2.6. Analysis of Short-Chain Fatty Acids
COLD, ALDRICH
In total, seven SCFAs, succinate, and the medium-chain fatty acid caproate were purchased; formate (Scharlau), acetate (Honeywell), sodium propionate, isobutyrate, and valerate (Alfa Aesar), sodium butyrate, isovalerate and caproate (Sigma Aldrich, Saint Louis, MA, USA), and sodium succinate (Acros). Derivatizing reagents 3-nitrophenylhydrazine hydrochloride (3-NPH), N- (3-dimethylaminopropyl)-N’-ethylcarbidimide hydrochloride (EDC-6), D- (-) quinic acid, HPLC grade pyridine, LiChrosolv methanol hypergrade for LC-MS, and LiChrosolv LC-MS grade water were purchased from Sigma Aldrich, and LC-MS grade acetonitrile from Fisher Scientific. 13C6-3NPH-HCl was synthesized by IsoSciences Inc. (King of Prussia, PA, USA) (catalogue 13309).Stock solutions of 4 mM acetic and formate, 2 mM propionate, and 1 mM for the remaining SCFA were prepared in 75% MeOH. Thereafter, a standard mix with 1:10 dilution (10% MeOH) of the SCFA was made. Formate and acetate standards were prepared in the concentration 3.2 µM-0.63 nM, propionate 3.2 µM-0.31 nM and the remaining SCFAs in the range 0.8 µM- 0.16 nM. In total, five blanks (LiChrosolv) and three quality control (QC) samples in triplicates were included in each batch. Two QC samples were long-term pooled plasma samples, the third was pooled plasma of samples from the current dataset. The reagents 3-NPH 200 mM, quinic acid 200 mM, and 120 mM EDC-6 in 6% pyridine were prepared in 75% MeOH. The 13C-labelled internal standard for all SCFAs was prepared by weighing 1 g of 13C6-3NPH HCl, adding 50 μL of the 1:10 standard mix solution, 25 μL of 120 mM EDC-6 and 25 μL 75% MeOH. The dilution was mixed and shaken in a cold room for 4 h. When finished, 25 μL quinic acid was added, and the sample was shaken at 1600 rpm (multitube vortexer DUX-2500) in RT for 45 min. Finally, it was diluted to 100 mL with 10% MeOH.