An Optimized High Throughput Clean-Up Method Using Mixed-Mode SPE Plate for the Analysis of Free Arachidonic Acid in Plasma by LC-MSMS

Abstract EN

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.