Abstract
A high throughput sample preparation method was developed utilizing mixed-mode solid phase extraction (SPE) in 96-well plate format for the determination of free arachidonic acid in plasma by LC-MS/MS. Plasma was mixed with 3% aqueous ammonia and loaded into each well of 96-well plate. After washing with water and methanol sequentially, 3% of formic acid in acetonitrile was used to elute arachidonic acid. The collected fraction was injected onto a reversed phase column at 30°C with mobile phase of acetonitrile/water (70 : 30, v/v) and detected by LC-MS/MS coupled with electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The calibration curve ranged from 10 to 2500 ng/mL with sufficient linearity (r 2 = 0.9999). The recoveries were in the range of 99.38% to 103.21% with RSD less than 6%. The limit of detection is 3 ng/mL.
Highlights
Arachidonic acid is a ω-6 long chain polyunsaturated fatty acid, a senior unsaturated fatty acid
The traditional techniques such as protein precipitation (PPT), liquid-liquid extraction (LLE), and solid phase extraction (SPE) with reversed phase packing material have been reported for clean-up of plasma samples, it is clear that these techniques are insufficient to remove interferences of phospholipids and proteins in plasma [22,23,24], while the combination of ionic interaction and reversed phase interaction was reported to remove phospholipids and proteins more sufficiently in the clean-up process for plasma samples [25]
It is well known that phospholipids in plasma will result in matrix effect on mass spectrometry [19, 20]
Summary
Arachidonic acid is a ω-6 long chain polyunsaturated fatty acid, a senior unsaturated fatty acid. There are many methods to detect arachidonic acid in plasma, such as gas chromatography coupled with mass spectrometry, liquid chromatography with precolumn derivation fluorescence detection, and ELISA method [6,7,8,9,10]. These methods are either too complicated to operate or lack of good reproducibility. The matrix effect of both phospholipids and proteins on the recovery of arachidonic acid was investigated and the final method was applied for the assay of some human plasma samples
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