Abstract：
Single-pass intestinal perfusion (SPIP) method is a widely used experimental model to determine the intestinal permeability of drugs. These studies are performed in the presence of a reference standard (metoprolol, MT) and a zero permeability marker (phenol red, PR). Therefore, it is important to develop a validated method for simultaneous determination of the investigated compound along with MT and PR. The aim of this study was to develop a reversed phase high-performance liquid chromatography (RP-HPLC) method with UV-detection for the simultaneous determination of atenolol (ATN), MT, and PR in the perfusion medium used in SPIP experiments. Separation of compounds were performed using an InertSustain C18 (250 × 4.6 mm, 5 µm) HPLC column at 35 °C. The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 7.0, 12.5 mM) in gradient elution, and was delivered at a flow rate of 1 mL/min. The acetonitrile ratio of the mobile phase increased linearly from 10 to 35 % over 15 min. The injection volume was 20 µL, and ATN, MT and PR were detected at 224 nm. The retention times under optimum HPLC conditions were 5.028 min, 12.401 min, and 13.507 min for ATN, MT and PR, respectively. The developed RP-HPLC method was validated for selectivity, specificity, calibration curve and range, accuracy and precision, carry-over effect, stability, reinjection reproducibility, recovery and robustness. The method was linear for ATN (0.76-50 μg/mL), MT (1.14-50 μg/mL), and PR (0.47-20 μg/mL) with determination coefficients of 0.9999, 0.9994 and 0.9998, respectively. The results obtained for all validation parameters of the developed RP-HPLC method met the required limits of the ICH M10 Guideline.
摘要：
单程肠灌注(SPIP)方法是一种广泛使用的测定药物肠道通透性的实验模型。这些研究是在参考标准（美托洛尔，MT)和零渗透率标记(酚红，公关）。因此,重要的是开发一种经过验证的方法来同时测定所研究的化合物以及MT和PR。这项研究的目的是开发一种反相高效液相色谱（RP-HPLC）方法，该方法具有UV检测功能，用于同时测定阿替洛尔（ATN）。MT,和PR在SPIP实验中使用的灌注培养基中。使用InertSustainC18（250×4.6mm，5µm）HPLC色谱柱，温度为35°C。流动相为乙腈和磷酸盐缓冲液(pH7.0,12.5mM)的梯度洗脱混合物,并且以1mL/min的流速递送。流动相的乙腈比率在15分钟内从10％线性增加到35％。进样体积为20微升,ATN,在224nm检测MT和PR。最佳HPLC条件下的保留时间为5.028min,12.401min,ATN为13.507分钟，MT和PR，分别。验证了所开发的RP-HPLC方法的选择性，特异性，校准曲线和范围，准确度和精密度，结转效应,稳定性,再注射再现性，恢复性和鲁棒性。该方法对ATN（0.76-50μg/mL）呈线性关系，MT（1.14-50μg/mL），和PR（0.47-20μg/mL），测定系数分别为0.9999、0.9994和0.9998。所开发的RP-HPLC方法的所有验证参数的结果均符合ICHM10指南的要求限值。
