Department of Pharmaceutical Chemistry, College of Pharmacy, Taif University, PO Box 11099, Taif 21944, Saudi Arabia.
Analytical Toxicology Laboratory, Forensic Medicine Authority, Ministry of Justice, Cairo 11647, Egypt.
J Chromatogr Sci. 2021 May 20;59(6):536-547. doi: 10.1093/chromsci/bmab031.
First analytical methods were herein developed for determination of pregabalin (PGB) and amitriptyline (AMT) as an active binary mixture used for management of neuropathic pain whether in pure forms or in human biological fluids (plasma/urine). First method is green high-performance liquid chromatography-diode array detector (HPLC-DAD) after derivatization of PGB with ninhydrin (NIN) on a reversed-phase C18 column using a mobile phase consisting of ethanol:water (97:3%, v/v) pumped isocratically at 0.8 mL/min; AMT were scanned at 215 nm, whereas PGB-NIN was scanned at 580 nm. Second method is High-performance thin-layer chromatography (HPTLC), where PGB and AMT were separated on silica gel HPTLC F254 plates, using ethanol:ethyl acetate:acetone:ammonia solution (8:2:1:0.05, by volume) as a developing system. AMT peaks were scanned at 220 nm, whereas PGB peaks were visualized by spraying 3% (w/v) ethanolic NIN solution and scanning at 550 nm. Linear calibration curves were obtained for human plasma and urine spiked with PGB and AMT over the ranges of 5-100 μg/mL and 0.2-2.5 μg/band for PGB, and 1-100 μg/mL and 0.1-2.0 μg/band for AMT for HPLC-DAD and HPTLC methods, respectively. The suggested methods were validated according to Food and Drug Administration guidelines for bioanalytical methods validation and they can be applied for routine therapeutic drug monitoring for the concerned drugs.
本文建立了用于测定普瑞巴林（PGB）和阿米替林（AMT）的分析方法，这两种药物以活性二元混合物的形式用于治疗神经病理性疼痛，无论是纯品形式还是在人体生物体液（血浆/尿液）中。第一种方法是在反相 C18 柱上用茚三酮（NIN）衍生 PGB 后，采用由乙醇：水（97:3%，v/v）组成的流动相以 0.8 mL/min 的等度泵入进行高效液相色谱-二极管阵列检测（HPLC-DAD）；在 215nm 处扫描 AMT，而 PGB-NIN 在 580nm 处扫描。第二种方法是高效薄层色谱（HPTLC），其中 PGB 和 AMT 在硅胶 HPTLC F254 板上分离，使用乙醇：乙酸乙酯：丙酮：氨溶液（8:2:1:0.05，体积比）作为展开系统。在 220nm 处扫描 AMT 峰，而 PGB 峰通过喷洒 3%（w/v）乙醇 NIN 溶液并在 550nm 处扫描来可视化。对于 HPLC-DAD 和 HPTLC 方法，人血浆和尿液中添加 PGB 和 AMT 的线性校准曲线分别在 5-100μg/mL 和 0.2-2.5μg/band 以及 1-100μg/mL 和 0.1-2.0μg/band 的范围内获得。根据美国食品和药物管理局生物分析方法验证指南对建议的方法进行了验证，它们可用于相关药物的常规治疗药物监测。
