Honda Akira, Yamashita Kouwa, Miyazaki Hiroshi, Shirai Mutsumi, Ikegami Tadashi, Xu Guorong, Numazawa Mitsuteru, Hara Takashi, Matsuzaki Yasushi
Center for Collaborative Research, Tokyo Medical University, Kasumigaura Hospital, Ami, Ibaraki 300-0395, Japan.
J Lipid Res. 2008 Sep;49(9):2063-73. doi: 10.1194/jlr.D800017-JLR200. Epub 2008 May 23.
We have developed a highly sensitive and specific method for the analysis of serum sterol profiles. Sterols in 1 mul of dried serum were derivatized into picolinyl esters (3beta-picolinate) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the electrospray ionization (ESI) mode. In addition to cholesterol, 19 cholesterol precursors, cholestanol, campesterol, sitosterol, and sitostanol were identified simultaneously. Quantitative analyses for the picolinyl esters of 11 available sterols were performed, and detection limits were found to be less than 1 pg on-column. Reproducibilities and recoveries of 8 noncholesterol sterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.6% to 8.2% and 2.5% to 16.5%, respectively. The recovery experiments were performed using 1 mul aliquots of normal human serum spiked with 1 ng to 6 ng of sterols, and recoveries of the sterols ranged from 88.1% to 102.5% with a mean recovery of 98.1%. The present method provides reliable and reproducible results for the identification and quantification of neutral sterols, especially in small volumes of blood samples, which is useful for serological diagnosis of inherited disorders in cholesterol metabolism and for noninvasive evaluation of cholesterol biosynthesis and absorption in humans.
我们开发了一种用于分析血清甾醇谱的高灵敏度和特异性方法。将1微升干燥血清中的甾醇衍生化为吡啶基酯（3β-吡啶甲酸酯），并使用电喷雾电离（ESI）模式通过液相色谱-串联质谱（LC-MS/MS）进行分析。除胆固醇外，还同时鉴定出19种胆固醇前体、胆甾烷醇、菜油甾醇、谷甾醇和谷甾烷醇。对11种可用甾醇的吡啶基酯进行了定量分析，发现柱上检测限小于1皮克。分别根据单向布局和多项式方程验证了8种非胆固醇甾醇的重现性和回收率。计算得出该方法在样品制备之间和测量之间的方差分别为1.6%至8.2%和2.5%至16.5%。使用添加了1纳克至6纳克甾醇的1微升正常人血清等分试样进行回收率实验，甾醇的回收率范围为88.1%至102.5%，平均回收率为98.1%。本方法为中性甾醇的鉴定和定量提供了可靠且可重现的数据，尤其是对于少量血液样本，这对于胆固醇代谢遗传性疾病的血清学诊断以及人类胆固醇生物合成和吸收的非侵入性评估很有用。
