Quantitation of Poly Unsaturated Fatty Acids in the OPPERA Study
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 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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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). Time %A %B 0 65 35 2 45 55 3 45 55 7 20 80 8 5 95 10 0 100 13 65 35 16 65 35 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.