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 NH-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/NH 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 H 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.

Importance of ICPMS for speciation analysis is changing: future trends for targeted and non-targeted element speciation analysis

“…The authors believe that in particular marine organisms such as ascidians or seaweed may contain a large number of brominated compounds still waiting to be identified [ 41 ]. Additionally, brominated drugs have been studied by HPLC–ICPMS–MS in a non-targeted analysis approach [ 42 , 43 ]. However, there seems to be no direct driving force for the study of the wealth of natural organobromine compounds, although the detection limits of ICPMS for bromine (below micrograms of Br per litre) are comparable to or even superior to those of other, cheaper but less selective methods such as electron-capture detection for volatile bromine compounds.…”

Multielement Detection of Nonmetals by Barium-Based Post-ICP Chemical Ionization Coupled to Orbitrap-MS

“…An attractive approach for standard-free quantitation is to utilize the heteroatoms in compounds as quantitative elemental tags. Notably, halogen (F, Cl, Br, and I), S, and P elements have been widely incorporated into drug motifs to enhance drug stability, metabolism, and selectivity. − These heteroatoms are also prevalent in environmental contaminants (e.g., halogenated disinfection byproducts and phosphorylated flame retardants). , Moreover, the appearance of F in drugs and environmental contaminants with health-related consequences has surged in the past decades. , These considerations have fueled the development of elemental MS methods for F, Cl, Br, I, S, and P detection to offer species-independent calibration and rapid quantitation without compound-specific standards. − …”

“…This along with specificity of isobaric removal strategies via ion-neutral reactions make detection of F and Cl in a multielement method with other heteroatoms challenging. The multielement capability is notable because each heteroatom provides independent quantitation for compounds that have several measurable heteroatoms, adding an internal check for accuracy of quantitation results . Moreover, parts of analytes may be cleaved in transformation reactions, leading to loss of some heteroatoms.…”

“…The multielement capability is notable because each heteroatom provides independent quantitation for compounds that have several measurable heteroatoms, adding an internal check for accuracy of quantitation results. 16 Moreover, parts of analytes may be cleaved in transformation reactions, leading to loss of some heteroatoms. Therefore, a better coverage of transformation products is achieved with multielement methods.…”