Abstract
Objective: Separation of tricyclic compounds sets the keystone for determining parent drug to metabolite concentration ratios and analysing impurities. The combined effects of acetonitrile composition and pH of the mobile phase on the separation of amitriptyline and nortriptyline by reversed-phase high-performance liquid chromatography (RP-HPLC) are presented.Methods: A series of RP-HPLC triplicate runs were carried out using acetonitrile and a phosphate buffer as the mobile phase and a Kinetex® C18 LC Column as the stationary phase using an Agilent 1260 Infinity Series® II liquid chromatography system with UV/visible detection. The stationary phase, column temperature, injection volume and flow rate were kept unchanged during analysis. Mobile phase composition and pH were varied to observe impact on peak shape, resolution and retention time, taking into consideration green analytical chemistry aspects.Results: Optimal chromatographic outcomes were achieved when using the mobile phase made up of 35% acetonitrile and 65% buffer at a pH of 5.6. These conditions resulted in nortriptyline and amitriptyline eluting at 4.66 min and 5.92 min respectively. Increasing the organic modifier content of the mobile phase to 40% completed separation within a run time of 4 min with comparable resolution. The 2 min gained by increasing 5% acetonitrile may not be justified due to potential implications on greening laboratory practices.Conclusion: Reversed-phase chromatography embodies a simple method for the separation of compounds that are similar in structure. Attuning the percentage of organic modifier and buffer pH provides acceptable retention times, without compromising resolution between neighbouring peaks.
Highlights
Tricyclic antidepressants (TCAs) have been in clinical use for over fifty years and, owing to their efficacy and low cost, are still prescribed for the treatment of major depression and other psychiatric conditions as well as in bulimia, migraine prevention, neuropathic pain management, fibromyalgia, and enuresis [1, 2]
The sodium or potassium salts of phosphoric acid are commonly used in buffer systems for RPHPLC
No more than 50% organic solvent should be used in the mobile phase, depending on the concentration of the specific buffer employed
Summary
Tricyclic antidepressants (TCAs) have been in clinical use for over fifty years and, owing to their efficacy and low cost, are still prescribed for the treatment of major depression and other psychiatric conditions as well as in bulimia, migraine prevention, neuropathic pain management, fibromyalgia, and enuresis [1, 2]. Modern-day methods for routine analysis of these drugs, their metabolites and potential impurities in pharmaceutical preparations and bio fluids are of paramount significance. High-performance liquid chromatography (HPLC) is the prevalent technique in published methods for the analysis of TCAs [3]. Amitriptyline, 3-(10,11-dihydro-5H-dibenzo [a, d] cyclohepten-5ylidene)-propyl dimethylamine, is a basic lipophilic drug, with a molecular weight of 277.39, made up of a hydrophobic skeleton and a basic tertiary amino group as the main functional group. The salt, amitriptyline hydrochloride, is freely soluble in water and alcohol and dissolved in polar solvents such as acetonitrile [4]. Water solubility of amitriptyline hydrochloride is pH-dependent with the highest solubility in an acidic environment. The solubility is significantly reduced when the protonation of the tertiary amino group is suppressed
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