Lakeev Alexander P, Yanovskaya Elena A, Tsuran Daria V, Bryushinina Olga S, Abdrashitova Natalia Yu, Zyuz'kova Yulia G, Udut Vladimir V, Kiselev Vsevolod I, Kuznetsov Igor N
Goldberg Research Institute of Pharmacology and Regenerative Medicine, Tomsk National Research Medical Center, Russian Academy of Sciences, Tomsk, Russia.
National Research Tomsk State University, Tomsk, Russia.
Biomed Chromatogr. 2022 Mar;36(3):e5296. doi: 10.1002/bmc.5296. Epub 2021 Dec 20.
Indole-3-carbinol is the subject of ongoing biomedical research owing to its potential antiatherogenic, anticarcinogenic and antioxidant effects. The antitumor properties are mainly associated with its major metabolite, i.e. 3,3'-diindolylmethane (DIM). Typically, the biological activity of the chemical compound is manifested in the ng/ml concentration range. Consequently, the development of highly sensitive analytical methods to determine DIM in various biological samples is an urgent issue. In this study, an HPLC-MS/MS method was established for the quantification of DIM in human plasma. The developed method was validated according to the European Medicines Agency guidelines. Sensitivity, selectivity, accuracy and precision were good, allowing DIM quantification in the concentration range of 5-500 ng/ml. The limit of detection and the lower limit of quantification were 1 and 5 ng/ml, respectively. 4-Methoxy-1-methylindole was used as an internal standard (IS). The analytes were extracted from the human plasma by the acetonitrile-induced protein precipitation method with the addition of 3 mol/L ammonium sulfate as a salting-out agent, which is a facile and efficient approach for high-throughput bioanalysis. The chromatographic separation was performed on the Synergi Fusion-RP C column (50 × 2.0 mm, 4 μm, 80 Å) under isocratic elution at 40°C. The mobile phase consisting of acetonitrile and water (0.1% formic acid; 85:15, v/v) was delivered at a flow rate of 0.20 ml/min. DIM and the IS were eluted at 2.36 ± 0.04 and 2.43 ± 0.03 min, respectively. The total analysis time was 3.20 min. Atmospheric pressure chemical ionization was carried out using multiple reaction monitoring in the positive polarity mode. The ion transitions were set to m/z 247.1 → 130.1 (DIM) and 162.1 → 147.1 (IS). The method was successfully applied to the analysis of plasma samples after a single oral administration of the Indinol® Forto drug (200 mg) to healthy female Russian volunteers. Also, the developed method was used for the analysis of rabbit plasma samples after a single oral dose of DIM (20 mg/kg).
吲哚 - 3 - 甲醇因其潜在的抗动脉粥样硬化、抗癌和抗氧化作用而成为当前生物医学研究的对象。其抗肿瘤特性主要与其主要代谢产物，即3,3'-二吲哚甲烷（DIM）有关。通常，该化合物的生物活性在纳克/毫升浓度范围内表现出来。因此，开发高灵敏度分析方法以测定各种生物样品中的DIM是一个紧迫的问题。在本研究中，建立了一种用于定量人血浆中DIM的HPLC - MS/MS方法。所开发的方法根据欧洲药品管理局的指南进行了验证。灵敏度、选择性、准确性和精密度良好，可在5 - 500纳克/毫升浓度范围内对DIM进行定量。检测限和定量下限分别为1和5纳克/毫升。4 - 甲氧基 - 1 - 甲基吲哚用作内标（IS）。通过乙腈诱导的蛋白沉淀法，添加3摩尔/升硫酸铵作为盐析剂，从人血浆中提取分析物，这是一种用于高通量生物分析的简便高效方法。在Synergi Fusion - RP C柱（50×2.0毫米，4微米，80埃）上于40℃等度洗脱条件下进行色谱分离。由乙腈和水（0.1%甲酸；85:15，v/v）组成的流动相以0.20毫升/分钟的流速输送。DIM和内标分别在2.36±0.04和2.43±0.03分钟洗脱。总分析时间为3.20分钟。在正离子模式下使用多反应监测进行大气压化学电离。离子跃迁设置为m/z 247.1→130.1（DIM）和162.1→147.1（内标）。该方法成功应用于对健康俄罗斯女性志愿者单次口服Indinol® Forto药物（200毫克）后的血浆样品进行分析。此外，所开发的方法还用于对兔子单次口服剂量的DIM（20毫克/千克）后的血浆样品进行分析。
