Villani Salvatore, Dematteis Giulia, Tapella Laura, Gagliardi Mara, Lim Dmitry, Corazzari Marco, Aprile Silvio, Del Grosso Erika
Department of Pharmaceutical Sciences, University of Piemonte Orientale, Largo Donegani 2, 28100 Novara, Italy.
Department of Health Science (DSS), Center for Translational Research on Autoimmune and Allergic Disease (CAAD) & Interdisciplinary Research Center of Autoimmune Diseases (IRCAD), University of Piemonte Orientale, 28100 Novara, Italy.
Pharmaceuticals (Basel). 2023 Feb 14;16(2):298. doi: 10.3390/ph16020298.
In recent years, 4-phenylbutyric acid (4-PBA), an FDA-approved drug, has increasingly been used as a nonspecific chemical chaperone in vitro and in vitro, but its pharmacodynamics is still not clear. In this context, we developed and validated a Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) method to quantify 4-PBA in NeuroBasal-A and Dulbecco's Modified Eagle widely used cell culture media. Samples were injected on a Luna 3 µm PFP(2) 100 Å (100 × 2.0 mm) column maintained at 40 °C. Water and methanol both with 0.1% formic acid served as mobile phases in a step gradient mode. The mass acquisition was performed by selected ion monitoring (SIM) in negative mode for a total run time of 10.5 min at a flow rate of 0.300 mL/min. The analogue 4-(4-Nitrophenyl)-Butyric Acid served as internal standard. Validation parameters were verified according to FDA and EMA guidelines. The quantification ranges from 0.38-24 µM. Inter and intraday RSDs (Relative Standard Deviations) were within 15%. The developed LC-HRMS method allowed the estimation of 4-PBA absorption and adsorption kinetics in vitro in two experimental systems: (i) 4-PBA improvement of protein synthesis in an Alzheimer's disease astrocytic cell model; and (ii) 4-PBA reduction of endoplasmic reticulum stress in thapsigargin-treated melanoma cell lines.
近年来，4-苯基丁酸（4-PBA）作为一种已获美国食品药品监督管理局（FDA）批准的药物，越来越多地在体内外被用作非特异性化学伴侣，但其药效学仍不明确。在此背景下，我们开发并验证了一种液相色谱-高分辨率质谱（LC-HRMS）方法，用于定量测定广泛使用的NeuroBasal-A和杜氏改良伊格尔细胞培养基中的4-PBA。样品注入到一根Luna 3 µm PFP(2) 100 Å（100×2.0 mm）色谱柱中，该色谱柱保持在40℃。均含有0.1%甲酸的水和甲醇作为流动相，采用梯度洗脱模式。通过选择离子监测（SIM）在负离子模式下进行质量采集，总运行时间为10.5分钟，流速为0.300 mL/min。类似物4-(4-硝基苯基)-丁酸用作内标。根据FDA和EMA指南验证了验证参数。定量范围为0.38 - 24 µM。日内和日间相对标准偏差（RSD）均在15%以内。所开发的LC-HRMS方法能够在两个实验系统中体外评估4-PBA的吸收和吸附动力学：（i）在阿尔茨海默病星形胶质细胞模型中4-PBA对蛋白质合成的改善作用；以及（ii）在毒胡萝卜素处理的黑色素瘤细胞系中4-PBA对内质网应激的减轻作用。
