Tetranor-PGDM Quantification in Artificial Urine

Artificial urine (Supplementary Table 1) was spiked with tetranor-PGDM in distilled water at four levels (1.25, 2.5, 5.0, and 10.0 ng/mL). For solid-phase extraction (SPE), 0.4 mL of the urine sample was diluted to 0.8 mL with 0.1% (v/v) formic acid. The mixed solutions were applied to an SPE cartridge (HLB μElution plate, Waters, Massachusetts, USA) preconditioned with 200 μL acetonitrile and distilled water. After washing with 200 μL distilled water and 200 μL hexane, the lipid fractions were eluted with 50 μL acetonitrile. The eluate was collected and dried in vacuo. The resulting residue was reconstituted in 0.4 mL of assay solution. The sample solution containing tetranor-PGDM was then introduced into the EIA. At the same time, tetranor-PGDM was measured by LC-MS/MS as previously described [2 (link), 3 (link)]. The recovery from the SPE procedure was found to be 77.1% by LC-MS/MS (Supplementary Table 2).