Application Note Metabolomics GC/MS Detection of Short Chain Fatty Acids from Mammalian Feces Using Automated Sample Preparation in Aqueous Solution Authors Abstract Takeshi Furuhashi and This Application Note presents a method for the profiling of short chain fatty Genki Ishihara acids (SCFAs) in mammalian feces using the Agilent 5977B GC/MSD equipped Anicom Specialty Medical with an Agilent 7890B GC and an Agilent 7693A autosampler. Despite the Institute Inc., 8-17-1, Nishi importance of profiling and quantifying SCFAs in feces, technical difficulties in the Shinjuku, Shinjuku-ku, analysis remain due to the volatility and hydrophilicity of SCFAs. We used isobutyl Tokyo, Kuniyo Sugitate, chloroformate/isobutanol derivatization in aqueous solution without drying the Agilent Technologies samples. Using this protocol, a method for C1 to C7 (formic to heptanoic) acids was Japan developed, and more than seven SCFAs were successfully identified and quantified from mammalian feces samples by GC/MS. Introduction Experimental Bertin Technologies, France) at 6,000 rpm for 20 seconds, twice, with a Short chain fatty acid (SCFA) profiling Sample pretreatment is important, 30 second interval. Each sample was has been a major topic in gut bacteria especially in the case of lipid-rich then centrifuged at 21,000 g for five studies1,2. However, the high volatility biological samples such as feces, minutes, and 675 μL of the supernatant and hydrophilic characteristics of SCFAs because lipids can influence the was transferred to a new tube. Next, to makes the detection of these analytes derivatization process. Therefore, remove monitor recoveries, a 20 μg aliquot of difficult. GC/MS can be a suitable the many lipophilic compounds in 3-methylpentanoic acid was added to technique, offering advantages for the biological samples by phase separation the supernatant. Then, 125 μL of 20 mM detection of volatile compounds. These before the derivatization process. This NaOH solution and 400 μL of chloroform highly polar species need derivatization helps to avoid blocking the derivatization were added to the tube, and the sample for proper GC/MS analysis. due to contaminants, and removes was vortexed and centrifuged at 21,000 g Derivatization can involve silylation the contaminant peaks in the GC for two minutes. A 400 μL aliquot of the (that is, trimethylsilyl (TMS))3, alkylation4, chromatogram. upper aqueous phase was transferred 5 into a new tube, then 80 μL isobutanol and esterification . Many derivatization Sample preparation protocols require nonaqueous and 100 μL pyridine were added to the The freshly collected feces samples conditions, calling for removing water tube along with ultrahigh quality water (human and cat, 100–150 mg fresh from the biological samples before the to adjust to 650 μL total volume. To weight) were placed into 2-mL screw cap derivatization reaction. SCFAs can be minimize foaming, one boiling chip was tubes with ceramic beads (KT03961-1, adversely affected during the drying placed into the tube. The sample can be Bertin Technologies, France). After process, resulting in inaccurate results. stored in a freezer after this step. the addition of 1 mL 10 % isobutanol, In previous studies, esterification the samples were homogenized using propyl chloroformate/propanol mechanically (Precellys Evolution; was used for derivatization under aqueous conditions6. Unfortunately, this derivatizing reagent could not be Sample preparation chromatographically separated from the formic acid, making identification and 675 µL of the 400 µL of the supernatant aqueous phase detection of formic acid difficult. Boiling chip An improved GC/MS derivatization protocol was developed using isobutyl 100–150 mg 650 µL Final volume chloroformate/isobutanol, which allows fresh weight sample Can be stored in freezer proper separation, identification, and 125 µL, 20 mM detection of 14 SCFAs, using sensitive NaOH solution, 80 µL isobutanol and Ceramic beads 400 µL chloroform GC/MS analysis. This Application Note 1 mL 10 % 100 µL pyridine plus introduces an SCFA profiling platform isobutanol/water ultra–high purity water to 650 µL for mammalian feces samples using GC/MS. Derivatization 50 µL Chloroformate 150 µL n-Hexane isobutyl extraction GC/MS Figure 1. Sample preparation and derivatization process. 