Determination of docosahexaenoic acid and arachidonic acid concentrations

The concentrations of docosahexaenoic acid (DHA) and arachidonic acid (ARA) in serum samples were determined by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

A standard solution of ARA (50 μg/mL) and of DHA (500 ng/mL, both obtained from Cayman Chemical, Ann Arbor, USA) was prepared in methanol. This solution was further diluted with methanol/butylhydroxytoluol (BHT) (100:0.1 v/v) to obtain working standard solutions covering the following concentration ranges: 2.5 ng/mL to 500 ng/mL for ARA and 0.025 ng/mL to 5 ng/mL for DHA.

Calibration standard samples and quality control (QC) samples were prepared by spiking 50 μL PBS with 20 μL standard working solution and 20 μL internal standard solution ([²H₈]- ARA (125 ng/mL) and [²H₅]-DHA (10 ng/mL both obtained from Cayman Chemical, Ann Arbor, USA)). Serum samples were diluted by factor 5 and 10 and spiked with 20 μL methanol/BHT (100:0,1 v/v) and 20 μL internal standard solution.

Afterwards, analytes were extracted from the prepared standard, QC and serum samples by liquid-liquid extraction with ethyl acetate. 600 μL of ethyl acetate were added to the prepared sample, the mixture was vortexed and centrifuged at 20,000 g for 5 minutes. The organic phase was transferred to a new tube. The extraction step was repeated and the combined organic phases were evaporated at a temperature of 45°C under a gentle stream of nitrogen. The residues were reconstituted with 50 μL of methanol/water/BHT (50:50:10–4, v/v/v), centrifuged for 2 minutes at 10,000 g and then transferred to glass vials prior to injection into the LC-MS/MS system.

DHA and ARA were analyzed using a Gemini NX column (150 x 2 mm I.D., 5 μm particle size and 110 Å pore size from Phenomenex, Aschaffenburg, Germany) coupled to a hybrid triple quadrupole tandem mass spectrometer 5500QTRAP (Sciex, Darmstadt, Germany). Separation was carried through under gradient conditions with solvent A (0.01% ammonia in water) and solvent B (0.01% ammonia in acetonitrile). The following gradient was employed: 0 min 85% A, 4 min 60% A, 6–7 min 10% A, 7.5–11.5 min 85% A. MS parameters were set as follows: Ionspray voltage -4500 V, source temperature 550°C, curtain gas 35 psi nebulizer gas 50 psi, heating gas 50 psi, collision gas 9 psi. The analysis was done in multiple reaction monitoring (MRM) mode with all quadrupoles running at unit resolution. The following precursor-to-product ion transitions were used for quantification: m/z 327.3 → 229.0 for DHA (declustering potential -140 V, collision energy -17 V) and m/z 303.0 → 40.9 for ARA (declustering potential -175 V, collision energy -80 V).

Data acquisition was done using Analyst Software V.1.6.2, quantitation was performed with MultiQuant Software V3.02. (Sciex, Darmstadt, Germany) using the internal standard method (isotope-dilution mass spectrometry). Ratios of analyte peak area and internal standard peak area (y-axis) were plotted against concentration (x-axis) and calibration curves for each analyte were calculated by linear regression with 1/concentration weighting.