Table 1. Summaries of sample preparations that rely on lipid-lipid extractions (LLE) to enrich lipids prior to MS analysis.

See “List of Abbreviations” for a key to those reagents abbreviated. Comments in italics were not in the original manuscripts but are clarifications by the authors here. Only the first author and publication year are listed in the table, though the full reference is available at the end of the Chapter.

Author, year	Sample type	eCB Lipids Measured	Methods that use LLE plus evaporation/reconstitution as the primary means of lipid enrichment
Ney, 2019	Saliva, plasma	2-AG, AEA	Saliva and plasma samples were frozen at −20C until processed. After thawing then centrifugation, 400μL of saliva supernatant was added to 1600μL of 1:1 methanol: acetone. 400μL of plasma was added to 1mL of ethyl acetate: cyclohexane 1:1. 20ng/mL of d5–2-AG and d4-AEA were added as internal standards and solutions vortexed. Saliva was frozen (−20°C, 1h) and then both saliva and plasma were centrifuged at 4°C. Each was evaporated under nitrogen and reconstituted with either 150μL methanol for saliva or 100μL acetonitrile for plasma. Samples were evaporated again and reconstituted in 50% MeOH for saliva or acetonitrile for plasma to a final volume of 30μL.
Ulu, 2021	Plasma	AEA, DHEA, OEA 2-AG, 2-DG,	To analyze plasma eCBs, they collected blood in K2EDTA tubes, purified lipids as reported in Argueta, 2017.
Argueta, 2017	Plasma	2-AG, AEA	Collected blood via cardiac puncture and kept it in EDTA tubes on ice, then centrifuged 1,500xG, 10min at 4°C to separate plasma and stored at −80°C. Plasma (0.1mL*) was combined with 0.9% saline (0.9 ml) then added to 2mL chloroform. The organic phase was separated on open-bed silica gel chromatography, the resulting eluate dried under nitrogen, resuspended in 0.1mL MeOH: chloroform (9:1), which was used for MS analysis. *amount of plasma used was inferred by the statement that “0.1 mL plasma at the expense of saline” in reference to the 1ml volume of saline. Otherwise, no clear statement on the amount of plasma per sample was used.
Martin-Perez, 2021	Plasma	2-AG, AEA, POEA	Collected blood in K2-EDTA tubes, centrifuged at 1,700xG, 15min, 4°C, then stored at −80°C. Plasma (0.5mL) was spiked with deuterium labeled standards in 0.5mL of 0.1M ammonium acetate buffer. Tert-butyl methyl ether (6.0mL) was added to the spiked plasma solution and the organic layer separated then evaporated at 39°C under nitrogen. Sample was reconstituted in 100μL water: acetonitrile (1:9, v/v) that contained 0.1% formic acid, and used for MS analysis.
Sempio, 2021	Plasma	NAEs, 2-AG, DH-g-LEA, NADA, 2-AGE, ODA	200μL of plasma was spiked with deuterium-labeled standards and added to 800μL acetonitrile. The solution was mixed via vortex for 10min, then centrifuged (25,000xG, 4°C, 10min). 750μL of the supernatant was added to 450μL of 0.1% formic acid in water and mixed via vortex. 400μL of the solution was used for MS analysis.
De Icco, 2021	Plasma	AEA, PEA	500μL of plasma was mixed with 1mL of methanol + d4-AEA and d4-PEA and added to 2mL of chloroform, centrifuged at 3000xRPM (no g indicated) for 15min at 4°C. The organic phase was transferred to glass vials, and the aqueous fraction extracted again with chloroform (1 mL) and 500μL phosphate buffered saline. The 2 resulting organic phases were combined, dried under N2, and the residues dissolved in 2mL chloroform. Silica Gel G columns were used to fraction the extracts, eluting into 2mL chloroform/MeOH (9:1), then 2mL chloroform/MeOH (8:2). The solutions were evaporated under nitrogen and reconstituted in 75μL of 9:1 chloroform/MeOH to be analyzed via MS.
