Quantitation of Poly Unsaturated Fatty Acids in the OPPERA Study

Published: 4 July 2022| Version 3 | DOI: 10.17632/gpptkyynwr.3
Diane Weatherspoon,


This is a set of quantitative LC-MS/MS data acquired on extracted human red blood cells form the OPPERA study. An assortment of Omega 3 and 6 fatty acids were monitored as well as their corresponding lipid mediators: Eicosapentaenoic acid, α-linolenic acid, Docosahexaenoic acid, Arachidonic acid, Linolenic acid, Resolvin 1, Prostaglandin E2, Protectin D1, Maresin 1, Maresin 2, 18-HEPE, 9-HODE, and 17-HDHA. The analysis was performed in 7 different batches over the course of 3 weeks on a Thermo TSQ Vantage triple quadrupole mass spectrometer coupled to a Waters Acquity UPLC. The purpose of this analysis was to relate the amount of free poly unsaturated fatty acids to the absence or presence of pain conditions in humans.


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Sample Collection: At the OPPERA-II study visit, a non-fasting 20 mL sample of circulating blood was obtained by venipuncture and collected into tubes containing EDTA that were promptly centrifuged for 10 min at 4°C. After removing the supernatant plasma, erythrocytes were washed with sodium perborate, vortexed and again centrifuged. After removing the sodium perborate supernatant, erythrocytes were aliquoted into 400 µL cryotubes prior to storage at -80C. Sample Extraction: Red blood cells were stored at -80°C until extraction. To 150 µL of red blood cells, 1 mL of 90:10 methanol to water was added. Samples were vortexed then centrifuged at 20,000 rcf for 10 minutes. The supernatant was dried down and resuspended in 150 µL of 90:10 methanol to water containing deuterated internal standards (50 ng/mL). Two quality control standards (20 and 500 ng/mL) as well as the deuterated internal standard mixture were analyzed at least twice per batch of 96 samples to ensure accuracy and reproducibility of the LC-MS/MS analysis. The calibration curve was analyzed twice per batch of 96 samples and averaged to minimize error across the analysis, using standard deviation as error bars. Instrument Methods: Samples were analyzed with a Waters Acquity Ultra-Performance Liquid Chromatography system tandem to a ThermoScientific TSQ Vantage. Separations were performed on a 150 mm x 2.1 mm BEH C18 with a flow rate of 0.25 mL/min and an injection volume of 10 µL. The column was heated to 45 C while the autosampler was chilled to 10 C. Initial mobile phase composition was 65% A (water with 30 mM ammonium formate) and 35% B (80% acetonitrile 20% methanol). A linear decrease was performed to 45% A at 2 min with a hold for 1 min. Another linear decrease to 20% A was performed at 7 min followed by a decrease to 5% A at 8 min and another at 10 min to 0% A. The gradient was returned to starting conditions at 13 min and held for 3 min for a total run time of 16 min (shown in tale below). Multiple reaction monitoring (MRM) was performed in negative mode. The peak width was set to 0.7 Da with a scan time of 0.05 sec per transition. The transitions are provided in Table 1. The source conditions were as follows: spray voltage 3200 V, sheath gas 50 units, auxiliary gas 15 units, vaporizer temp 300 °C, S-Lens 35 V and capillary temp 270°C. The scan settings for each MRM were as follows: scan time 0.05 sec, scan width (m/z)= 0.7, peak width Q1 0.7, and peak width Q3 0.7. Argon was used as the collision cell gas. MRM scans were separated into 3 segments. The first segment is 0-3.7 min and includes Resolvin 1 and Prostaglandin E2. The second segment takes place between 3.7-8 min and includes 9-HODE, 18-HEPE, 17-HDHA, Maresin 2, Maresin 1, Protectin D1, and Maresin 1-D5. The lasts segment is 8-16 min and includes EPA, ALA, LA, AA, DHA, EPA-D5, DHA-D5, and LA-D4. Compounds were quantified over a range of 1000 ng/mL to 0.975 ng/mL with an internal standard concentration of 50 ng/mL.


University of North Carolina at Chapel Hill


Omega 3 Fatty Acid, Polyunsaturated Fatty Acid, Quadrupole Mass Spectrometry, Biological Science Quantitative Method