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.

中文翻译：

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QTrap方法的开发和验证，用于1-磷酸鞘氨醇的灵敏定量

鞘氨醇1-磷酸（S1P）是一种生物活性磷脂，是五个G蛋白偶联的细胞表面受体S1PR1-5的配体。确定低水平的S1P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三重四极杆质谱仪的线性离子阱模式的技术。使用改良的Folch提取程序从酸化的生物样品中提取S1P。加入C17-鞘氨醇作为内标后，采用逐步梯度液相色谱法在反相C18 MultoHigh分析柱上分离分析物。通过多反应监测（MRM）检测到内标C17-鞘氨醇后，将检测模式切换为增强产物离子（EPI）模式以检测S1P。再次将模式切换回MRM，以检测其他分析物。使用这种QTrap方法，我们对S1P的检测限为1 nM，定量限为3 nM，其灵敏度是使用同一仪器的MRM模式的30倍。根据加标的S1P浓度，日内精度介于−3.8和6.3％之间，日间精度介于−13.8和3.3％之间。