Development and Validation of a Densitometric-High-Performance Thin-Layer Chromatographic Method for Quantitative Analysis of Amitriptyline in Gastric Lavage

Abstract

Drug overdose is the most common method in suicide attempts. Amitriptyline (AMT), a tricyclic antidepressant agent, known for potentially lethal cardiovascular and neurological effects, is used especially by intoxicated patients. The aim of this study was to validate a method for the determination of AMT in gastric lavage samples by high-performance thin-layer chromatography (HPTLC). Artificial gastric lavage was used to obtain a better extraction procedure for AMT. The method was also applied to patients who attempted suicide with AMT. A validated, accurate, and rapid HPTLC-based method was developed for quantitation of AMT in gastric content of intoxicated patients. Extraction was done from 3 mL gastric lavage sample by liquid-liquid extraction procedure with ethyl acetate-n-heptane (1:1, w/w) in alkaline pH with 10.8. The mobile phase was an isocratic solvent system consisting of methanol-ammonia (25%) (98.5:1.5, v/v). 3,4-Methylenedioxy-N-methamphetamine (MDMA) was used as internal standard (IS). Analytes were quantified by TLC Scanner operating under 209 nm. The retardation factors of AMT and MDMA were determined as 0.49 and 0.25, respectively. The analytical range was set as 10-250 ng spot(-1) for AMT. Calibration was linear within the selected range in gastric lavage (r > 0.9995). Limit of detection and limit of quantification of AMT were found to be 5.1 and 17.3 ng spot(-1), respectively. Recoveries of 25 ng spot(-1) and 100 ng spot(-1) concentrations were found 83.12% and 91.89%; bias% values were 10.91 and 3.19, respectively. The validated method was also applied on samples of five patients who attempted suicide by AMT, and concentrations in gastric lavage samples of 5 patients were found 7.1, 8.3, 9.9, 14.6, and 36.6 ng mL(-1). This successfully validated method was applied to suicide cases, and it can be conveniently employed on both antemortem and postmortem cases suspected by amitriptyline.