D5 propionic acid

Formic acid of analytical grade, acetonitrile, and isopropanol LiChrosolv were purchased from Merck (Darmstadt, Germany). Acetic, propionic, butyric and isobutyric acid, pyridine, N,N-dimethylglycine (DMG), and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) were purchased from Sigma Aldrich Chemie GmbH (Steinheim, Germany). The methanol LC-MS Chromasolv was purchased from Honeywell Riedel-de Haën (Seelze, Germany). [¹³C,D₃]-Acetic acid, [D₅]-propionic acid, and [D₇]-butyric acid were from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Stock solutions of the SCFAs, including the labeled compounds, were prepared in water and were stored at −80 °C.

SCFA were quantified by LC-MS/MS after derivatization to their 3-nitrophenylhydrazones as described by Han et al.^{54 (link)}. In brief, 10 µl of an internal standard mixture containing 20 µg/ml each D₄-acetic acid, D₅-propionic acid, D₇-butyric acid (Cambridge Isotope Laboratories, USA) were added to 10 µl of portal vein plasma and mixed. 100 µl acetonitrile were added and centrifuged after mixing. 50 µl of the supernatant were derivatized for 30 min at 40 °C with each 20 µl of 200 mM 3-nitrophenylhydrazine hydrochloride and 120 mM N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride. The reaction was quenched by addition of 200 µl 0.1 % formic acid. LC separation was performed using a Kinetex® 2.6 µm XB-C18, 50×2.1 mm (Phenomenex, Torrance, CA, US) with water as mobile phase A and acetonitrile as mobile phase B both containing 0.1% formic acid. Gradient elution started with 90% A with a linear increase to 81% A at 0.3 min follow by an increase to 78%A at 2.5 min. Column was cleaned with 100% B from 2.6–3.0 min and re-equilibrated from 3.1 to 4 min with 90% A. The column flow was 500 µl at 60 °C and 10 µl sample were injected. The method included acetic acid, propionic acid, butyric acid and iso-butyric acid. Butyric acid and iso-butyric acid were separated by LC. For quantification a calibration lines were generated by addition of SCFA to human plasma.