Chromatography

Liquid chromatography tandem mass spectrometry (LC-MS) was carried out with a Shimadzu Prominence HPLC system and SCIEX 4000 mass spectrometer using a Kinetix C18 column (2.6μm, 100 × 3 mm) at 50°C. Mobile phases were A) 10 mM NH₄Formate, 0.1% Formic acid in Water and B) 80% methanol/20% isopropanol w/0.1% Formic acid at a flow rate of 0.4 mL/min. A binary gradient starting at 30% B was linearly increased to 60%B at 4 minutes; then 75%B at 4.9 minutes, then 95%B at 5.2 minutes. A hold of 95%B continues till 5.5 minutes and returned to initial conditions at 5.6 minutes and re-equilibrated until 8 minutes. The total analysis time was 8 min/sample, and injection volumes were 20 μl.

Extraction Protocols:

Extraction protocols for sample processing are dependent on the matrices involved. Please see extraction methods section for more specific instructions.

References

Method derived from the following

[1] Anal Biochem. 2014 Nov 15;465:134-47. doi: 10.1016/j.ab.2014.07.027. Epub 2014 Aug 4.Derivatization of the tricarboxylic acid intermediates with O-benzyl hydroxylamine for liquid chromatography-tandem mass spectrometry detection. Tan B, Lu Z, Dong S, Zhao G, Kuo MS.

[2] J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Apr 15;1083:137-145. doi: 10.1016/j.jchromb.2018.02.040. Epub 2018 Mar 8. Fast quantification of short chain fatty acids and ketone bodies by liquid chromatography-tandem mass spectrometry after facile derivatization coupled with liquid-liquid extraction. Zeng M1, Cao H2.

Resources

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