In elemental speciation, the different chemical forms in which an element occurs, are separated from one another and an attempt is made to identify and quantify the species present. This is accomplished at our lab by coupling high-performance liquid chromatography (HPLC) and ICP-mass spectrometry with the aim of developing methods for quantitative metabolite profiling of pharmaceutical drugs.

The activities of the A&MS research group in this context are illustrated below via a couple of case studies published in the international literature.

For more applications, you are invited to check our list of publications.

Speciation analysis of bromine-containing drug metabolites in feces samples from a human in vivo study by means of HPLC/ICP-MS combined with on-line isotope dilution

During the development of a new drug compound, its metabolism needs to be unraveled. For quantification of the metabolites formed, the drug under investigation is traditionally synthesized with a radiolabel (¹⁴C or³H) and the metabolites present in different matrixes (blood, urine, feces) upon drug administration are determined by means of high-performance liquid chromatography (HPLC) coupled to radiodetection. However, in some cases, the use of a radiolabeled compound in human in vivo studies is not advisable, e.g., for drug compounds or their metabolites showing a long plasma or tissue half-life. In cases where the candidate drug molecule contains an element detectable by means of inductively coupled plasma-mass spectrometry (ICP-MS), HPLC/ICP-MS is a promising alternative approach.

The aim of this work was speciation analysis of metabolites in feces samples collected within a clinical study during which a bromine-containing anti-tuberculosis drug (TMC207) was administered to patients with multidrug-resistant tuberculosis infection. Owing to slow elimination of the drug, no ¹⁴C label could be used within this study. Quantification of the bromine species was accomplished using HPLC/ICP-MS in combination with on-line isotope dilution (on-line ID), while structural elucidation of the species was performed using HPLC coupled to electrospray ionization–mass spectrometry. The limit of detection (1.5 mg TMC207 L^-1) was of the same order of magnitude as that for HPLC/radiodetection. Based on the excellent figures of merit, the “cold” HPLC/ICP-MS approach could be deployed for the actual human in vivometabolism study, such that exposure of the human volunteers to the ¹⁴C radiolabel was avoided.

More information: Meermann et al., Analytical and Bioanalytical Chemistry, 2012, 402, 439-448.

HPLC/ICP-MS in combination with “reverse” on-line isotope dilution in drug metabolism studies

During the development of a new drug compound, its metabolism needs to be unraveled. For quantification of the metabolites formed, the drug under investigation is traditionally synthesized with a radiolabel (¹⁴C or ³H) and the metabolites present in different matrixes (blood, urine, feces) upon drug administration are determined by means of high-performance liquid chromatography (HPLC) coupled to radiodetection. This approach allows for quantification of the metabolites formed and enables a straightforward distinction between exogenous (i.e., drug-related) and endogenous species (as only the radiolabeled species are detected). For pharmaceutical drugs containing an element detectable via ICP-MS, HPLC/ICP-MS can be a viable alternative as also ICP-MS provides a signal intensity independent of the chemical form in which the target element occurs. However, HPLC/ICP-MS lacks specificity when a distinction between drug-related species and endogenous compounds containing the same target element needs to be accomplished. As a result, we have developed an HPLC/ICP-MS-based method combined with “reverse” online isotope dilution (“reverse” online ID) for metabolite quantification. The methodology was evaluated by the analysis of feces samples from rats dosed with a ⁸¹Br-labeled drug compound. The method allows for both (i) valid quantification of the drug metabolites and (ii) distinction among endogenous, exogenous, and “mixed” species, based on their isotopic “fingerprint”.

More information: Meermann et al., Analytical Chemistry, 2012, 84, 2395-2401.

Quantitative metabolite profiling of an amino group containing pharmaceutical in human plasma via pre-column derivatization and high-performance liquid chromatography-inductively coupled plasma-mass spectrometry

Quantitative determination of a 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 pre-column derivatization followed by HPLC-inductively coupled plasma-mass spectrometry (ICP-MS), was developed and validated. To introduce a suitable “heteroelement” (defined here as an element that is detectable with ICP-MS), 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 ICP-MS 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.

More information: Li et al., Analytical Chemistry, 2017, 89, 1907-1915.

A pre-column derivatization method allowing quantitative metabolite profiling of carboxyl and phenolic hydroxyl group containing pharmaceuticals in human plasma via liquid chromatography-inductively coupled plasma-tandem mass spectrometry (LC-ICP-MS/MS)

Within the context of this study, an accurate, simple and sensitive HPLC-ICP-MS/MS method for the quantitative metabolite profiling of diclofenac in human plasma based on the pre-column derivatization of the carboxylic and phenolic –OH groups present in the parent drug and its major metabolite, 4’-hydroxy-diclofenac, was developed and validated. A cost-effective and commercially available derivatization reagent, p-bromophenacyl bromide (p-BPB), was applied for the introduction of Br into the drug molecule and its major metabolite, enabling the element-selective detection and quantification based on the Br-signal. The presence of Cl in both diclofenac and 4’-hydroxy-diclofenac allowed an additional validation via simultaneous monitoring of the Cl-signal by using a state-of-art ICP-MS/MS instrument equipped with a collision/reaction cell. The reaction conditions were successfully optimized to achieve a quantitative formation of the corresponding derivatization products, while the baseline separation of the target compounds in a typical biological matrix (i.e. human plasma) was achieved using gradient reversed phase high-performance liquid chromatography (RP-HPLC). A fit-forpurpose accuracy (recovery between 85–115%) and precision (repeatability ≤7.2% RSD) were achieved. The limits of quantification (LOQ) are ≈50 mg L^-1 for Br and ≈80 mg L^-1 for Cl, corresponding to ≈0.2 mg L^-1 and ≈0.4 mg L^-1 of diclofenac, respectively. 

More information: Li et al., Journal of Analytical Atomic Spectrometry, 2018, 33, 274-282.