID set to 280 °C. The fatty acids were identified by comparing the retention times

with those of four purified standard mixtures of fatty acid methyl esters (4-7801; 47085-U; 49453-U and 47885-U from Sigma Chemical Co.). Peak areas were calculated as area % of total fatty acids. PS content was determined according to Laakso (2005). Lipids extracts containing about 1–2 mg of PS were mixed with 2 mg of internal standard (5β-cholestan-3α-ol; epicoprostanol) and evaporated to dryness under a nitrogen stream.

VX-809 datasheet A hot saponification was carried out by adding 2.5 mL of KOH 2.0 M in methanol followed by extraction with heptane. Sterols were derivatized with 200 μL of bis(trimethylsilyl)-trifluoracetamide (BSTFA) containing 1 g/100 g trimethylchlorosilane (TMCS) (99:1) and 100 μL of pyridine, at 70 °C for 15 min. An aliquot of 1.0 μL of derivatized sample solution was injected into the column selleck at 250 °C with a split injector (split ratio 1:50). Sterols were separated at 300 °C and detected with flame ionization detector (FID) at 280 °C. The carrier gas was helium at flow rate of 1.0 mL/min. Reference standards were used to identify the peaks. Quantification was calculated based

on standard curve prepared with β-sitosterol/IS area ratio, as a linear function (r = 0.9983) of sterol concentration (0.5–5.0 mg). About 20 mg of the chocolate were placed in a tube glass with 19-hydroxycholesterol (0.58 mg in n-hexane:isopropanol (3:2, mL/mL)) used as internal standard for the quantification of POPs. The solvent was evaporated under nitrogen and 30 mL of 2 mol equiv/L KOH solution in methanol were added to perform a cold saponification at room temperature for 18 h in darkness and under continuous agitation ( Sander, Addis, Park, & Smith, 1989). The unsaponifiable material was extracted with diethyl ether. PD184352 (CI-1040) For determination of POPs, 70 g/100 g of the unsaponifiable matter was purified by silica solid-phase extraction (SPE) according to Guardiola, Codony, Rafecas, and Boatella (1995). After cartridge activation with hexane (5 mL), PS and impurities were removed with hexane (5 mL) and diverse solvent mixtures of n-hexane:diethyl ether (10, 30 and 10 mL of 95:5, 90:10, 80:20 (v/v), respectively). POPs were finally eluted with acetone (10 mL), then subjected to silylation, dried under nitrogen stream and dissolved in 40 μL of n-hexane. One μL of the TMSE derivatives was analyzed by GC–MS (GCMS-QP2010 Plus (Shimadzu, Kyoto, Japan)), using a Fast GC–MS method suggested by Cardenia, Rodriguez-Estrada, Baldacci, Savioli, and Lercker (2012), with minor modifications. The system was fitted with a capillary RTX-5 Restek column (10 m × 0.10 mm i.d. × 0.