Manca, 2021	Plasma	NAEs 2-acyl glycerols	Same protocol as Turcotte 2020 to extract lipids (below)
Turcotte, 2020	Plasma	2-acyl glycerols LPA; free fatty acids including AA; AEA	200μL plasma was added to 300μL TRIS buffer, then added 2mL toluene + internal standards. Solution was mixed via vortex and then separated via centrifuge at 4,000xG for 5min. Samples were then placed in an ethanol-dry-ice bath (−80°C) to freeze the aqueous phase (bottom). The organic phase (top) was then collected and evaporated to dryness under a stream of nitrogen. Samples were reconstituted in 25μL of HPLC solvent A (H2O with 0.05% acetic acid and 1 mM NH4+) and 25μL of solvent B (MeCN/H2O, 95/5, v/v, with 0.05% acetic acid and 1 mM NH4+). A 25μL aliquot was used for MS analysis.
Brunk-horst Kanaan, 2021	Plasma	AEA, 2-AG, 1-AG, OEA, PEA	Blood was collected in K+-EDTA tubes on ice, centrifuged at 3000RCF (no G provided), for 4°C, 10min, then plasma stored at −80°C until processing. Plasma was thawed and internal standard added. Ethyl acetate/hexane was added to the plasma solution as 9:1 (v:v). Organic phase was removed, evaporated, then reconstituted in acetonitrile for MS analysis.
Kirkwood, 2016	Serum	AEA, OEA, PEA, 2/1-AG	Collected blood was “spun” to separate serum, then frozen in aliquots at −80°C. Sample preparation protocol was adapted from Zoerner, 2012. 50μL of serum was added to 1mL of toluene + 100pg/mL internal standards, mixed via vortex for 15min, medium speed at room temperature. 400μL of water + 3% formic acid was added and mixed via vortex for 5min. Samples were held at −80°C for 2hr, then moved to 4°C to thaw and centrifuged at 3,000xG for 15min at 4°C. Organic phase was evaporated under nitrogen, resuspended in 40μL of 50/50 acetonitrile/MeOH, then mixed via vortex for 2min. 60μL water + 0.2% formic acid was added and mixed via vortex for 2min and then used for MS analysis.
Siebers, 2021	Plasma	AEA, 2-AG, PEA, AA	Collected blood in a tube coated with EDTA and centrifuged 10min, 2000xG, at 4°C then stored at −85°Cs. eCBs were measured from 100μL plasma. Detailed methods were not provided and authors referenced Post 2020, Lerner 2019, and Lerner 2018.
Post et al 2020	N/A	N/A	This reference is to a “Jove” video presentation that is still inaccessible with a notice that “Video is coming soon” in a Jan 2022 search, so it was not accessible to evaluate.
Lerner, 2019	Brain, plasma	2-AG, AEA, free fatty acids including AA, LA, and EPA,	40μL of plasma was combined with 1000μL MTBE/methanol (10:3), spiking solution, and 250 μL water+ 5μM THL/URB597 and 10ug/mL BHT. The authors then reference the methods of Lerner 2016; however, there was no “Lerner 2016” that matched the reference; however, this was likely due to an online publication date, as there is a Lerner 2017 that matches the title. (see below).
Lerner, 2017	Brain, liver, plasma	NAEs including OEA, PEA, AEA; 2-AG; AA; eicosanoids	Blood was collected in chilled 500μL EDTA tubes containing 10μM indomethacin and 25 μM tetradydrolipstatin/URB, and centrifuged 2,000xG, 4°C 10min to separate plasma which was removed and stored at −80°C. To extract lipids they used 100μL of plasma + 1200μL ethylacetate/n-hexane (9:1) + internal standards + 600μL of 0.1M formic acid. They vortexed for 2 min at 4°C, then centrifuged 13,000xRPM for 20min at 4°C, froze them for 10 min, then evaporated under nitrogen at 37°C and reconstituted in 50μL ACN/water (1:1).