Biological sample collection and analysis
Participants provided an overnight fasted blood sample and a spot urine sample that was their first void of the day.For FLAV and methylxanthine analyses, the metabolites were purified from 50 μL human urine by 96-well plate solid phase extraction (Strata™-X Polymeric Reversed Phase, microelution 2 mg/well). The solid phase extraction treated samples were chromatographically separated and quantified using Exion high-performance ultrahigh performance LC-tandem MS SCIEX QTRAP 6500+ enhanced ultrahigh performance hybrid triple quadrupole-linear ion trap MS with electrospray IonDrive Turbo-V Source. The samples were injected into a Kinetex PFP UPLC column (1.7 μm particle size, 100 Å pore size, 100 mm length, 2.1 mm internal diameter; Phenomenex) with oven temperature maintained at 37°C. Mobile phase A and mobile phase B consisted of 0.1% v/v formic acid in water (Optima grade, Fisher Scientific) and 0.1% v/v formic acid in LC-MS grade acetonitrile (Honeywell Burdick and Jackson) respectively, with a binary gradient ranging from 2% B to 90% B over 30 min and flow rate gradient from 0.55 mL/min to 0.75 mL/min. MS/MS scanning was accomplished by advanced scheduled multiple reaction monitoring using polarity switching between positive and negative ionization mode in Analyst (v.1.6.3, SCIEX) and with quantitation conducted using MultiQuant (v.3.0.2, SCIEX) software platforms. Internal standards included L-tyrosine-The targeted metabolite analysis protocol was optimized and validated to detect 43 analytes, which were quantified relative to 12 authentic commercial and synthetic standards. Where reference standards for metabolites (including FLAV Phase II conjugates) were not available (31 analytes), identification was based on fragmentation profiling involving the precursor structure and 3 to 5 product transitions and confirmed in pooled samples. These metabolites were then quantified relative to their closest structural reference standard with similar ionization intensities (For the fatty acid analysis, plasma samples were analyzed for AA, EPA, and DHA using free/NEFA and lysophosphatidylcholine (LPC) fractions. NEFA and LPC were chosen because they are quantitatively the most important and efficient respectively at delivering DHA to the brain, across the blood brain barrier [BDNF was quantified using a commercially available ELISA assay in serum samples, according to the manufacturer’s instructions (ab212166 – Human BDNF SimpleStep ELISA Kit, Abcam).