利用液相層析串聯質譜儀偵測培養基中溶血磷脂酸的含量   免費試閱

Quantification of Lysophosphatidic Acid in Medium by LC-MS/MS

本研究開發以液相層析串聯質譜儀（LC-MS/MS）偵測細胞培養基中溶血磷脂酸（lysophosphatidic acid, LPA）含量的方法。以C-8 管柱（2.1 × 100 mm i.d.,3.5 μm）搭配分別含有0.5 mM 醋酸銨之水及甲醇移動相進行梯度流洗，流速為0.22 mL/min；質譜條件使用負離子模式搭配多重反應監測進行（multiple reactionmonitoring, MRM）偵測，可得LPA 的母離子為m/z 435、子離子碎裂為m/z 153的訊號。並使用正丁醇進行液—液萃取後，取有機層吹乾以甲醇回溶即可上機偵測；此方法之線性範圍為1 μM ～ 20 μM，R2 為0.9968。偵測極限（limit ofdetection, LOD）為0.3 μM，定量極限（limit of quantitation, LOQ）為1 μM。將此最佳化條件實際應用於培養基中溶血磷脂酸的萃取，可得到105.0% ～ 113.5%之回收率。此方法可以有效並準確地偵測培養基中溶血磷脂酸的含量，並可應用於其他生物樣品中LPA 含量之偵測。

In this study, we develop a new method to detect the concentration of lysophosphatidic acid (LPA) in cell culture medium with LC-MS/MS. The C-8 column (2.1 × 100 mm i.d., 3.5 μm) was used as the separation column and 0.5 mM ammonium acetate in water and methanol were used as mobile phase with flow rate of 0.22 mL/min. Using the negative mode and multiple reaction monitoring (MRM) in mass spectroscopy, we can find the mother ion of LPA at m/z 435 as well as the daughter ion of LPA at m/z 153. The LPA in cell culture medium could be extracted by acidic n-butanol. After evaporated and then redissolved in methanol, the sample could be injected into the msss spectroscopy directly. The linear range of this method was from 1μM ～ 20 μM with R2 of 0.9968. And the limit of detection (LOD) was 0.3 μM, and the limit of quantitation (LOQ) was 1 μM with the recovery from 105.0 to 113.5%. This method not only can detect the LPA in the cell culture accurately and efficiently, but also can be used in the analysis of other bio-samples.