Quantitation of Poly Unsaturated Fatty Acids in the OPPERA Study
Description
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.
Files
Steps to reproduce
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.