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

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• 作者：Sanwang Li ; Balázs Klencsár ; Lieve Balcaen ; Filip Cuyckens ; Frederic Lynen ; Frank Vanhaecke
• 刊名：Analytical Chemistry
• 出版年：2017
• 出版时间：February 7, 2017
• 年：2017
• 卷：89
• 期：3
• 页码：1907-1915
• 全文大小：450K
• ISSN：1520-6882

文摘

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/NH₂ ratio. 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 H₂ as 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.