α-GalCer is detected in serum using SFC/HRMS/MS. (A) Separation of the C:M = 19:1 fraction from serum by SFC. Samples, including HexCer, with a RT of 9.5–13.5 min were fractionated every 0.5 min. The other parts of the samples were separated into four subfractions: 0–9.5 (#1), 13.5–24 (#2), 24–34.5 (#3), and 34.5–45 min (#4). (B) HRMS chromatograms of the synthesized four diastereomers of HexCer (upper) and HexCer in serum (lower) (d18:1/16:0 and d18:1/24:1) obtained using SFC/HRMS. The mass error tolerance of the precursor ions (m/z 700.5722 and m/z 810.6817) was <7 ppm. (C) Monoisotopic (m/z 702.5858) and ¹³C₁ isotopic spectra (m/z 703.5911) of α-GalCer (d18:0/16:0) candidate in serum. (D) The HRMS chromatograms were plotted from the theoretical m/z ± 7 ppm of candidate HexCer molecular species in serum obtained using SFC. The colored shadows indicate the peaks coincident with the theoretical RT of β-GlcCer (green) and β-GalCer (yellow), respectively. (E) Representative HRMS/MS spectra of four synthesized diastereomers of HexCer (d18:0/16:0) (m/z 702.5878, as [M+H]⁺) obtained using HRMS/MS. From top to bottom, collision energy settings are −10 eV (+), −20 eV (++), −30 eV (+++), and −40 eV (++++). Data are representative of three independent experiments (B–E).