Arachidonic Acid (AA) is ω-6 long chain polyunsaturated fatty acid, a senior unsaturated fatty acid. There is high content of AA in vivo. AA and its metabolites have a strong biological activity and can regulate a variety of physiological processes, such as the regulation of lipid and glucose, prevention of cardiovascular disease, chemoprevention of cancer cells, while exciting uterus, improve memory, vision and so on. Therefore, it is important to determine the concentrations of AA in human plasma for medical research and clinical diagnosis.
The plasma components are complex, containing protein and phospholipid which will interfere the detection. Therefore the sample pre-treatment method is crucial. This experiment presents 3 kinds of methods to extract Arachidonic Acid (AA) from plasma, which involve Cleanert® MAS-M 96-well plate.
Figure 1 Chemical structure of Arachidonic Acid
AA standard substance was diluted by methanol to required concentration.
(1) Cleanert® PPT plate
100 μL of plasma was placed into the well of protein precipitation plate and mixed with 400 μL ACN on vortex shaker, and then centrifuged under 6000r/min for 5 min. The elution was collected and then analyzed by LC-MS/MS.
(2) Cleanert® MAS-M 96-well plate
A mixed-phase sorbent of RP adsorption, cation exchange interaction and anion exchange interaction was packed into the well of Cleanert® MAS-M 96-well plate.
Activation: 600 μL of Methanol and 600 μL of Water were added into the well of Cleanert MAS-M plate successively.
Sample Loading: 100 μL of plasma sample diluted with 100 μL of 3 % ammonium hydroxide solution was added into the activated well.
Washing: 600 μL of water and then 600μL of methanol was used to wash the well.
Elution: 600 μL of ACN with 3% formic acid was used to eluted the well.
Then, the elution was collected for further analysis on LC-MS/MS.
Instrumentation: LC-MS/MS, API 4000+
HPLC Column: Venusil® ASB C18, 2.1 mm × 150 mm, 3 μm, 150 Å
Mobile Phase: Acetonitrile:Water = 70:30(v/v)
Flow rate: 0.2 mL/min
Injection: 5 μL
Ion source: ESI Negative
Scan mode: MRM
Table 1 Precursor/Product Ions of AA
Results and Discussion
Table 2 Recoveries and Precision
The experiment employed 3 kinds of sample pre-treatment to extract AA from plasma. The recoveries of AA on Cleanert® MAS-M 96-well plate were 99.19 %~106.38 which ensured an extraction procedure without reconstitution to support a rapid, high throughput assay of AA in plasma.