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

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.