Sensitive and Rapid determination of Trientine and N1-Acetyl Trientine in Human Plasma by LC-MS/MS for bioequivalence study

  • Sheeba Nair
  • Bhavesh Dasandi
  • Dharmesh Parmar
  • , Shivprakash
  • Denish Karia


A simple and robust method for simultaneous determination of Trientine and N1-Acetyl Trientine in human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated. The analyte and internal standard were extracted from 200 μL plasma by liquid phase extraction. Chromatographic analysis was carried out on column Xtimate, C18 (4.6 x 50 mm) 5 μm with a flow rate of 1 mL/min, at 40˚C temperature. An isocratic elution method was applied using (A) Acetonitrile - 80% and (B) 10mM Ammonium Acetate in water - 20%.  Detection and quantitation was done by multiple reactions monitoring in positive ionization with Q3 LCMS-8050, Shimadzu. Mass parameters 1035.45/1030.55 m/z and 855.15/859.50 m/z on a triple quadrupole mass spectrometer were chosen for analysis of Trientine and N1-Acetyl Trientine. Linearity was established in human plasma covering the concentration range 10.009 ng/mL to 1000.571 ng/mL for Trientine and 10.009 ng/mL to 1000.628 ng/mL for N1-Acetyl Trientine. Correlation coefficient was consistently greater than 0.99 for Trientine and N1-Acetyl Trientine using Trientine-D4 and N1-Acetyl Trientine Trihydrochloride D4 as internal standards. Different parameters such as linearity, range, precision, accuracy, ruggedness and robustness, limit of detection (LOD) and limit of quantification (LOQ) were used for a full validation of this method. The results were found to be acceptable as per the guidelines of International Conference on Harmonization (ICH), CDER, EMA1,2,3,4,5. The developed and validated method was successfully applied to estimate Trientine and N1-Acetyl Trientine in a bioequivalence study in healthy human volunteers. Assay reproducibility was checked by reanalysis of samples near the Cmax and the elimination phase in the pharmacokinetic profile of the drug.

Keywords: Trientine and N1-Acetyl Trientine, LC-MS/MS, Validation, ICH.


Download data is not yet available.


[1] Draft Guidance on Trientine Hydrochloride (2013).
[2] EMA/308946/2017 CHMP assessment report Cuprior International non-proprietary name: Trientine tetrahydrochloride Procedure No. EMEA/H/C/004005/0000; 21 April 2017.
[3] FDA Guidance; Guidance for Industry, Bioanalytical Method Validation, US Department of Health and Human Services Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM),; September 2013.
[4] Guidance for Industry (March 2003): Bioavailability and Bioequivalence Studies for Orally Administered Drug-Products-General Considerations. US Department of Health and Human Services, Food and Drug Administration Centre for Drug Evaluation and Research (CDER):
[5] Guideline on bioanalytical method validation; EMEA/CHMP/EWP/192217/2009 Rev. 1 Corr. 2** Committee for Medicinal Products for Human Use (CHMP) 21 July 2011.
[6] Hea-Young Cho, PhD, Robert A. Blum, PharmD, Tracey Sunderland, Garth J. S. Cooper, MB, PhD, and William J. Jusko PhD; Pharmacokinetic and Pharmacodynamic Modeling of a Copper-Selective Chelator (TETA) in Healthy Adults; Journal of Clinical Pharmacology, 2009; 49:916-928.
[7] Miyazaki K, S. Kishino, M. Kobayashi, S. Arashima, S. Matsumoto, T. Arita; Determination of Triethylenetetramine in plasma of patients by HPLC 1035; Chemical and Pharmaceutical Bulletin, 1990: 38.
[8] Marc Cerrada-Gimenez, Janne Weisell, Mervi T. Hyvo¨ nen, Myung Hee Park, Leena Alhonen, Jouko Vepsa¨ la¨ inen, and Tuomo A. Keina¨ nen; Complex N-Acetylation of Triethylenetetramine; (2011); Drug Metabolism Disposition; 0090-9556/11/3912-2242–2249.
[9] Lu J, Chan YK, Poppitt SD, Cooper GJ; Determination of triethylenetetramine (TETA) and its metabolites in human plasma and urine by liquid chromatography-mass spectrometry (LC-MS); Journal of Chromatography. B, 2007; 859(1):62-68.
[10] Othmana, Asma d, Jun Lua, Tracey Sunderland, Garth J.S. Cooper Development and validation of a rapid HPLC method for the simultaneous determination of triethylenetetramine and its two main metabolites in human serum; Journal of Chromatography B, 2007; 860 42–48.
[11] Hansen EB Jr, Rushing LG, Thompson HC Jr; Determination of triethylenetetramine dihydrochloride in aqueous solution by reversed-phase ion-pairing high performance liquid chromatography and conductivity detection; Journal of Analytical Toxicology, 1985; 167-71.
[12] B. K. Matuszewski, M. L. Constanzer, and C. M. Chavez-Eng; Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS; Analytical Chemistry, 2003; 75:3019-3030.
[13] Cooper GJ, Phillips AR, Choong SY, Leonard BL, Crossman DJ, Brunton DH, Saafi L, Dissanayake AM, Cowan BR, Young AA, C.J.; Development and validation of a rapid HPLC method for the simultaneous determination of triethylenetetramine and its two main metabolites in human serum; Journal of Chromatography. B. 2008; 860(1):42-8.
[14] Jun Lu, Sally D. Poppitt, Asma A. Othman, Tracey Sunderland, Katya Ruggiero, Michael S. Willett, Lisa E. Diamond; Wilfredo D. Garcia, Benno G. Roesch and Garth J. S. Cooper; An Open-Label Trial Pharmacokinetics, Pharmacodynamics, and Metabolism of Triethylenetetramine in Healthy Human Participants, Journal of Clinical Pharmacology, 2010; 50:647.
[15] Lu J; Triethylenetetramine Pharmacology and Its Clinical Applications; 2010; 10.1158/1535-7163.MCT-10-0523.
[16] Dams R, Huestis MA, Lambert WE, Murphy CM; Matrix effect in bio-analysis of Illicit drugs with LC-MS/MS: Influence of ionization type, sample preparation, and biofluid; Journal of the American Society for Mass Spectrometry, 2003; 14:1290-1294.
402 Views | 600 Downloads
How to Cite
Nair S, Dasandi B, Parmar D, Shivprakash , Karia D. Sensitive and Rapid determination of Trientine and N1-Acetyl Trientine in Human Plasma by LC-MS/MS for bioequivalence study. JDDT [Internet]. 15May2019 [cited 19Jun.2021];9(3):310-8. Available from: