Pyridine hydrochloride

Lyophilized metabolome samples were derivatized using a solution of ethoxyamine hydrochloride in pyridine as the oximation reagent followed by silylation with N-trimethyl-N-trimethylsilylacetamide as described by [18 (link)]. GC-MS-analysis of the derivatized samples was performed using temperature gradient from 70°C to 320°C at a rate of 10°C/min on an Agilent 6890 N GC (Palo Alto, CA, USA) and an Agilent 5973 mass selective detector. 1 μl aliquots of the derivatized samples were injected in the splitless mode on a DB5-MS capillary column. Detection was performed using MS detection in electron impact mode (70 eV).

For GC–MS analysis a protocol according to Weckwerth et al. was used (Weckwerth et al. 2004 (link)). Deep frozen plant material was ground to a fine powder using a mortar and pestle under constant adding of liquid nitrogen. About 45 mg of each replicate was transferred to pre-cooled reaction tubes. For the extraction process, 1 ml of ice cold extraction mixture (methanol:chloroform:water, 5:2:1, v:v:v) was subsequently added. Additionally, 10 μl of internal ¹³C₆-Sorbitol standard were added into each tube. Tubes were vortexed for several seconds and incubated on ice for 8 min to achieve a good extraction. Hereupon, the samples were centrifuged for 4 min at 14,000×g, separating the soluble compounds from remaining cell structure components. For phase separation, the supernatant was then carried over into a new tube containing 500 μl deionized water and 200 μl chloroform. After 2 min of centrifugation at 14,000×g, the water/methanol phase, containing the polar metabolites, was separated from the subjacent chloroform phase and completely dried out overnight.
Samples were derivatised by dissolving the dried pellet in 20 μl of a 40 mg methoxyamine hydrochloride per 1 ml pyridine solution and incubation on a thermoshaker at 30 °C for 90 min. After adding of 80 μL of N-methyl-N-trimethylsilyltrifluoroacetamid (MSTFA), the mixture was again incubated at 37 °C for 30 min with strong shaking.
A solution of even-numbered alkanes (Decane C10, Dodecane C12, Tetradecane C14, Hexadecane C16, Octadecane C18, Eicosane C20, Docosane C22, Tetracosane C24, Hexacosane C26, Octacosane C28, Triacontane C30, Dotriacontane C32, Tetratriacontane C34, Hexatriacontane C36, Octatriacontane C38, Tetracontane C40) was spiked into the derivatized sample before GC–MS analysis in order to infer the retention time and create the retention index.
For LC–MS analysis, frozen plant leaf material was ground as for GC–MS sample preparation, followed by addition of 1 ml pre-chilled 80/20 v:v MeOH/H₂O extraction solution containing each 1 μg of the internal standards Ampicillin and Chloramphenicol per 50 mg of fresh weight. Samples were hereupon centrifuged at 15,000×g for 15 min and the supernatant was placed into a new tube and completely dried out overnight. The resulting pellet was then dissolved in 100 μl of a 50/50 v:v MeOH/H₂O solution and centrifuged again for 15 min at 20,000×g. The remaining supernatant was then filtered through a STAGE tip (Empore/Disk C18, diameter 47 mm) into a vial with a micro insert tip. Before analysis lipid components were removed by adding 500 µl of chloroform, centrifugation and separation of the non-polar-phase to avoid contamination of the ESI ion transfer capillary.

Scopolamine hydrobromide and Donepezil hydrochloride were brought from Merck (Merck KGaA, Darmstadt, Germany).Trichloroacetic acid, acetylthiocholine iodide, 5,5′-dithiobisnitrobenzoic acid (DTNB), hydroxylamine hydrochloride, and ferric chloride, were brought from Sigma-Aldrich (St. Louis, MO, USA). Synthetic pyridine-2-one derivatives 28, 28a–e previously synthesized by our group were used in the study [11 (link)]. All other reagents and chemicals utilized in the investigation were of analytical grade.