Department of Gastroenterology and Hepatology, Cleveland Clinic, Cleveland, OH 44195, USA.
Anal Biochem. 2010 Jun 1;401(1):154-61. doi: 10.1016/j.ab.2010.02.023. Epub 2010 Feb 21.
We present an optimized and validated liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the simultaneous measurement of concentrations of different ceramide species in biological samples. The method of analysis of tissue samples is based on Bligh and Dyer extraction, reverse-phase high-performance liquid chromatography separation, and multiple reaction monitoring of ceramides. Preparation of plasma samples also requires isolation of sphingolipids by silica gel column chromatography prior to LC-ESI-MS/MS analysis. The limits of quantification were in a range of 0.01-0.50ng/ml for distinct ceramides. The method was reliable for inter- and intraassay precision, accuracy, and linearity. Recoveries of ceramide subspecies from human plasma, rat liver, and muscle tissue were 78 to 91%, 70 to 99%, and 71 to 95%, respectively. The separation and quantification of several endogenous long-chain and very-long-chain ceramides using two nonphysiological odd chain ceramide (C17 and C25) internal standards was achieved within a single 21-min chromatographic run. The technique was applied to quantify distinct ceramide species in different rat tissues (muscle, liver, and heart) and in human plasma. Using this analytical technique, we demonstrated that a clinical exercise training intervention reduces the levels of ceramides in plasma of obese adults. This technique could be extended for quantification of other ceramides and sphingolipids with no significant modification.
我们提出了一种优化和验证的液相色谱-电喷雾串联质谱（LC-ESI-MS/MS）方法，用于同时测量生物样本中不同神经酰胺种类的浓度。组织样品的分析方法基于 Bligh 和 Dyer 提取、反相高效液相色谱分离和神经酰胺的多重反应监测。血浆样品的分析前还需要通过硅胶柱色谱法分离鞘脂，然后再进行 LC-ESI-MS/MS 分析。不同神经酰胺的定量下限在 0.01-0.50ng/ml 范围内。该方法在精密度、准确度和线性方面具有良好的重现性。从人血浆、大鼠肝和肌肉组织中回收神经酰胺亚类的回收率分别为 78-91%、70-99%和 71-95%。使用两种非生理奇数链神经酰胺（C17 和 C25）内标，可在单次 21 分钟的色谱运行中分离和定量几种内源性长链和超长链神经酰胺。该技术已应用于定量不同大鼠组织（肌肉、肝脏和心脏）和人血浆中的不同神经酰胺种类。使用这种分析技术，我们证明了临床运动训练干预可降低肥胖成年人血浆中神经酰胺的水平。该技术可扩展用于定量其他神经酰胺和鞘脂，无需进行重大修改。
