Determination of sphingoid bases from hydrolyzed glucosylceramide in rice and wheat by online post-column high-performance liquid chromatography with O-phthalaldehyde derivatization

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  • Determination of sphingoid bases from hydrolyzed glucosylceramide in rice and wheat by online post-column high-performance liquid chromatography with <i>O</i>-phthalaldehyde derivatization

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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 (d18:14t), 4-hydroxy-sphinganine (t18:0), glucosylsphingosine (glc-d18:14t), and galactosylsphingosine (gal-d18:14t). 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-d18:14t standard was also hydrolyzed and analyzed by HPLC using the same procedure, and the d18:14t peak obtained from the hydrolyzed glc-d18:14t 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.

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