This is a LC-MS/MS method for determination of fatty acid in faeces by Cleanert MAS-M.
Arachidonic acid (AA) was chose to represent fatty acid.
Stock solution was prepared by dissolved 10 mg of AA in 100mL of methanol. The stock solution was diluted with a mixture of acetonitrile﹕water (70:30, v/v) to obtain work solution.
50mg of faeces sample was added to centrifuge tube, then mixed by 0.5mL 1% formic acid in water and vortexed for 2min, added 1.5mL methanol into the tube, ultrasonic for 10min, and centrifuged under 6000r/min for 5 min. The supernatant was collected into a clean vial. Added 1.5mL methanol into the tube and repeat the extract process. Combined supernatants and filtrated by PTFE filter (0.22μm), the filtrate was collected and then mixed with 0.6mL ammonium hydroxide.
The pretreatment sample was loaded into each well of Cleanert® MAS-M plate which was conditioned with 600μL methanol and 600μL water sequentially. The plate was washed with 600 μL of water followed by 600 μL of methanol. The target compounds were eluted with 600 μL of 3% formic acid in acetonitrile. The eluates were concentrated at 45℃ under a gentle stream of nitrogen to dryness. The residues were reconstituted with 100 μL of acetonitrile﹕water (70﹕30,v/v) separately for further analysis.
HPLC Column: Venusil® ASB C18 (2.1 ´ 50mm, 3μm, 150 Å)
Mobile Phase: Acetonitrile/Water = 70/30(v/v)
Flow rate: 0.2mL/min
LC Instrument: Shimadzu LC-20A
Detection: MS (ABI4000+) – see below for conditions
Ion source: ESI-
Scan mode: MRM
Table 1 MS parameters
Table 2 Precursor/Product Ions of AA
Results and Discussion
Figure 1 Chromatogram of 5ppm AA standard
Figure 2 Chromatogram of AA in faeces sample
Figure 3 Chromatogram of faeces sample spiked with 5ppm AA standard
There was approximately 3μg/g of AA in the mouse faeces in the experiment. Background should be subtracted from the response of AA in spiked samples to calculate the recovery data.
Table 4 Recovery data
In this experiment we use Cleanert® MAS-M to extra fatty acid from faeces and determination by LC-MS/MS. The recovery of fatty acid is 88.9% at 10μg/g. It should be a good method to determine fatty acid.