Department of Anesthesiology and Intensive Care Medicine, Jena University Hospital, Jena, Germany.
Center for Molecular Biomedicine, Jena University Hospital, Jena, Germany.
Biomed Chromatogr. 2021 Mar;35(3):e5004. doi: 10.1002/bmc.5004. Epub 2020 Nov 5.
Sphingosine 1-phosphate (S1P) is a bioactive phospholipid and ligand for five G protein-coupled cell-surface receptors designated S1PR1-5. The determination of low levels of S1P remains a challenge and usually requires sophisticated analytical instrumentation and methodology. This report describes a technique using the linear ion trap mode of a basic QTrap triple-quadrupole mass spectrometer. S1P was extracted from acidified biological samples using a modified Folch extraction procedure. After the addition of C17-sphingosine as an internal standard, a step gradient LC method was used to separate the analytes on a reversed-phase C18 MultoHigh analytical column. After the internal standard C17-sphingosine was detected by multiple reaction monitoring (MRM), the detection mode was switched to enhanced product ion (EPI) mode for the detection of S1P. The mode was switched back to MRM again for the detection of other analytes. Using this QTrap method, we reached a limit of detection of 1 nM and a limit of quantification of 3 nM for S1P, which was up to 30 times more sensitive than the MRM mode with the same instrument. Intra-day precision ranged between -3.8 and 6.3%, and inter-day precision was between -13.8 and 3.3%, depending on the spiked S1P concentration.
鞘氨醇 1-磷酸（S1P）是一种生物活性磷脂，也是五个 G 蛋白偶联细胞表面受体（S1PR1-5）的配体。S1P 的低水平测定仍然是一个挑战，通常需要复杂的分析仪器和方法。本报告介绍了一种使用基本 QTrap 三重四极杆质谱仪线性离子阱模式的技术。S1P 是通过改良的 Folch 提取程序从酸化的生物样品中提取的。在加入 C17-鞘氨醇作为内标后，采用阶梯梯度 LC 法在反相 C18 MultoHigh 分析柱上分离分析物。内标 C17-鞘氨醇被多重反应监测（MRM）检测到后，检测模式切换为增强产物离子（EPI）模式以检测 S1P。再次切换回 MRM 模式以检测其他分析物。使用这种 QTrap 方法，我们达到了 1 nM 的检测限和 3 nM 的定量限，这比相同仪器的 MRM 模式敏感 30 倍。日内精密度在 -3.8%至 6.3%之间，日间精密度在 -13.8%至 3.3%之间，具体取决于添加的 S1P 浓度。
