A comparison between HPLC-dynamic reaction cell-ICP-MS and HPLC-sector field-ICP-MS for the detection of glutathione-trapped reactive drug metabolites using clozapine as a model compound

Abstract

In this study, a novel method was developed for tracing down reactive drug metabolites, the formation of which in the human body constitutes an important health risk as a result of their capability to bind to body proteins and DNA. Clozapine was used as a model because this drug forms both reactive and stable metabolites. Glutathione, which forms complexes with reactive metabolites, was added in order to trap reactive species of clozapine, formed by degradation in an electrochemical cell, thereby mimicking the real drug metabolism process. The method developed is based on the use of reversed-phase HPLC as a chromatographic separation technique and inductively coupled plasma-mass spectrometry (ICP-MS) for monitoring of Cl and S. Via Cl-monitoring, all metabolites of the Cl-containing clozapine can be detected, whereas S-monitoring allows for the detection of the S-containing molecule glutathione and its conjugates with reactive metabolites. The spectral overlap of the signals of S-32(+) and S-34(+) with those of (OO+)-O-16-O-16 and (OO+)-O-16-O-18, respectively, was tackled in 2 ways. On one hand, a quadrupole-based ICP-MS instrument, equipped with a dynamic reaction cell, was used. O-2 was used as a reaction gas to convert the S+ ions to a sufficient extent into the corresponding SO+ species. This did not yield optimal results, due to pronounced ArC+ signals at mass 48 and 50 upon introduction of methanol into the ICP. On the other hand, a sector-field ICP-MS instrument operated at medium mass resolution permitted interference-free monitoring of the S+-signals. A new type of skimmer cone - termed X-skimmer - was evaluated and its use resulted in a 4-fold increase in the sensitivity in a methanolic environment, providing a limit of detection of 1 mu g L-1 for S. The chromatograms obtained via HPLC-SF-ICP-MS permitted differentiation between reactive and stable metabolites. As a result, the method developed looks very promising for the detection of glutathione conjugates and shows potential for their quantification in early stages of drug development when a radiolabeled compound is not yet available

Hyphenated techniques as tools for speciation analysis of metal-based pharmaceuticals: developments and applications

“…They contrasted HPLC/ (DRC)-ICP-MS [ 32 S 16 O] + with HPLC/HR-ICP-MS (medium resolution); the observed limits of detection for sulfur were in the low ppb range after applying HR-ICP-MS, while (DRC)-ICP-MS led to an LOD that was a factor of 10 higher [123].…”

Determination of S-containing drug metabolites from in vitro and in vivo metabolism studies by using LC-ICP/MS

“…The major isotope of sulfur 32 S (abundance 95.0 %) is interfered mainly by the polyatomic ion 16 O 16 O + which compromises importantly the sensitivity for 32 S detection. Improved responses could be obtained using sector field instruments which operate at higher mass resolution, which was not available with current instrument [19]. On the other hand, an attractive alternative for sulfur analysis relies on the detection of sulfur oxide species.…”

“…This element is present in more drugs than the halogens and drug metabolites with potential reactive electrophilicity may be effectively trapped with reduced GSH, a natural soft S-containing nucleophile, and important detoxifying agent. That is the case of the evaluation of in vitro reactive metabolites of clozapine using glutathione (GSH) as a trapping agent and further evaluation by using S-and Cl-monitoring [9,19].…”

Inductively coupled plasma- and glow discharge plasma-sector field mass spectrometry : Part II. Applications

“…374 De 34 S, the dynamic reaction cell instrument did not yield optimal results because of the pronounced ArC + formation at masses 48 and 50, interfering with the SO + species used for S determination. 375 Exposure of trivalent and hexavalent chromium species can have severe health effects in humans and in animals. [376][377][378] Thus, analysis of chromium species in tissues and also in environmental samples is of interest.…”