Formalin-fixed paraffin-embedded (FFPE) tissues play an irreplaceable role in cancer research. Although extensive research has been conducted for the detection of DNA, RNA and proteins in FFPE samples, literature dealing with the FFPE determination of small molecules is scarce. In this study, we aimed to explore the potential of targeted metabolomics in FFPE specimens. For that purpose, we developed a LC-MS/MS method for the quantification of acidic metabolites in FFPE samples. The method involves trimming tissue slices from FFPE blocks, deparaffinization, lysis of the tissue, o-benzyl hydroxylamine derivatization and LC-MS/MS detection. Deparaffinization and lysis steps were optimized to maximize the analytes extraction and to minimize the effect of the ubiquitous presence of some metabolites in the paraffin. Two validation approaches were applied: (i) using blank paraffin as matrix and (ii) using actual human FFPE tissue samples by standard additions. The method quantified 40 metabolites with appropriate accuracy (commonly 80–120%) and precision (CV 2–19%) in both validation approaches. LLOQs ranging 0.88–2001 pg mg⁻¹ with low-moderate matrix effects (commonly 85–115%) were obtained. FFPE samples from 15 patients with colorectal cancer were analyzed and metabolites concentrations in tumor vs matched normal FFPE tissues were compared. Results show that tumor tissues have a well-established fingerprint including an increase in ketogenesis, a decrease in lipogenesis and an imbalance in the tricarboxylic acid cycle.

福尔马林固定石蜡包埋（FFPE）组织在癌症研究中起着不可替代的作用。尽管已经进行了广泛的研究以检测FFPE样品中的DNA，RNA和蛋白质，但有关FFPE测定小分子的文献很少。在这项研究中，我们旨在探索FFPE标本中靶向代谢组学的潜力。为此，我们开发了一种LC-MS / MS方法来定量FFPE样品中的酸性代谢物。该方法包括从FFPE块中修剪组织切片，脱石蜡，裂解组织，邻苄基羟胺衍生化和LC-MS / MS检测。优化了去石蜡和裂解步骤，以最大程度地提高分析物的提取率，并最小化石蜡中某些代谢物普遍存在的影响。应用了两种验证方法：（i）使用空白石蜡作为基质，以及（ii）通过标准添加使用实际的人FFPE组织样品。该方法在两种验证方法中均以适当的准确度（通常为80–120％）和精确度（CV为2–19％）对40种代谢物进行了定量。LLOQ范围为0.88-2001 pg毫克^-1的基质效应较低（通常为85–115％）。分析了15例大肠癌患者的FFPE样品，比较了肿瘤与匹配的正常FFPE组织中的代谢产物浓度。结果表明，肿瘤组织具有完善的指纹，包括生酮增加，脂肪生成减少和三羧酸循环失衡。