IMPLICATIONS OF MOBILE PHASE COMPOSITION AND PH ON THE CHROMATOGRAPHIC SEPARATION OF AMITRIPTYLINE AND ITS METABOLITE NORTRIPTYLINE
DOI:
https://doi.org/10.22159/ijpps.2018v10i4.24817Keywords:
RP-HPLC, Acetonitrile composition, pH, Amitriptyline, NortriptylineAbstract
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.
Downloads
References
Dean L. Amitriptyline therapy and CYP2D6 and CYP2C19 genotype. In: Pratt V, McLeod H, Dean L, Malheiro A, Rubinstein W. editors. Medical Genetics Summaries [Internet]. Bethesda (MD): NCBI (US). Available from: https://www.ncbi.nlm.nih.gov/books/ NBK-425165/. [Last accessed on 10 Dec 2017].
Seema AM, Chinchu J, Akhil B, Prudence AR. A prospective study on geriatric prescribing pattern and medication adherence in a tertiary care hospital. Asian J Pharm Clin Res 2017;10:220-5.
Samanidou VF, Nika MK, Papadoyannis IN. HPLC as a tool in medicinal chemistry for the monitoring of tricyclic antidepressants in biofluids. Mini Rev Med Chem 2008;8:256-75.
Manzo RH, Olivera ME, Amidon GL, Shah VP, Dressman JB, Barens DM. Biowaiver monographs for immediate release solid oral dosage forms: amitriptyline hydrochloride. J Pharm Sci 2006;95:966–73.
Hansen S, Pedersen Bjergaard S, Rasmussen K. Analytical chemical characteristics of selected drug substances. In: Introduction to pharmaceutical chemical analysis. 1st ed. West Sussex: John Wiley and Sons, Ltd; 2012. p. 299-301.
ElHoussini OM, Zawilla NH. Chromatographic methods for the determination of fluphenazine, nortriptyline and its impurity amitriptyline in bulk and pharmaceutical formulations. J Anal Chem 2014;69:1187–92.
Dell’Aquila C. Separation of tricyclic antidepressants by capillary zone electrophoresis with N,N,Ǹ,Ǹ-tetramethyl-1,3-butanediamine (TMBD) as an effective electrolyte additive. J Pharm Biomed Anal 2002;30:341-50.
Galaon T, David V. The influence of mobile phase pH on the retention and selectivity of related basic compounds in reversed-phase liquid chromatography. Rev Roum Chim 2012;57:131-40.
Vella J, Mifsud M, Sammut Bartolo N, Ferrito V, Serracino Inglott A, Azzopardi LM, et al. The combined effect of pH and acetonitrile composition on the separation of two lincosamide antibiotics. Asian J Pharm Clin Res 2014;7:96-100.
Ashour S, Kattan N. Simultaneous determination of nortriptyline hydrochloride and fluphenazine hydrochloride in microgram quantities from low dosage forms by liquid chromatography–UV detection. J Pharm Anal 2012;2:437-42.
Rao G, Goyal A. An overview on analytical method development and validation by using HPLC. Pharma Chem J 2016;3:280-9.
Subirats X, Roses M, Bosch E. On the effect of organic solvent composition on the pH of buffered HPLC mobile phases and the pKa of analytes-a review. Sep Purif Rev 2007;36:231–55.
Neue UD, Mendez A, Tran K, Diehl DM. pH and selectivity in RP-chromatography. In: Kromidas S. editor. HPLC made to measure: a practical handbook for optimization. Weinheim: Wiley-VCH; 2006. p. 71-87.
Goeringer KE, McIntyre IM, Drummer OH. LC-MS analysis of serotonergic drugs. J Anal Toxicol 2003;27:30-5.
Kirkland JJ, Van Straten MA, Claessens HA. High pH mobile phase effects on silica-based reversed-phase high-performance liquid chromatographic columns. J Chromatogr A 1995;691:3-19.
Berges R, Sanz-Nebot V, Barbosa J. Modelling retention in liquid chromatography as a function of solvent composition and pH of the mobile phase. J Chromatogr A 2000;869:27-39.
Espinosa S, Bosch E, Roses M. Retention of ionizable compounds in high-performance liquid chromatography 14. Acid-base pK values in acetonitrile-water mobile phases. J Chromatogr A 2002;964:55-66.
Published
How to Cite
Issue
Section
