The identification and quantitative determination of amitriptyline by the HPLC-MWD method

Authors

DOI:

https://doi.org/10.24959/nphj.26.196

Keywords:

amitriptyline; high performance liquid chromatography-multi-wavelength detector; identification; quantitative determination.

Abstract

Aim. To develop the sensitive and specific HPLC-MWD method for the determination of amitriptyline suitable for use in bioanalytical studies in clinical and forensic toxicology.

Materials and methods. The analysis was performed on a “MiLiChrome A-02” microcolumn liquid chromatograph attached to a dual-beam multi-wave UV spectrophotometer under the following conditions: 2×75 mm column ProntoSIL 120-5-C18 AQ, 5 μm; the gradient elution mode with a linear gradient, eluent A (5 % acetonitrile and 95 % perchlorate buffer) to eluent B (100 % acetonitrile) for 40 min, then 100 % eluent B for 3 min; the mobile phase flow rate – 100 μL/min; the thermostat temperature – 40°C; detection was carried out at 8 wavelengths (210, 220, 230, 240, 250, 260, 280, 300 nm).

Results. The retention time of amitriptyline was 22.80 min; the absorbance ratios Sλ/S210 were also set as additional option to increase the reliability of identification. The calibration curve was represented by the dependence of a peak area (S, mm2) against concentration (c, μg/μL) and described by the regression equation у = 0.00514 × x. The method showed linearity in the range of 1.0–100.0 μg/mL. The correlation coefficient was lower than 0.99. The LOD and LOQ values were calculated from the parameters of the calibration curve, they were 0.3 μg/mlL and 0.9 μg/mL, respectively. The accuracy and precision of the method developed were determined at three concentration levels (low, middle, high) within one day (intraday); RSD did not exceed 1.0 % across the full concentration range studied when analyzing model solutions.

Conclusions. The sensitive and specific method for the quantitative determination of amitriptyline using the HPLC-MWD method has been developed. The method meets the requirements set to techniques recommended for use in clinical and forensic toxicology, and it has been confirmed by a number of the validation parameters. The HPLC-MWD method developed can be recommended for implementation into the practice of regional bureaus of forensic medical examination and clinical and diagnostic laboratories of regional drug addiction centers.

