Fig. 3. Composition of lipid classes in viable sperm cell subpopulations with low or high M540 fluorescence. (a) Separation of lipid classes on HPLC as detected by evaporative light scattering detection. Lipid classes were identified by comparison with lipid standards and online electrospray ionization mass spectrometry as triacylglycerols and cholesterol esters (1); cholesterol (2); diacylglycerol (3); ceramides (4); phosphoglycerolipids (5-7) with head-group ethanolamine (PE) (5), choline (PC) (6), serine (PS) (7); sphingomyelin (SM) (8); seminolipid (SGG) and phosphatidylinositol (PI) (9); and lysoPC (10). (b) Online identification of individual molecular species during the elution of PC. The distribution across the mass spectrum results from the variety in fatty radyl chain length, the degree of unsaturation and the type of linkage at the sn-1 position (ester versus ether) of the glycerol backbone. Peaks are labelled with their nominal masses (bottom) or the total number of carbon atoms in the radyl groups and the type of sn-1 linkage (top). PtdCho, 1,2 diacyl phosphatidylcholine; AlkCho, 1-alkyl 2-acyl phosphatidylcholine; PlasCho, 1-alk-1'-enyl 2-acyl phosphatidylcholine (Plasmalogen PC). The italic numbers indicate the total number of unsaturations (double bonds) in the fatty radyl chains. The top line was recorded during elution of lipids from low M540 fluorescent cells, the bottom line during elution of lipids from high M540 fluorescent cells. Note the occurrence of isotope peaks at odd m/z ratios, due to the natural occurrence of approximately 1% [¹³C]. Experiments were performed three times with similar results.