For the measurement of SCFAs by gas chromatography (GC)-mass spectroscopy (MS) without derivatization using a previously published protocol (Anaerobe 2018; 53: 64-73). Methyl tert-butyl ether (MTBE) was used for liquid-liquid extraction of SCFAs from acidified EBC. Negative controls including MTBE alone, MTBE used to extract acidified water and internal standards, a water wash of the EBC acquisition equipment, and a representative sample of normal saline acquired from the bronchoscopy suite were also assayed.
For our newly developed LC-MS assay, we applied the methods of Han, et al (Anal Chim Acta 2015; 854: 86-94). Briefly, samples were analyzed using an Agilent 6410 triple quadrupole LC-MS system. The chromatographic column was a Waters HSS T3, 2.1 mm x 100 mm, 1.7 µm particle size. Mobile phase A was 0.1% formic acid in water; mobile phase B was 0.1% formic acid in methanol. The gradient was as follows: linear ramp from 15% to 80% B from 0-12 min; step to 100%B from 12-12.1 min; hold 100%B from 12.1-16 min; step to 15%B from 16-16.1 min; hold 15%B from 16.1-20min. The injection volume was 5uL and the column temperature was 55 °C. MS parameters were as follows: gas temp 325 °C, gas flow 10 L/min, nebulizer 40 psi, capillary voltage 4000V, scan type MRM, negative ion mode, delta EMV 600.
A post-mortem porcine portal vein plasma sample was assayed as a positive control for both assays.
This was a small descriptive study to determine whether short chain fatty acids (SCFAs) are detectable in water. It is part of a larger study that assessed the utility of exhaled breath condensate (EBC) as a biofluid for microbiome assays.
- Yue, Min
- Kim, Jae Hyun
- Evans, Charles R.
- Kachman, Maureen
- Erb-Downward, John R.
- D’Souza, Jennifer
- Foxman, Betsy
- Adar, Sara D.
- Curtis, Jeffrey L.
- Stringer, Kathleen A.
- National Institutes of Health (NIH)
|Citations to related material
- Yue, M., Kim, J. H., Evans, C. R., Kachman, M., Erb-Downward, J. R., D’Souza, J., Foxman, B., Adar, S. D., Curtis, J. L., & Stringer, K. A. (2020). Measurement of Short-Chain Fatty Acids in Respiratory Samples: Keep Your Assay above the Water Line. American Journal of Respiratory and Critical Care Medicine, 202(4), 610–612. https://doi.org/10.1164/rccm.201909-1840LE