Schober Yvonne, Wahl Hans Günther, Renz Harald, Nockher Wolfgang Andreas
Institute of Laboratory Medicine and Pathobiochemistry, Philipps-University Marburg, Germany.
Institute of Laboratory Medicine and Pathobiochemistry, Philipps-University Marburg, Germany; Medizinisches Labor Wahl, Lüdenscheid, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Jan 1;1040:1-7. doi: 10.1016/j.jchromb.2016.11.019. Epub 2016 Nov 13.
Cellular fatty acid (FA) profiles have been acknowledged as biomarkers in various human diseases. Nevertheless, common FA analysis by gas chromatography mass spectrometry (GC-MS) requires long analysis time. Hence, there is a need for feasible methods for high throughput analysis in clinical studies. FA was extracted from red blood cells (RBC) and derivatized to fatty acid methyl esters (FAME). A method using gas chromatography tandem mass spectrometry (GC-MS/MS) with ammonia-induced chemical ionization (CI) was developed for the analysis of FA profiles in human RBC. We compared this method with classical single GC-MS using electron impact ionization (EI). The FA profiles of 703 RBC samples were determined by GC-MS/MS. In contrast to EI ammonia-induced CI resulted in adequate amounts of molecular ions for further fragmentation of FAME. Specific fragments for confident quantification and fragmentation were determined for 45 FA. The GC-MS/MS method has a total run time of 9min compared to typical analysis times of up to 60min in conventional GC-MS. Intra and inter assay variations were <10% for all FA analyzed. Analysis of RBC FA composition revealed an age-dependent increase of the omega-3 eicosapentaenoic and docosahexaenoic acid, and a decline of the omega-6 linoleic acid with a corresponding rise of the omega-3 index. The combination of ammonia-induced CI and tandem mass spectrometry after GC separation allows for high-throughput, robust and confident analysis of FA profiles in the clinical laboratory.
细胞脂肪酸（FA）谱已被公认为多种人类疾病的生物标志物。然而，通过气相色谱 - 质谱联用（GC-MS）进行常规FA分析需要较长的分析时间。因此，临床研究中需要可行的高通量分析方法。从红细胞（RBC）中提取FA并衍生化为脂肪酸甲酯（FAME）。开发了一种使用气相色谱串联质谱（GC-MS/MS）结合氨诱导化学电离（CI）的方法来分析人RBC中的FA谱。我们将该方法与使用电子轰击电离（EI）的经典单GC-MS方法进行了比较。通过GC-MS/MS测定了703个RBC样本的FA谱。与EI相比，氨诱导CI产生了足够量的分子离子，用于FAME的进一步裂解。确定了45种FA的用于可靠定量和裂解的特定碎片。与传统GC-MS中长达60分钟的典型分析时间相比，GC-MS/MS方法的总运行时间为9分钟。所有分析的FA的批内和批间变异均<10%。RBC FA组成分析显示，ω-3二十碳五烯酸和二十二碳六烯酸随年龄增加，ω-6亚油酸下降，同时ω-3指数相应上升。GC分离后结合氨诱导CI和串联质谱可在临床实验室中对FA谱进行高通量、稳健且可靠的分析。
