Department of Medical Services and Techniques, Yunus Emre Vocational School of Health Services, 26470 Eskişehir, Turkey.
Department of Pharmaceutical Technology, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir, Turkey.
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Jun 15;1147:122150. doi: 10.1016/j.jchromb.2020.122150. Epub 2020 May 7.
One of the most common techniques for assessing the intestinal absorption characteristics of drugs is single-pass intestinal perfusion (SPIP) method. Metoprolol tartrate (MT, reference standard) and phenol red (PR, zero permeability marker) are the compounds that are normally used in SPIP studies. The aim of this study was to develop a reverse phase high-performance liquid chromatography (RP-HPLC) method combined with UV-detection for the simultaneous determination of MT and PR in the perfusion medium used in SPIP experiments. Elution was performed using a Restek Raptor C18 column (5 μm, 4.6 mm × 250) at a temperature of 25 °C. The mixture of the mobile phase consisted of (MeOH):(Phosphate buffer solution, PBS), (20 mM, pH 3.0 adjusted with ortho-phosphoric acid),(55:45, v/v). Flow rate and column temperature were set at 1.2 mL min and 25 °C, respectively. MT and PR were injected as 20 µL into the HPLC system. UV detection was performed at 227 nm. The obtained retention times were reported as 2.89 and 3.80 min for MT and PR, respectively. The developed RP-HPLC method was validated according to Q2(R1) guideline of The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). The method was linear within the range of 2-50 μg mL for PR and 10-75 μg mL for MT. The developed RP-HPLC method was successfully applied on determination of MT and PR in perfusion medium. The developed method could be helpful for researchers working on in-situ rat intestinal permeability studies and it could be easily modified on further studies.
一种评估药物肠道吸收特性的常用技术是单次肠灌注（SPIP）法。酒石酸美托洛尔（MT，参比标准）和酚红（PR，零通透性标记物）是通常用于 SPIP 研究的化合物。本研究旨在开发一种反相高效液相色谱（RP-HPLC）法，结合紫外检测，用于同时测定 SPIP 实验中灌注介质中的 MT 和 PR。洗脱在 25°C 下使用 Restek Raptor C18 柱（5μm，4.6mm×250）进行。流动相混合物由（MeOH）：（磷酸盐缓冲溶液，PBS）（20mM，用邻磷酸调至 pH 3.0），（55：45，v/v）组成。流速和柱温分别设定为 1.2mL/min 和 25°C。将 MT 和 PR 以 20µL 注入 HPLC 系统。在 227nm 处进行紫外检测。MT 和 PR 的保留时间分别为 2.89 和 3.80min。所建立的 RP-HPLC 方法根据人用药物技术要求国际协调理事会（ICH）的 Q2(R1)指南进行了验证。该方法在 PR 的 2-50μg/mL 和 MT 的 10-75μg/mL 范围内呈线性。所建立的 RP-HPLC 方法成功应用于测定灌注介质中的 MT 和 PR。所建立的方法可有助于从事大鼠原位肠道通透性研究的研究人员，并且可以在进一步的研究中进行轻松修改。
