Development and validation of LC-MS/MS method for determination of very long acyl chain (C22:0 and C24:0) ceramides in human plasma
Development and validation of LC-MS/MS method for determination of very long acyl chain (C22:0 and C24:0) ceramides in human plasma
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DOI：
10.1007/s00216-013-7166-9
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发表时间：
2013-09-01
影响因子：
4.3
通讯作者：
Jiang, Xuntian
中科院分区：
文献类型：
--
作者：
Jiang, Hui;Hsu, Fong-Fu;Jiang, Xuntian
神经酰胺是鞘脂合成代谢和分解代谢途径中的关键代谢物。极长脂肪酰基链神经酰胺N-（二十二烷酰基）-sphing-4-enine（Cer（22：0））和N-（二十四烷酰基）-sphing-4-enine（Cer（24：0））具有多种生物学功能。这些鞘脂在组织和循环中的水平升高与胰岛素抵抗和糖尿病有关。为了便于对来自人类受试者的临床样本中的这些非常长链的神经酰胺进行定量，我们开发了一种灵敏、准确和高通量的测定方法，用于测定人血浆中的Cer（22：0）和Cer（24：0）。Cer（22：0）和Cer（24：0）及其氘代内标物通过蛋白沉淀提取并通过HPLC色谱分离。分析物及其内标物使用正离子电喷雾质谱电离，然后用串联质谱仪进行多反应监测检测。总液相色谱-串联质谱法（LC-MS/MS）运行时间为5 min。该测定法显示，人血浆中Cer（22：0）和Cer（24：0）的线性动态范围分别为0.02 - 4和0.08 - 16 μ g/ml，血浆中相应的绝对回收率分别为109和114%。Cer（22：0）和Cer（24：0）的定量下限分别为0.02和0.08 μ g/ml。对于校准曲线范围内的浓度，获得了可接受的精密度和准确度。通过半自动化的形式和较短的LC运行时间，可以轻松实现类似于200个样品/天的通量。
Ceramide is a key metabolite in both anabolic and catabolic pathways of sphingolipids. The very long fatty acyl chain ceramides N-(docosanoyl)-sphing-4-enine (Cer(22:0)) and N-(tetracosanoyl)-sphing-4-enine (Cer(24:0)) are associated with multiple biological functions. Elevated levels of these sphingolipids in tissues and in the circulation have been associated with insulin resistance and diabetes. To facilitate quantification of these very long chain ceramides in clinical samples from human subjects, we have developed a sensitive, accurate, and high-throughput assay for determination of Cer(22:0) and Cer(24:0) in human plasma. Cer(22:0) and Cer(24:0) and their deuterated internal standards were extracted by protein precipitation and chromatographically separated by HPLC. The analytes and their internal standards were ionized using positive-ion electrospray mass spectrometry, then detected by multiple-reaction monitoring with a tandem mass spectrometer. Total liquid chromatography-tandem mass spectrometry (LC-MS/MS) runtime was 5 min. The assay exhibited a linear dynamic range of 0.02-4 and 0.08-16 mu g/ml for Cer(22:0) and Cer(24:0), respectively, in human plasma with corresponding absolute recoveries from plasma at 109 and 114 %, respectively. The lower limit of quantifications were 0.02 and 0.08 mu g/ml for Cer(22:0) and Cer(24:0), respectively. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges. With the semi-automated format and short LC runtime for the assay, a throughput of similar to 200 samples/day can easily be achieved.