RESUMO
Background: Imipramine, desipramine, amitriptyline and nortriptyline are widely prescribed antidepressant drugs in Asia because of their low price and high efficacy. The concentrations of these drugs in plasma need to be monitored for achieving efficient therapy. A simple, rapid and practical analytical method is required. Objective: To develop a plasma deproteinization process for rapid determination of imipramine, desipramine, amitriptyline or nortriptyline in plasma using high-performance liquid chromatography (HPLC). Methods: A deproteinizing agent, with an acceptable accuracy and sensitivity, to precipitate plasma protein containing imipramine, desipramine, amitriptyline and nortriptyline was investigated along with the HPLC condition for resolving each of these compounds. The developed method was verified by performing a bio-analytical method validation, and by analyzing plasma samples from volunteers who received imipramine or amitriptyline. Results: Using clomipramine as an internal standard, the concentrations of imipramine, desipramine, amitriptyline and nortriptyline in plasma could easily be determined by precipitating plasma protein with acetonitrile and concentrated aqueous potassium phosphate solution prior to HPLC analysis. The mobile phase comprised of acetonitrile and 70 mM phosphate buffer (pH 6.1) (60/40 v/v). A separation was achieved on a C₁₈ column, and the effluent was measured by UV absorption at 251 nm or by EC detection at +1.0 V of glassy carbon against silver/silver chloride reference electrode. The chromatographic separation was excellent, without interference from endogenous plasma constituents. All compounds were resolved in a run time of 12 minutes. The method was suitable for quantifying drug concentrations in the ranges of 40-900 ng/ml for UV detection or 4-900 ng/mL for EC detection. The relative standard intra-day and inter-day deviations for each compound in the plasma were less than 8%. The percentage of bias was within \±4% which confirmed the accuracy of the method. The method proved to be efficient and practical for determining the plasma concentrations of each compound, following the administration of imipramine or amitriptyline to volunteers. Conclusion: This developed method is very simple, rapid and inexpensive and is practical for routine therapeutic drug monitoring and forensic toxicological screening.
RESUMO
Background: Simultaneous screening of ephedrine with amphetamine or methamphetamine in drug abusers is useful in countries, such as Thailand, that prohibit the use of ephedrine. The lack of an adequate screening test kit suitable for this purpose is a significant obstacle in the detection of ephedrine abusers. A reliable analytical method for the simultaneous detection of amphetamine, methamphetamine and ephedrine is needed. Objective: To develop a process for the detection of amphetamine, methamphetamine and ephedrine by enzyme-linked immunosorbent assay (ELISA), based on the polyclonal antibody and heterology principle. Methods: The 3-aminopropyl (3AP) and 4-aminobutyl (4AB) derivatives of amphetamine (A), methamphetamine (M) and ephedrine (E) were chemically synthesized. They were used for the preparations of immunogens and hapten tracers. Direct competitive ELISA of matrix combinations of antisera and hapten tracers were performed using amphetamine, methamphetamine and ephedrine as the analytes. Only the competitive reactions with specified sensitivity and specificity are selected. Results: The study discovered three assay combinations that demonstrated concentration-dependent competition of analyte (single or multiple). They passed the confirmation test for the cut-off concentration and had no cross-reactivity with other amines or structured related compounds. The assay combinations of 4ABA-Ab with 3APA-PO and 4ABE-Ab with 3APM-PO were specific for the detection of amphetamine with ephedrine and methamphetamine with ephedrine, respectively. The third assay combination of 4ABE-Ab with 3APE-PO was highly specific to ephedrine with negligible cross-reactivity from other structure-related compounds. Direct competitive ELISA of 4ABE-Ab with 3APM-PO has been proven useful in field tests for the detection of methamphetamine in urine samples from Thai truck drivers suspected of drug abuse. Conclusion: By using heterology, these three assay combinations could be used separately or simultaneously for drug abuse screening.