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2 protocols using α linolenic acid d14

1

Lipid Extraction and Derivatization Protocol

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HPLC grade water, chloroform, methanol, formic acid, and anhydrous acetonitrile required for lipid extraction were purchased from VWR (West Chester, PA, United States). Hydrochloric acid and butylated hydroxytoluene (BHT) were purchased from Sigma (St. Louis, MO, United States). Linoleic Acid-d4, α-Linolenic Acid-d14, Arachidonic Acid-d8, Eicosanoic Acid-d3, Eicosapentaenoic Acid-d5, Docosanoic Acid-d43, and Docosahexaenoic Acid-d5 (Avanti Polar Lipids, Alabaster, AL, United States) were used as internal standards.to monitor PUFA extraction recovery and for quantification. Non-deuterated FA standards containing a mixture of 50 free fatty acids were purchased from NuChek Prep (Elysian, MN, United States). Pentafluorobenzyl bromide (PFBBr) from Thermo Fisher Scientific (Bellafonte, PA, United States) and NN-diisopropylethanolamine (DIPE) from Sigma-Aldrich were used for synthesizing PFBBr-derivatives of PUFAs.
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2

Quantification of Free Fatty Acids

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HPLC grade water, chloroform, methanol, formic acid, and anhydrous acetonitrile were purchased from VWR (West Chester, PA). Hydrochloric acid and butylated hydroxytoluene (BHT) were purchased from Sigma (St Louis, MO). Deuterated fatty acid standards (Decanoic Acid-d19, Hexadecanoic Acid-d4, Oleic Acid-d17, Linoleic Acid-d4, α-Linolenic Acid-d14, Arachidonic Acid-d8, Eicosanoic Acid-d3, Eicosapentaenoic Acid-d5, Docosanoic Acid-d43, and Docosahexaenoic Acid-d5) were purchased from Avanti Polar Lipids (Alabaster, AL). Non-deuterated standard containing a mixture of 50 free fatty acids was purchased from NuChek Prep (Elysian, MN). Derivatizing reagents Pentafluorobenzyl Bromide (PFBBr) was purchased from Thermo Scientific (Bellafonte, PA) and 99.5% redistilled NN-diisopropylethanolamine (DIPE) from Sigma Aldrich.
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