2D-TLC was performed on silica gel 60, 10×10 cm thin-layer plates. The solvents used were chloroform/methanol/concentrated ammonia/water, 65 : 30 : 2.5 : 2.5 (by vol.), in the first dimension and chloroform/methanol/acetic acid/water, 80 : 18 : 12 : 5 (by vol.), in the second dimension. Acid hydrolysis of lipids on TLC plates using HCl fumes for detection of plasmalogens has been described previously.Amine-containing lipids were detected using 0.3 % ninhydrin in ethanol, followed by heating at 120 °C for 5–10 min. Phosphorus-containing lipids were detected with 0.3 % (w/v) molybdenum blue (Sigma). Standards of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL) from Sigma-Aldrich, and of glycerol acetal of plasmenylethanolamine (GAPlaE) isolated from C. butyricum, were run alongside the lipid samples in both the first and second dimensions. The standards were spotted in two lanes at the left side of the plate for the first dimension run and at the right side of the top of the plate for the second dimension. HCl hydrolysis of plasmalogens results in the formation of the corresponding lyso lipid, i.e. with the remaining sn-2 acyl chain and long-chain aldehydes. HCl hydrolysis of the GAPlaE results in the formation of lyso-PE, and hydrolysis of phosphatidylglycerol acetal of plasmenylethanolamine (PGAPlaE) results in the formation of lyso-PE and PG in addition to long-chain aldehydes