Department of Biochemistry, Cancer Biology, Neuroscience & Pharmacology, Meharry Medical College, 1005 Dr. D.B. Todd Blvd., Nashville, TN 37208, USA.
Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, Lubbock, TX 79409, USA.
J Chromatogr A. 2020 Jul 5;1622:461126. doi: 10.1016/j.chroma.2020.461126. Epub 2020 Apr 27.
Since metabolism is implicated in the carcinogenesis of toxicants, an efficient extraction method together with an analytical method is warranted to quantify tissue burdens of a carcinogen and/or its metabolites. Therefore, the aim of this study was to validate a pressurized liquid extraction (PLE) method for measuring metabolites of benzo(a)pyrene [B(a)P; a food-borne carcinogen] from tissue samples. The sample extraction was performed separately by PLE and liquid-liquid extraction (LLE). PLE followed by high-performance liquid chromatography coupled to online fluorescence detector (HPLC-FLD) was used to quantify separated analytes; and by ultra-high-performance liquid chromatography (UHPLC) coupled to atmospheric pressure chemical ionization tandem mass spectrometry (UHPLC-APCI-MS/MS) were used for confirmation purposes. The UHPLC-MS/MS was set-up in the atmospheric pressure chemical ionization (APCI) positive interface with selective reaction monitoring (SRM). The analytical performance characteristics of the PLE technique was assessed at different temperatures, pressure, number of cycles and solvent types. A methanol + chloroform + water mixture (30:15:10, v/v/v) yielded greater recoveries at an extraction temperature range of 60-80°C, pressure of 10 MPa and an extraction time of 10 min. The PLE method was validated by the analysis of spiked tissue samples and measuring recoveries and limits of quantitation for the analytes of interest using HPLC-FLD equipment. The optimized PLE-HPLC-FLD method was used to quantify the concentrations of B(a)P metabolites in liver samples obtained from a colon cancer animal model. Overall, PLE performed better in terms of extraction efficiency, recovery of B(a)P metabolites and shortened sample preparation time when compared with the classic LLE method.
由于代谢在毒物致癌作用中起作用，因此需要一种有效的提取方法和分析方法来定量检测致癌物及其代谢物在组织中的含量。因此，本研究的目的是验证一种加压液体萃取（PLE）方法，用于测量组织样品中苯并（a）芘（一种食物来源的致癌物）的代谢物。通过 PLE 和液液萃取（LLE）分别进行样品提取。PLE 后采用高效液相色谱-在线荧光检测器（HPLC-FLD）用于定量分离分析物；并通过超高效液相色谱（UHPLC）与大气压化学电离串联质谱（UHPLC-APCI-MS/MS）联用进行确证。UHPLC-MS/MS 设置在大气压化学电离（APCI）正接口，采用选择反应监测（SRM）模式。在不同的温度、压力、循环次数和溶剂类型下评估了 PLE 技术的分析性能特征。甲醇+氯仿+水（30:15:10，v/v/v）混合物在 60-80°C 的提取温度范围内、压力为 10 MPa 和提取时间为 10 min 时，可获得更高的回收率。通过分析加标组织样品并用 HPLC-FLD 设备测量感兴趣分析物的回收率和定量限，对 PLE 方法进行了验证。优化后的 PLE-HPLC-FLD 方法用于定量测定结肠癌动物模型肝脏样本中苯并（a）芘代谢物的浓度。总体而言，与经典的 LLE 方法相比，PLE 在提取效率、苯并（a）芘代谢物回收率和缩短样品制备时间方面表现更好。
