Table 2. Summaries of sample preparations that rely on Solid Phase Extraction partial purification techniques to enrich specific lipid species 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

Author, year	Sample type	Measuring	Methods that use SPE columns to partially purify and enrich samples with specific lipid classes
Balvers, 2009	Plasma	2-AG, 2-AG ether, AEA, DEA, NADA, DLE, NAGly, O-AEA, OLDA, OEA, PEA, SEA	Collected blood in K₃EDTA tubes on ice, centrifuged at 2,000xG, 15min, 4°C, added phenylmethanesulfonyl fluoride (PMSF) to plasma then stored at −80°C. 1mL plasma was added to 4mL acetonitrile + d8–2-AG, d8-AEA, d8-NADA, d8-NAGly + 100μM PMSF, then centrifuged at 3,000xG for 5min, and decanted into 15mL water + 0.133% trifluoro acetic acid (TFA). Partial purification was performed on C8-SPE columns. Columns were initially washed with 1mL methanol, then 1mL water, sample was loaded, washed with 2mL of 20% acetonitrile/ 80% water / 0.1% TFA, and eluted with 2mL of 80% acetonitrile/20% water/0.1% TFA. This elution was evaporated with a vacuum and stored at −80°C until analysis. At the time of MA analysis, sample was reconstituted the in 100μL acetonitrile/0.1% TFA spiked with d4-PEA. 5μL was used for MS analysis.
Taglia-monte, 2021	Plasma	2-AG, AEA, OEA, LEA, PEA	Blood was collected by venipuncture in tubes containing EDTA and kept on ice. Plasma samples were centrifuged 3,000RPM (information on g not provided), 15min, 4°C within 15min of collection, then stored at −80 °C. On day of processing, samples were thawed at 4°C and then kept on ice. 500μL of plasma was diluted 1:2 in distilled water + 50μL of 200ηg/mL d8-AEA. Samples were vortexed, then centrifuged at 21,1000xG, 5min, 4°C. Supernatant partially purified on HLB Oasis solid phase extraction columns that had been conditioned with 1mL MeOH and 1mL water, were washed with 1mL 40% MeOH, and eluted with 1mL of acetonitrile. The eluate was dried under nitrogen, reconstituted in 100μL of acetonitrile: water (50:50), which was used for MS analysis.
Gaisber-ger, 2021	Plasma	AEA	Collected blood in serum- and plasma-specific tubes, then froze and stored at −80°C until processing. Samples were thawed and prior to partial purification via C18 solid phase extraction column separation. Columns were conditioned with 1mL acetonitrile (0.1% formic acid), and 1mL water (0.1% formic acid). 500μL of plasma spiked with 1μL of 1ηg/mL d4-AEA was added to the column. The column was washed with 1mL of 20% acetonitrile (0.1% formic acid), then eluted with 400μL of acetonitrile (0.1% formic acid), which was used for MS analysis.
Biernacki, 2021	Plasma	AEA, 2-AG	Blood was collected in tubes with EDTA and butylhydroxytoluene, centrifuged at 3,000xG 4°C to collect plasma. Luque-Cordoba 2018 is referenced the as protocol for sample preparation and MS analysis.
Luque-Cordoba, 2018	Serum	DHEA, AEA, NAGly, NADA, 2-AG, PEA, DEA, OEA, 2-AG ether, PALDA, OLDA/ODA, SEA, STEARDA	Samples were thawed on ice then centrifuged at 20,200×g for 10 min, then filtrated by a nylon filter with a 0.2 μm pore size. 200μL of the filtrated sample is introduced in an opaque vial with a glass insert and located in the autosampler of the SPE (C18 solid phase extraction) system, which is maintained at 4°C. SPE column is conditioned with 10mL of methanol, then 1mL of methanol, followed by two solvation steps each with 1 mL of 25% (v/v) ACN in water containing 0.7% of formic acid. The 100μL sample loop is filled with the analytical sample that is then loaded in the sorbent cartridge with 1.5mL of solvation solution. In this process, the analytes are retained in the sorbent and the sample matrix removed to the waste. Then, the analytes are eluted with the chromatographic mobile phase (elution time 90 s), and the cartridge is cleaned by five rinsing steps with 5 mL of water, methanol, water, 30% (v/v) ACN in water, and water.
Bashashati et al, 2021	Plasma	AEA, OEA, LEA, DEA, SEA, PEA, 2-AG, 2-PG, 2-LG, 2-OG, AA, LA, OA	Plasma was aliquoted into 150μL volumes for each participant and then added to 2mL of (HPLC)-grade methanol. Solutions were spiked with 500 picomoles deuterium labeled N-arachidonoyl glycine (d8-NAGly; Cayman Chemical) and centrifuged at 19,000 × g for 20 minutes at 24°C. Supernatants were diluted with HPLC water to make a 15% supernatant solution. Lipid extractions were performed using C18 solid-phase extraction columns. SPE columns were conditioned with 5 mL HPLC methanol followed by 2.5 mL HPLC water. Then, the supernatant/water solution was loaded onto the column, followed by 2.5 mL HPLC water. A series of 5 elutions with 1.5 mL 40%, 60%, 75%, 85%, and 100% methanol were collected for MS analysis. #Extractions and MS analysis performed in Bradshaw lab.
Gaitan, 2018	Milk	AA, DHA, EPA, AEA, PEA, OEA, DHEA, EPEA, EEA, 2-acyl glycerols	Participants expressed milk at home and kept in the fridge for a maximum of 24h until transferred to lab on ice. In lab, samples were heated with a 37°C water bath, gently mixed, and aliquoted into 15mL to store at −80°C. To process, samples were thawed in 37°C water bath, vortexed 10sec and then added to an un-specified volume of acetonitrile + PBS + internal standards then centrifuged at 14,000xG, 4°C for 5min. The supernatant was mixed with 4 volumes of 5% phosphoric acid before partial purification on solid phase extraction columns (OASIS HLB reverse phase cartridges). SPEs were conditioned with MeOH and water. Sample loaded, they washed with 40% MeOH, then eluted with acetonitrile. The elution was the evaporated under nitrogen, reconstituted in EtOH, vortexed, sonicated, and centrifuged prior to MS analysis.
Bruun, 2018	Milk	OEA, SEA, PEA	Milk was collected in lab, aliquoted to 10mL, and stored at 5°C for a maximum of 3 days. The sample was then centrifuged3,600RPM, 21°C, 5min, the fat, skimmed and solid fractions separated, and stored at −80°C. To extract lipids, they centrifuged to remove particles, added 20μL of a 20ηg/mL d4-OEA, d3-SEA, d4-PEA, and then passed them through solid phase extraction columns. They washed with 5% MeOH, 0.1% acetic acid, then eluted into 2mL of acetonitrile and 2mL MeOH. Then they dried w/speed vacuum, reconstituted in 100μL MeOH, added 10μL recovery (0.025ug/mL 12-[[cyclohexylamino)carbonyl amino] dodecanoic acid), and centrifuged again using an Eppendorf tube filter prior to MS analysis.