Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia.
Medicina (Kaunas). 2023 Feb 23;59(3):441. doi: 10.3390/medicina59030441.
This study presents the development and validation of the 96-microwell-based spectrofluorimetric (MW-SFL) and high performance liquid chromatography (HPLC) with fluorescence detection (HPLC-FD) methods for the quantitation of alectinib (ALC) in its bulk powder form and in urine samples. The MW-SFL was based on the enhancement of the native fluorescence of ALC by the formation of micelles with the surfactant cremophor RH 40 (Cr RH 40) in aqueous media. The MW-SFL was executed in a 96-microwell plate and the relative fluorescence intensity (RFI) was recorded by utilizing a fluorescence plate reader at 450 nm after excitation at 280 nm. The HPLC-FD involved the chromatographic separation of ALC and ponatinib (PTB), as an internal standard (IS), on a C column and a mobile phase composed of methanol:potassium dihydrogen phosphate pH 7 (80:20, /) at a flow rate of 2 mL min. The eluted ALC and PTB were detected by utilizing a fluorescence detector set at 365 nm for excitation and 450 nm for emission. Validation of the MW-SFL and HPLC-FD analytical methods was carried out in accordance with the recommendations issued by the International Council for Harmonization (ICH) for the process of validating analytical procedures. Both methods were efficaciously applied for ALC quantitation in its bulk form as well as in spiked urine; the mean recovery values were ≥86.90 and 95.45% for the MW-SFL and HPLC-FD methods, respectively. Both methodologies are valuable for routine use in quality control (QC) laboratories for determination of ALC in pure powder form and in human urine samples.
本研究建立了 96 孔板基质荧光分光光度法（MW-SFL）和高效液相色谱法（HPLC）-荧光检测法（HPLC-FD），用于定量分析原料药阿来替尼（ALC）和尿液中的阿来替尼。MW-SFL 基于 ALC 在水相介质中与表面活性剂聚氧乙烯蓖麻油 RH40（Cr RH40）形成胶束，增强其固有荧光。MW-SFL 在 96 孔板中进行，在 280nm 激发下，利用荧光板读数器记录 450nm 处的相对荧光强度（RFI）。HPLC-FD 涉及 ALC 和泊那替尼（PTB）（作为内标）在 C 柱上的色谱分离，流动相由甲醇：磷酸二氢钾 pH7（80:20，/）组成，流速为 2mL/min。洗脱的 ALC 和 PTB 通过荧光检测器检测，激发波长为 365nm，发射波长为 450nm。MW-SFL 和 HPLC-FD 分析方法的验证按照国际协调委员会（ICH）发布的关于分析程序验证过程的建议进行。两种方法均有效地用于 ALC 原料药的定量分析以及尿液中的加标实验；MW-SFL 和 HPLC-FD 方法的平均回收率分别为≥86.90%和 95.45%。这两种方法都可用于纯粉末形式和人尿液样品中 ALC 的常规 QC 实验室检测。
