Silyl 991

All buffer constituents and solvents were of reagent or HPLC grade and obtained from either Roth (Karlsruhe, Germany), Sigma-Aldrich (St Louis, MO, USA) or J. T. Baker (Deventer, The Netherlands). Coumarin standards were purchased from PhytoLab (Vestenbergsgreuth, Germany; scopolin, isofraxidin, fraxin), and Sigma-Aldrich (esculin, esculetin, fraxetin, 4-methyl-umbelliferon, scopoletin). [2,2,4,4-²H]citric acid and [2,3,3-²H]malic acid were from CDN isotopes (Point Claire, QC, Canada), [2,2,3,3-²H]succinic acid and methoxyamine HCl from Sigma-Aldrich, and Silyl-991 from Macherey-Nagel (Düren, Germany).

n-Propanol (for synthesis), squalene (99%) and a technical β-sitosterol product (55.5% β-sitosterol, 34.2% campesterol [16 (link)]) were purchased from Merck (Darmstadt, Germany). Silica gel 60 (for column chromatography), 3-phenyl propionic acid (99%), 5α-cholestane (97%), cholesterol (> 99%) and stearic acid (99%) were ordered from Sigma-Aldrich (Steinheim, Germany). Dichloromethane (99.8%), mEthanol (HPLC gradient grade) and n-hexane (HPLC gradient grade) were purchased from Th. Geyer (Renningen, Germany). Ethanol (distilled before use, 7.4% water content), sodium chloride (99%), pyridine (99%) and sulphuric acid (96%) were obtained from Carl Roth (Karlsruhe, Germany). Tripalmitin (95%, containing traces of dipalmitoylglycerol), cholesteryl stearate (97%) and elaidic acid (99%) were from Fluka (Taufkirchen, Germany). Palmitic acid (98%) was purchased from Riedel de Häen (Seelze, Germany). 8-Phenyl octanoic acid (97%) and n-hexanol (99%) were from Thermo Fisher Scientific (Kandel, Germany). The trimethylsilylating reagent SILYL-991 was ordered from Macherey–Nagel (Düren, Germany). Nitrogen and helium 5.0 quality originated from Westfalen company (Münster, Germany). Dihydrolanosterol [19 ] and α-tocopherol [23 (link)] were isolated autonomously with CCC. Demineralised water was obtained through the in-house supply (Hohenheim, Germany).

Nitrogen (99.95% purity) and helium (99.999% purity) were from Westfalen Company (Münster, Germany). Ethanol (technical grade, distilled prior to use) was from BASF (Ludwigshafen, Germany), n-hexane (HPLC grade) was from Th. Geyer (Renningen, Germany), whereas potassium hydroxide (> 85%) and sodium sulfate (> 99%) were from Carl Roth (Karlsruhe, Germany). Stearic acid (18:0; 99%), lignoceric acid (24:0; > 99%), cerotic acid (26:0; ≥ 90%), and pyridine were from Sigma-Aldrich (Steinheim, Germany). Pyrrolidine (> 98%), heneicosanoic acid (21:0, ≥ 98%), tricosanoic acid (23:0, ≥ 95%), and pentacosanoic acid (25:0, ≥ 95%) were from TCI (Zwijndrecht, Belgium). Nonadecanoic acid (19:0, ≥ 99%), arachidic acid (20:0, ≥ 99%), and behenic acid (22:0, ≥ 97%) were from Fluka (Buchs, Switzerland). A semipure β-sitosterol standard (total sterols, 95%; unsaturated sterols calculated as β-sitosterol, 85%) was from Merck (Darmstadt, Germany). 5α-Cholestane (98%) was from Acros Organics (Geel, Belgium), and the silylating agent consisting of N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS), 99:1, v/v (SILYL-991), was from Macherey–Nagel (Düren, Germany).