N methyl n trimethylsilyl trifluoroacetamide

The samples were extracted for metabolite profiling (LC–MS and GC–MS) as described in Weckwerth et al. (2004) (link). Briefly tissue was grounded using RETCH-mill (Retsch Gmbh, 42787 Haan, Germany) with pre-chilled holders and grinding beads. Forty milligram frozen dried powder were extracted in a pre-chilled methanol/chloroform/water extraction solution (2.5/1/1 v/v/v). Internal standards, i.e., 0.2 mg/mL ribitol in water, 1 mg/mL ampicillin in water and 1 mg/mL corticosterone in methanol were subsequently added. The mixture was then briefly vortexed and ultra-sonicated for 10 min to release the cell components. Subsequently, samples were centrifuged for 10 min at 14000 RPM (micro centrifuge 5417R). The supernatant was mixed with equal volumes (300 μl) of chloroform and Millipore Direct-Q3 UV system purified water, vortexed, and then centrifuged at 14,000 RPM for 5 mins. Finally, 1 mL of water/methanol phase was transferred to UPLC vials for LC–MS and 75 μL were derivatized for GC–MS analysis with micro filtered retention time standard alkane mixture (0.029% v/v n-dodecane, n-pentadecane, n-nonadecane, n-docosane, n-octacosane, n-dotracontane, and n-hexatriacontane dissolved in pyridine (0.0075% H₂O), purchased from Sigma–Aldrich (Jerusalem, Israel) and MSTFA, N-methyl-N-[trimethylsilyl] trifluoroacetamide purchased from Macherey-Nagel GmbH & Co. KG, Düren, Germany.

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50 μL body fluid (for analysis in triplicates), such as plasma, serum, saliva or CSF (fresh or stored at −80 °C)

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Methanol (High purity, LC–MS grade) at −20 °C

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Internal standard [U-¹³C]Ribitol (Omicron Biochemicals, ALD-062)

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Sample collection tubes, such as sterile collection tubes (for CSF and saliva), EDTA and serum-separating tubes (for blood)

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Wet ice

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Methoxyamine hydrochloride 98% (Sigma-Aldrich, 226904)

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Pyridine 99.8% (Sigma-Aldrich, 270970)

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N-methyl-N-trimethylsilyl-trifluoroacetamide (Macherey-Nagel, 701270.110)

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Alkane standard mixture for performance tests of GC-systems (Sigma-Aldrich, 68281-10ml-F)

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GC glass vials with micro insert (gastight) 5–250 μL (various suppliers)

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(Magnetic) caps for GC glass vials (various suppliers)

All aqueous solutions used throughout this protocol should be prepared with MilliQ or deionized water (18.2 MΩ cm, <3 ppb TOC).
Note: The proper selection of the collection tubes is highly relevant for GC–MS based metabolomics analyses. In Section Choice of collection tubes we review and recommend several types of collection tubes for the different body fluid types. In general, we recommend the use of sterile tubes to reduce the risk of sample contamination.

A pulverized leaf sample (5 mg) was homogenized using a ribolyzer (3 × 45 s, 6.5 ms^–1; Peqlab) with 0.5 g of silica beads (0.5 mm diameter; Roth) and 1 ml 80% methanol containing 10 µM ribitol as internal standard. A 600-µl aliquot of supernatant was dried in a stream of nitrogen gas and derivatized at 37 °C by addition of 75 µl methoxylamin-hydrochloride (20 mg ml^–1 in pyridine; Sigma Aldrich) for 90 min and 75 µl N-methyl-N-(trimethylsilyl) trifluoroacetamide (Macherey–Nagel) for 30 min.

All consumables were from VWR (Ismaning, Germany). Derivatization reagents trifluoroacetic anhydride (TFAA), 1-(trimethylsilyl)imidazole (TSIM), and N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) were from Macherey-Nagel (Düren, Germany). Deionized water was prepared with an in-house ion-exchanger. 1,4-Dioxane and methyl tert-butyl ether (MtBE) were distilled before use. β-Glucuronidase/sulfatase from Helix pomatia type HP-2, 5α-cholestane (≥97%), pregnenolone (>98%), pregnenolone sulfate sodium salt (>98%), and cholesteryl sulfate sodium salt (>99%) were purchased from Sigma-Aldrich (Schnelldorf, Germany). Dehydroepiandrosterone sulfate sodium salt (>99%) and 25-hydroxycholesteryl sulfate sodium salt (>99%) were from Avanti Polar Lipids (Alabaster, AL, USA). All other sterol sulfate sodium salts were from Steraloids (Newport, RI, USA). All other reagents and solvents were purchased in HPLC grade or in pro analysis quality from Sigma-Aldrich (Schnelldorf, Germany).