Studying showing: Develop a GC-MS method for the analysis of three short chain fatty acids (SCFA) in human feces.

1. Determination of Short Chain Fatty
Acids in Human Feces by Direct
Inject GC/MS
Pine Lake Laboratories

2. Study Objectives
• Develop a GC-MS method for the analysis of
three short chain fatty acids (SCFA) in human
feces
• The three SCFA were acetic acid, butyric acid
and propionic acid

3. Sample Preparation
• Human feces samples from 20 individuals were combined and
homogenized.
• Approximately 100 mg of sample was weighed into a 15 mL
falcon tube.
• To each tube 8 mL of water was added. Tubes were mixed
vigorously by shaking or vortexing for approximately 5
minutes.
• Samples were centrifuged for approximately 10 minutes at
approximately 4000 rpm.
• 1 mL of the supernatant was then added to 950 µL of
methanol in a 5 mL centrifuge tubeand spiked with 50 µL of
internal standard (acetic acid-d4 and proprionic acid-d2).
• Samples were mixed thoroughly by vortexing.

4. GC-MS Conditions
Equipment:
Column: Initial temperature:
Injection Volume: Ramp:
Final Temperature:
Split Ratio: Run Time:
Purge Flow:
Temperature Linear velocity
Components m/z Approximate RT (min)
Ionization Mode: Acetic Acid 60 2.39
Scan Mode: Butyric Acid 60 3.51
Scan Cycle Time: Propionic Acid 74 2.90
Threshold: Acetic Acid d3 63 2.35
Transfer Line Temperature: Propionic Acid d2 76 2.90
Oven Temperature Program:
MS Detector:
Approximately 80 cm/sec
300ºC
80 °C for 1 minute
To 200 °C @ 15 °C/min
200 °C for 6 min
15min
250°C
EI
Scanning: m/z: 20-200
Approximately 7 scan/sec
20
Agilent 6890 GC and 5973 MSD
Nukol capillary column, 15 m x 0.32 mm, 0.25 mm
GC Inlet:
1 µL
None (Splitless)
Approximately 15.5 mL/min Gas Flow Rates:

5. Example Chromatograms – Acetic Acid and Butyric Acid
Acetic Acid (RT 2.37 min) and Butyric Acid (RT 3.51 min) at 150 µg/mL in 50:50 MeOH:H2O
(top) and matrix (bottom)

6. Example Chromatogram – Proprionic Acid
Proprionic acid at 150 µg/mL in 50:50 MeOH:H2O (top) and matrix
(bottom)

7. Method Evaluation
• Linearity was evaluated from 5 to 150 µg/mL.
• Three analyses were performed between two
analysts
• Matrix was spiked at 3 levels.
• Spiked recoveries were calculated by subtracting
the average endogenous amounts of each SCFA
as determined in the matrix blanks (n=6).

8. Representative Linearity (Acetic Acid)

9. Endogenous Levels of SCFA in Matrix

10. Recoveries of Spiked Matrix (Low Level)

11. Recoveries of Spiked Matrix (Mid Level)

12. Recoveries of Spiked Matrix (High Level)
• Table 3: Matrix spiked recoveries (High Spike) of SCFA’s across three
analyses

13. Summary of Method
• Linearity for all three SCFA was linear from 5 to
150 µg/mL with R2 values greater than 0.990.
• Spiked recoveries of each analysis on three
different levels averaged 85-110% recovery.
• Precision of the spiked recoveries was less than
17%.