Center for Mitochondrial Diseases, Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
J Lipid Res. 2010 Apr;51(4):856-65. doi: 10.1194/jlr.D002857. Epub 2009 Oct 30.
An improved high-performance liquid chromatography-mass spectrometry method for the separation and characterization of cardiolipin molecular species is presented. Reverse-phase ion pair chromatography with acidified triethylamine resulted in increased chromatographic retention and resolution when compared with chromatography without acidified triethylamine. Using a hybrid triple quadrupole linear ion trap mass spectrometer to generate MS/MS spectra revealed three regions within each spectrum that could be used to deduce the structure of the cardiolipin molecular species: the diacylglycerol phosphate region, the monoacylglycerol phosphate region, and the fatty acid region. Cardiolipin standards of known composition were analyzed and exhibited expected chromatographic and mass spectral results. Two minor components in commercial bovine heart cardiolipin, (with the same molecular weight but different chromatographic retention times), were shown to differ by fatty acid composition: (C18:2)(2)(C18:1)(2) versus (C18:2)(3)(C18:0)(1). These compounds were then analyzed by HPLC-MS(3) to examine specific diacylglycerol phosphate generated fatty acid fragmentation. Also, two commercial sources of bovine heart cardiolipin were shown to have minor differences in cardiolipin species content. Cardiolipin isolated from rat liver, mouse heart, and dog heart mitochondria were then characterized and the relative distributions of the major cardiolipin species were determined.
呈现了一种改进的高效液相色谱-质谱法，用于分离和鉴定心磷脂分子种类。与没有酸化三乙胺的色谱相比，酸化三乙胺的反相离子对色谱导致色谱保留和分辨率增加。使用混合三重四极杆线性离子阱质谱仪生成 MS/MS 谱揭示了每个谱图中的三个区域，可用于推断心磷脂分子种类的结构：二酰基甘油磷酸酯区域、单酰基甘油磷酸酯区域和脂肪酸区域。分析了具有已知组成的心磷脂标准品，表现出预期的色谱和质谱结果。商业牛心心磷脂中的两种次要成分（具有相同的分子量但不同的色谱保留时间）通过脂肪酸组成不同：(C18:2)(2)(C18:1)(2)与(C18:2)(3)(C18:0)(1)。然后通过 HPLC-MS(3)分析这些化合物，以检查特定的二酰基甘油磷酸酯生成的脂肪酸片段化。此外，还表明两种商业来源的心磷脂在心磷脂种类含量上存在微小差异。然后对来自大鼠肝、小鼠心和狗心线粒体的心磷脂进行了表征，并确定了主要心磷脂种类的相对分布。
