title: Determination of sphingoid bases from hydrolyzed glucosylceramide in rice and wheat by online post-column high-performance liquid chromatography with O-phthalaldehyde derivatization.
abstract: We developed a determination method for sphingoid bases using online post-column high-performance liquid chromatography (HPLC) with O-phthalaldehyde (OPA) derivatization. Good separation was achieved using a reversed-phase column and eluting with 50% acetonitrile containing formic acid and heptafluorobutyric acid. Using these conditions, an excellent linearity (R² > 0.999) was achieved using standard solutions of sphinganine (d18:0), sphingosine (d18:1(4t)), 4-hydroxy-sphinganine (t18:0), glucosylsphingosine (glc-d18:1(4t)), and galactosylsphingosine (gal-d18:1(4t)). Plant glucosylceramides were hydrolyzed with 1 M aqueous HCl in methanol for 18 h at 90°C, followed by extraction of sphingoid bases with diethyl ether in preparation for analysis using the proposed HPLC conditions. The glc-d18:1(4t) standard was also hydrolyzed and analyzed by HPLC using the same procedure, and the d18:1(4t) peak obtained from the hydrolyzed glc-d18:1(4t) standard was used as a reference for calculation. We also confirmed the applicability of this method to the analysis of sphingoid bases in rice and wheat, obtaining relative standard deviations of 8.0% for rice and 4.6% for wheat. The recoveries of spiked rice and wheat samples were 104% and 106%, respectively. Our proposed method enables the straightforward determination of sphingoid bases without expensive facilities, employing fluorescence detection of OPA derivatives.
authors.affiliation: Japan Food Research Laboratories, Nagoya Branch, Nagoya, Japan. gotouh@jfrl.or.jp
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Goto
authors.forename: Keiko
authors.initials: K
authors.lastname: Nishikawa
authors.forename: Noriko
authors.initials: N
authors.lastname: Shionoya
authors.forename: Makoto
authors.initials: M
authors.lastname: Taniguchi
authors.forename: Tomoji
authors.initials: T
authors.lastname: Igarashi
mesh_terms: D002851:Chromatography, High Pressure Liquid; D005963:Glucosylceramides / Q000737:chemistry*; D006868:Hydrolysis; D020407:Internet; D015394:Molecular Structure; D012275:Oryza / Q000737:chemistry*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000032:analysis*; D014908:Triticum / Q000737:chemistry*; D009764:o-Phthalaldehyde / Q000031:analogs & derivatives* / Q000737:chemistry*
chemical_list: D005963:Glucosylceramides; D009764:o-Phthalaldehyde; D013110:Sphingosine