A convenient method for the determination of the stereochemical configuration and alkyl groups of natural 1-
O-alkylglycerols was developed. For this purpose, 1-
O-alkylglycerols were converted into bis-3,5-dinitrophenylurethane (DNPU) derivatives, followed by chiral-phase high-performance liquid chromatography (HPLC). Complete enantiomer separation was achieved using two columns (25 cm × 4.6 mm i.d., 5 μm particle size) having chiral phases of opposite configurations,
N -[(
S)-1-(1-naphthyl)ethylaminocarbonyl]-(
S)-
tert-leucine and
N -[(
R)-1-(1-naphthyl)ethylaminocarbonyl]-(
R)-
tert-leucine, which caused a reversal in the order of enantiomer elution. The analysis was optimized by using a ternary mobile phase, hexane-dichloromethane-ethanol, and detection at 254 nm. Chiral-phase HPLC in conjunction with electrospray ionization mass spectrometry (ESI-MS) of the derivatives was carried out using an ion-trap mass spectrometer. Post-column addition of 2-propanol was used to assure electrospray ionization in the negative mode. The spectra showed a prominent fragment [M−DNPU]
− ion, by which individual molecular species could be identified. This method was standardized with synthetic 1-
O-, 2-
O- and 3-
O-hexadecyl-
sn-glycerols (16 : 0) and was applied to the identification and quantification of individual molecular species of enantiomeric 1-
O-alkylglycerols derived from muscle lipids from a deep-sea teleost fish,
Stromateus Stellatus. The results clearly showed that the glycerol moieties in all of the alkylglycerols have the S-configuration (1-
O-alkyl-
sn-glycerols), and that the major alkyl groups are 16 : 0, 18 : 1, 14 : 0 and 18 : 0. The method demonstrates that the chiral-phase HPLC in conjunction with ESI-MS provides unambiguous information about the chirality and the alkyl groups of natural alkylglycerols.
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