2 Calibration standards preparation Chloroformate RCOOH: carboxylic acid O O R OH: alcohol The desired concentration of the SCFAs R 1 R OH 2 R COOCl: alkyl chloroformate standard with 125 μL 20 mM NaOH, Cl O 2 100 μL pyridine, and 80 μL isobutanol O were combined in a tube. The final R O– volume was adjusted to 650 μL with water. To avoid foaming, one boiling chip was added into the tube. O– O O O R O R 2 2 R O O R1 Derivatization process R O O R O Cl Calibration standards and samples were R1 OH subjected to the same derivatization R2 OH + CO2 procedure. A 50-μL aliquot of isobutyl Figure 2. Derivatization reaction mechanism. chloroformate was carefully added to the 650 μL sample or standard solution. Table 1. GC/MS Analytical method. To release the gases generated by the reaction, the tube lid was kept open Parameter Value for 1 minute, then the lid was closed GC/MS System Agilent 7890B GC/5977 MSD and the sample vortexed. Next, 150 μL Column VF-5ms 30 m × 0.25 mm, 0.5 μm (p/n CP8945) hexane was added, and the tubes were Column flow 1.0 mL/min centrifuged at 21,000 g for 2 minutes. Liner Ultra Inert liner, Universal, Low PSI drop, Wool (p/n 5190-2295) The upper hexane-isobutanol phase was Injection mode Split (50:1) transferred into an autosampler vial for Injection temperature 260 °C GC/MSD analysis. Preslit screw caps Oven temperature 40 °C for 5 minutes, 10 °C/min to 310 °C (p/n 5185-5824) are recommended for Transfer line temperature 280 °C Scan the vials because CO2 gas is produced. MS mode Figure 2 shows the reaction mechanism Scan range m/z 30–350 of the process. Ion source temperature 250 °C Quad. temperature 150 °C Instrumentation For SCFA quantification, GC/MS Table 2. List of analytes, their retention times, and quantification ions. measurements were carried out on an Agilent 5977B GC/MSD single No. Compound RT (min) m/z quadrupole mass spectrometer equipped 1 Formic acid 4.28 56 with an Agilent 7890B GC and an 2 Acetic acid 7.30 56 Agilent 7693A autosampler. Table 1 lists 3 Propionic acid 9.83 57 the GC/MS analytical conditions. 4 Isobutanoic acid 10.88 71 Results and discussion 5 Butanoic acid 11.75 71 6 2-Methylbutanoic acid 12.67 85 Chromatographic separation 7 Isovaleric acid 12.75 85 Table 2 lists the monitored 14 SCFAs 8 Pentanoic acid (valeric acid) 13.59 85 with their retention times and 9 3-Methylpentanoic acid (IS) 14.26 99 quantification ions. 10 Isocaproic acid 14.58 99 Figures 3 and 4 show that the modified 11 Hexanoic acid (caproic acid) 14.67 99 derivatization process allowed the 12 2-Methylhexanoic acid 15.23 113 derivatization reagent to be completely 13 4-Methylhexanoic acid 15.70 113 separated from the early eluting SCFAs, 14 Heptanoic acid 16.33 113 formic acid, and acetic acid. 3 ×106 4.0 Peak ID 3.8 1. Formic acid 2. Acetic acid 3.6 3. Propionic acid 3.4 4. Isobutanoic acid 3.2 5. Butanoic acid 3.0 6. 2-Methylbutanoic acid 7. Isovaleric acid 2.8 8. Pentanoic acid (valeric acid) 2.6 9. 3-Methylpentanoic acid (IS) 2.4 10. Isocaproic acid 2.2 11. Hexanoic acid (caproic acid) 12. 2-Methylhexanoic acid 2.0 Counts 13. 4-Methylhexanoic acid 1.8 10 14. Heptanoic acid 1.6 1.4 5 9 11 1.2 14 7 1.0 3 4 8 13 1 12 0.8 2 0.6 6 0.4 0.2 0 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Acquisition time (min) Figure 3. Total ion current chromatogram (TICC) of the SCFAs. 5 ×10 1 2 *4.275 2 *7.298 1 m/z 56 Counts 0 ×105 4 3 *9.83 2 m/z 57 Counts 0 ×105 4 4 5 *10.88 *11.75 2 m/z 71 Counts 0 ×105 7 8 2 6 *12.67 *12.75 *13.59 1 m/z 85 Counts 0 10 ×105 *14.67 3 9 11 2 *15.23 m/z 99 *14.58 1 Counts 0 ×105 13 14 2 12 *16.33 *15.70 *16.75 1 m/z 113 Counts 0 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Acquisition time (min) Figure 4. Extracted ion chromatogram (EIC) of the quantitation ions. 4 The large peaks around 6.5 minutes and Table 3. Excellent calibration results were obtained both for 14.3 minutes are from the derivatizing the naturally occurring high and trace concentration ranges. reagent. However, they do not interfere Range with the proper identification and No. Compound (on-column) R2 quantitation of the SCFAs. 1 Formic acid 40 pg–1 ng 0.9963 2 Acetic acid 20 pg–1 ng 0.9979 Quantitative performance 3 Propionic acid 20 pg–1 ng 0.9992 Calibration was carried out to 4 Isobutanoic acid 10 pg–1 ng 0.9993 accommodate the naturally occurring concentrations in mammalian feces. 5 Butanoic acid 10 pg–1 ng 0.9993 Since C1 to C4 SFCAs are detected 6 2-Methylbutanoic acid 10 pg–1 ng 0.9987 in biological samples at much 7 Isovaleric acid 10 pg–1 ng 0.9985 higher concentrations than higher 8 Pentanoic acid (valeric acid) 10 pg–1 ng 0.9995 carbon SCFAs, calibration started at 9 3-Methylpentanoic acid (IS) – – higher concentrations for the early 10 Isocaproic acid 4 pg–1 ng 0.9995 eluting analytes than the later eluting 11 Hexanoic acid (caproic acid) 4 pg–1 ng 0.9996 compounds.