title: Quantitative Metabolite Profiling of an Amino Group Containing Pharmaceutical in Human Plasma via Precolumn Derivatization and High-Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry.
abstract: Quantitative determination of the candidate drug molecule and its metabolites in biofluids and tissues is an inevitable step in the development of new pharmaceuticals. Because of the time-consuming and expensive nature of the current standard technique for quantitative metabolite profiling, i.e., radiolabeling followed by high-performance liquid chromatography (HPLC) with radiodetection, the development of alternative methodologies is of great interest. In this work, a simple, fast, sensitive, and accurate method for the quantitative metabolite profiling of an amino group containing drug (levothyroxine) and its metabolites in human plasma, based on precolumn derivatization followed by HPLC-inductively coupled plasma mass spectrometry (ICPMS), was developed and validated. To introduce a suitable "heteroelement" (defined here as an element that is detectable with ICPMS), an inexpensive and commercially available reagent, tetrabromophthalic anhydride (TBPA) was used for the derivatization of free NH2-groups. The presence of a known number of I atoms in both the drug molecule and its metabolites enabled a cross-validation of the newly developed derivatization procedure and quantification based on monitoring of the introduced Br. The formation of the derivatives was quantitative, providing a 4:1 stoichiometric Br/NH2ratio. The derivatives were separated via reversed-phase HPLC with gradient elution. Bromine was determined via ICPMS at a mass-to-charge ratio of 79 using H2as a reaction gas to ensure interference-free detection, and iodine was determined at a mass-to-charge ratio of 127 for cross-validation purposes. The method developed shows a fit-for-purpose accuracy (recovery between 85% and 115%) and precision (repeatability <15% RSD). The limit of quantification (LoQ) for Br was approximately 100 μg/L.
authors.affiliation: Department of Analytical Chemistry, Ghent University , Campus Sterre, Krijgslaan 281-S12, 9000 Ghent, Belgium.
authors.forename: Sanwang
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Analytical Chemistry, Ghent University , Campus Sterre, Krijgslaan 281-S12, 9000 Ghent, Belgium.
authors.forename: Balázs
authors.initials: B
authors.lastname: Klencsár
authors.affiliation: Department of Analytical Chemistry, Ghent University , Campus Sterre, Krijgslaan 281-S12, 9000 Ghent, Belgium.
authors.forename: Lieve
authors.initials: L
authors.lastname: Balcaen
authors.affiliation: Pharmacokinetics, Dynamics & Metabolism, Janssen R&D , Turnhoutseweg 30, 2340 Beerse, Belgium.
authors.forename: Filip
authors.initials: F
authors.lastname: Cuyckens
authors.affiliation: Department of Organic and Macromolecular Chemistry, Ghent University , Campus Sterre, Krijgslaan 281-S4-bis, 9000 Ghent, Belgium.
authors.forename: Frederic
authors.initials: F
authors.lastname: Lynen
authors.affiliation: Department of Analytical Chemistry, Ghent University , Campus Sterre, Krijgslaan 281-S12, 9000 Ghent, Belgium.
authors.forename: Frank
authors.identifier: 0000-0002-1884-3853
authors.initials: F
authors.lastname: Vanhaecke
mesh_terms: D000588:Amines / Q000378:metabolism*; D001966:Bromine / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007455:Iodine / Q000737:chemistry; D057230:Limit of Detection; D013058:Mass Spectrometry / Q000379:methods*; D004364:Pharmaceutical Preparations / Q000097:blood*; D010796:Phthalic Anhydrides / Q000737:chemistry; D015203:Reproducibility of Results; D013974:Thyroxine / Q000097:blood
chemical_list: D000588:Amines; D004364:Pharmaceutical Preparations; D010796:Phthalic Anhydrides; D007455:Iodine; D013974:Thyroxine; C016133:tetrabromophthalic anhydride; D001966:Bromine