Orthogonal Comparison of GC–MS and ¹H NMR Spectroscopy for Short Chain Fatty Acid Quantitation

Abstract

Short chain fatty acids (SCFAs) are important regulators of host physiology and metabolism and may contribute to obesity and associated metabolic diseases. Interest in SCFAs has increased in part due to the recognized importance of how production of SCFAs by the microbiota may signal to the host. Therefore, reliable, reproducible, and affordable methods for SCFA profiling are required for accurate identification and quantitation. In the current study, four different methods for SCFA (acetic acid, propionic acid, and butyric acid) extraction and quantitation were compared using two independent platforms including gas chromatography coupled with mass spectrometry (GC-MS) and H nuclear magnetic resonance (NMR) spectroscopy. Sensitivity, recovery, repeatability, matrix effect, and validation using mouse fecal samples were determined across all methods. The GC-MS propyl esterification method exhibited superior sensitivity for acetic acid and butyric acid measurement (LOD< 0.01 μg mL, LOQ < 0.1 μg mL) and recovery accuracy (99.4%-108.3% recovery rate for 100 μg mL SCFA mixed standard spike in and 97.8%-101.8% recovery rate for 250 μg mL SCFAs mixed standard spike in). NMR methods by either quantitation relative to an internal standard or quantitation using a calibration curve yielded better repeatability and minimal matrix effects compared to GC-MS methods. All methods generated good calibration curve linearity (R > 0.99) and comparable measurement of fecal SCFA concentration. Lastly, these methods were used to quantitate fecal SCFAs obtained from conventionally raised (CONV-R) and germ free (GF) mice. Results from global metabolomic analysis of feces generated by H NMR and bomb calorimetry were used to further validate these approaches.

Structural and Functional Analysis of the Gut Microbiome for Toxicologists

“…A standard one-dimensional pulse sequence noesyprld (recycle delay-90°-t1–90°-tm-90°-acquisition) is used with a 90-pulse length of approximately 10 μsec (−9.6 dbW) and 64 transients are recorded into 32k data points with a spectral width of 9.6 KHz. For quantitation purposes, arelaxation delay (5s) and a recycle delay (4s) are added to the cycle to ensure the total repetition time (relaxation time, recycle delay and acquisition time) is more than 5 times the longitudinal relaxation time (T1) of the compounds (Cai et al, 2017). Quantitation analysis is performed based on either TSP-d4 reference with known concentration (Dai, Xiao, Liu, & Tang, 2010) or calibration curve.…”

“…If the spectra are not overlaid properly, repeat 1b to improve spectra phase, baseline, and calibration.Bucketing:Click “Amix-Tools-Bukcets,Statistics-Statistics-Bucket Table-New”.Choose 1D NMR and simple rectangular buckets.Change bucket width to 0.004 ppm (2.4 Hz).Change the scaling mode to either scaling to total intensity (non-quantitative purpose) to compensate the overall concentration differences (Cai et al, 2016), or no scaling then normalize to the tissue weight later (for quantitative purpose) (Cai et al, 2017). …”

“…Change the scaling mode to either scaling to total intensity (non-quantitative purpose) to compensate the overall concentration differences (Cai et al, 2016), or no scaling then normalize to the tissue weight later (for quantitative purpose) (Cai et al, 2017). …”

Simultaneous determination of short‐chain fatty acids in human feces by HPLC with ultraviolet detection following chemical derivatization and solid‐phase extraction segmental elution

“…In addition, complicated working process also limits the application of HPLC‐UV analysis, which includes alkaline freeze‐drying of sample, sulfonated polystyrenedivinylbenzene column with temperature of 60°C and mobile phase of 0.2 M sulfuric acid (pH 3.1) , or diethyl ether extraction and acid‐base neutralization . Besides, the column contamination and complex sample clean‐up procedure are also problematic for GC–MS analysis .…”

Multiplatform Physiologic and Metabolic Phenotyping Reveals Microbial Toxicity

“…Metabolomic approaches have been important for exploration of the metabolic effects of pharmaceuticals ( 25 , 26 ), environmental contaminants ( 27 , 28 ), and dietary factors ( 29 , 30 ), as well as microbe-derived metabolites and microbiome-host cometabolites, to understand the metabolite chatter between the host and the gut microbiome ( 31 – 34 ). Mass spectrometry and NMR are widely used platforms for metabolomics, with each technique exhibiting its own merits and limitations ( 35 , 36 ). Microbial metabolism determined by MS- and NMR-based metabolomics provides a functional readout of the microbial community, thus providing complementary insight into the characteristic changes in the metabolic activity, enzymatic pathways, and networks within the microbiota ( 37 , 38 ).…”