Short Chain Fatty Acid Analysis
To support microbiome research, Pine Lake Laboratories has developed an assay for short chain fatty acids (acetic, butyric, and propionic acid) in feces. These targeted metabolites can be used as an indicator of microbiome activity in research subjects and as biomarkers to evaluate the effect of various pharmaceutical drug treatments during pre-clinical and clinical trials. The method involves extracting the short chain fatty acids form feces then analysis by direct injection GC-MS.
This white paper will describe the method and highlight the performance of the method.
- Method Description
A. Sample Preparation
B. Standard and QA Preparation
Standards and QCs are prepared by spiking known levels of each short chain fatty acid into feces. The endogenous level of each short chain acid in the feces used to prepare the standards and QCs had previously been determined by standard addition. The final concentration is then the total of the amount added and the endogenous level.
Standards and QCs are then prepared for analysis same as the samples.
C. Instrumental Analysis
Samples are analyzed by GC-MS using a Nukol capillary column (15 m x 0.23 mm, 0.25 µm). Details available upon request.
A. Linearity and Range
For all three short chain fatty acids, the range of the method was 5 to 150 μg/mL with R2 > 0.990. Representative calibration curves can be found in Figures 1-3.
B. Precision in Matrix
A representative lot of feces was assayed in triplicate for all three short chain fatty acids. Results are in Table 1. Precision on all matrix samples (n=6) was excellent with %RSD values less than 5% for each SCFA within each of the three analyses. The total precision of the grand average was less than 10% for acetic and propionic acid. Butyric acid was higher at 26.6% but was still acceptable at less than 30%.
C. Accuracy and Precision
A representative lot of feces was spiked at three separate levels with known amounts of each of the three short chain fatty acids. This was repeated in three separate analyses. Results are in Table 2-4. Accuracy was acceptable for all three short chain fatty acids with average recovery within ± 15.0% of target for each level in each analysis and in the grand average. Similarly precision was acceptable at ≤15.0% for each level in each analysis and in the grand average.
This simple yet effective GC/MS analysis can be used to accurately determine the concentration of acetic, butyric, and propionic acid in feces. The method is specific, accurate, and precise with a large quantifiable range for short chain fatty acids. Additional short chain fatty acids not included in the method evaluation reported in this white paper can also be detected and quantitated using this method.