Department of Biotechnology, Chemistry, and Pharmacy (DBCF), University of Siena, 53100 Siena Italy.
Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy; Department of Life Sciences, University of Siena, 53100 Siena, Italy.
J Pharm Biomed Anal. 2023 Oct 25;235:115599. doi: 10.1016/j.jpba.2023.115599. Epub 2023 Jul 23.
Short-chain fatty acids (SCFAs), the end products of gut microbial fermentation of dietary fibers and non-digestible polysaccharides, act as a link between the microbiome, immune system, and inflammatory processes. The importance of accurately quantifying SCFAs in plasma has recently emerged to understand their biological role. In this work, a sensitive and reproducible LC-MS/MS method is reported for SCFAs quantification in three different matrices such as human, rat and mouse plasma via derivatization, using as derivatizing agent O-benzylhydroxylamine (O-BHA), coupled with liquid-liquid extraction. First, the instrumental parameters of the mass spectrometer and then the chromatographic conditions were optimized using previously SCFAs derivatives synthetized and used as standards. After that, the best conditions for derivatization and extraction from plasma were studied and a series of determinations were performed on human, rat, and mouse plasma aliquots to validate the overall method (derivatization, extraction, and LC-MS/MS determination). The method showed good performance in terms of recovery (> 80%), precision (RSD <14%), accuracy (RE < ± 10%) and sensitivity (LOQ of 0.01 µM for acetic, butyric, propionic and isobutyric acid) in all plasma samples. The method thus developed and validated was applied to the quantification of major SCFAs in adult and aged mice, germ-free mice and in germ-free recipient mice subjected to fecal transplant from adult and aged donors. Results highlighted how plasma concentrations of SCFAs are correlated with age further highlighting the importance of developing a method that is reliable for the quantification of SCFAs to study their biological role.
短链脂肪酸（SCFAs）是肠道微生物对膳食纤维和不可消化多糖发酵的终产物，它们作为微生物群、免疫系统和炎症过程之间的联系。准确量化血浆中 SCFAs 的重要性最近已经显现出来，以了解它们的生物学作用。在这项工作中，我们报道了一种灵敏且可重现的 LC-MS/MS 方法，用于通过衍生化，使用邻苯甲羟胺（O-BHA）作为衍生化剂，结合液液萃取，从人、大鼠和小鼠血浆的三种不同基质中定量 SCFAs。首先，使用先前合成的 SCFAs 衍生物作为标准，优化了质谱仪的仪器参数，然后优化了色谱条件。之后，研究了从血浆中衍生化和提取的最佳条件，并对人、大鼠和小鼠血浆等分试样进行了一系列测定，以验证整个方法（衍生化、提取和 LC-MS/MS 测定）。该方法在所有血浆样品中的回收率（>80%）、精密度（RSD<14%）、准确性（RE<±10%）和灵敏度（醋酸、丁酸、丙酸和异丁酸的 LOQ 为 0.01µM）方面表现出良好的性能。所开发和验证的方法应用于成年和老年小鼠、无菌小鼠以及接受来自成年和老年供体粪便移植的无菌受体小鼠中主要 SCFAs 的定量。结果强调了血浆中 SCFAs 的浓度与年龄的相关性，进一步强调了开发一种可靠的 SCFAs 定量方法以研究其生物学作用的重要性。