References

  1. Schmidt, J. J., Bertram, A., Kühn-Velten, W. N., Suhling, H., Wiesner, O., Schneider, A., & Kielstein, J. T. (2015). Treatment of amitriptyline intoxications by extended high cut-off dialysis. Clinical Kidney Journal, 8(6), 796–799. https://doi.org/10.1093/ckj/sfv094
  2. Kamasak, T., Kader, Ş., Mutlu, M., Özkaya, K., Erduran, E., Orhan, F., Dilber, E., Kalyoncu, M., Karagüzel, G., Cakir, M., Bahat, E., & Cansu, A. (2021). Amitriptyline Intoxication in Children: Twenty Years’ Experience in a Tertiary Care Center in Turkey. Pediatric Emergency Care, 37(12), e1377–e1381. https://doi.org/10.1097/pec.0000000000002055
  3. Nguyen, T., Le, L., Vu, T., Nguyen, A., Doan, D. B., Pham, D., Pham, T., Vu, C., & Nguyen Vo, M. H. (2024). Resuscitation from Respiratory Arrest Due to Life-Threatening Ventricular Arrhythmias in a Patient with Amitriptyline Intoxication: An Old Problem in a New Era. International Medical Case Reports Journal, 17, 949–957. https://doi.org/10.2147/imcrj.s478761
  4. Siddique, A., Khokhar, M. M., Waheed, A., Naeem, U., & Aziz, S. A. (2025). Effect of combination therapy of methylfolate with antidepressants in patients with depressive disorder. BMC Pharmacology and Toxicology, 26(1), e14. https://doi.org/10.1186/s40360-025-00846-x
  5. Farag, H. M., Yunusa, I., Goswami, H., Sultan, I., Doucette, J. A., & Eguale, T. (2022). Comparison of Amitriptyline and US Food and Drug Administration – Approved Treatments for Fibromyalgia. JAMA Network Open, 5(5), e2212939. https://doi.org/10.1001/jamanetworkopen.2022.12939
  6. Hieronymus, F., Lisinski, A., & Eriksson, E. (2024). Impact of sedative and appetite-increasing properties on the apparent antidepressant efficacy of mirtazapine, selective serotonin reuptake inhibitors and amitriptyline: an item-based, patient-level meta-analysis. eClinicalMedicine, 77, 102904. https://doi.org/10.1016/j.eclinm.2024.102904
  7. Brueckle, M.-S., Thomas, E. T., Seide, S. E., Pilz, M., Gonzalez-Gonzalez, A. I., Dinh, T. S., Gerlach, F. M., Harder, S., Glasziou, P. P., & Muth, C. (2023). Amitriptyline’s anticholinergic adverse drug reactions–A systematic multiple-indication review and meta-analysis. PLOS ONE, 18(4), e0284168. https://doi.org/10.1371/journal.pone.0284168
  8. Li, X., Xin, L., Yang, L., Yang, Y., Li, W., Zhang, M., Liao, Y., Sun, C., Li, W., Peng, Y., & Zheng, J. (2024). Identification of an Epoxide Metabolite of Amitriptyline In Vitro and In Vivo. Chemical Research in Toxicology, 37(6), https://doi.org/10.1021/acs.chemrestox.4c00008
  9. Nishimura, T., Maruguchi, H., Nakao, A., & Nakayama, S. (2017). Unusual complications from amitriptyline intoxication. BMJ Case Reports, 2017, bcr-2017-219257. https://doi.org/10.1136/bcr-2017-219257
  10. Allibe, N., Eysseric-Guerin, H., Kintz, P., Bartoli, M., Bost-Bru, C., Grenier, F., Scolan, V., & Stanke-Labesque, F. (2015). Amitriptyline poisoning of a baby: How informative can hair analysis be? Forensic Science International, 249, 53–58. https://doi.org/10.1016/j.forsciint.2015.01.012
  11. Paland, M. (2021). Use of CytoSorb in cases of acute amitriptyline intoxication. Journal of Clinical Pharmacy and Therapeutics, 46(5), 1476–1479. https://doi.org/10.1111/jcpt.13373
  12. van de Wint, T., de Vries Schultink, A. H. M., Meinders, A. J., Harmsze, A., & Bruins, P. (2022). Prolonged coma due to amitriptyline overdose and genetic polymorphism: a case report. Journal of Medical Case Reports, 16(1), e112. https://doi.org/10.1186/s13256-022-03294-x
  13. El Zaher, A. A., El Kady, E. F., EL Messiry, H. M., El Ghwas, H. E., & El Houssini, O. M. (2021). Synchronous LC-MS/MS determination of pantoprazole and amitriptyline in rabbit plasma: application to comparative in vivo pharmacokinetic study of novel formulated effervescent granules with its marketed tablet dosage form. Heliyon, 7(8), e07752. https://doi.org/10.1016/j.heliyon.2021.e07752
  14. Ma, W., Gao, X., Guo, H., & Chen, W. (2021). Determination of 13 antidepressants in blood by UPLC-MS/MS with supported liquid extraction pretreatment. Journal of Chromatography B, 1171, 122608. https://doi.org/10.1016/j.jchromb.2021.122608
  15. Sabaghi, M., Ezoddin, M., Ghazi‐Khansari, M., Javadi, M. S., & Abdi, K. (2025). Development of Air‐assisted Liquid‐Liquid Microextraction in the Primary Amine‐based Supramolecular Solvent for Determination of Tricyclic Antidepressants Prior to High‐Performance Liquid Chromatography‐Ultraviolet Detection. Journal of Separation Science, 48(10), e70254. https://doi.org/10.1002/jssc.70254
  16. Zamani, R., & Yamini, Y. (2023). On-Chip Electromembrane Surrounded Solid Phase Microextraction for Determination of Tricyclic Antidepressants from Biological Fluids Using Poly(3,4-ethylenedioxythiophene)–Graphene Oxide Nanocomposite as a Fiber Coating. Biosensors, 13(1), 139. https://doi.org/10.3390/bios13010139
  17. Khan, A. A. P. (2020). Electrocatalytic Behavior and Determination of Amitriptyline Drug with MWCNT@Celllulose Composite Modified Glassy Carbon Electrode. Materials, 13(7), 1708. https://doi.org/10.3390/ma13071708
  18. Derzhavne pidpryiemstvo «Naukovo-ekspertnyi farmakopeinyi tsentr» (2008). Derzhavna farmakopeia Ukrainy. Dopovnennia 2 (1-e vyd.). [State Pharmacopoeia of Ukraine. Supplement 2]. Naukovo-ekspertnyi farmakopeinyi tsentr. [Ukraine].
  19. Scientific Working Group for Forensic Toxicology (SWGTOX) Standard Practices for Method Validation in Forensic Toxicology. (2013). Journal of Analytical Toxicology, 37(7), 452–474. https://doi.org/10.1093/jat/bkt054

Downloads

Published

2026-03-31

Issue

Vol. 111 No. 1 (2026): News of Pharmacy

Section

Articles

License

Copyright (c) 2026 National University of Pharmacy

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 International License.