Identifying and overcoming bioanalytical challenges associated with chlorine-containing dehydrogenation metabolites

Abstract

Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) is a widely utilized analytical tool for quantifying small molecules in complex biological matrices. In certain situations the mass-selection capabilities of the tandem mass spectrometer may be insufficient to discriminate between the analyte of interest and its metabolites, particularly those metabolites that are isobaric with the analyte. One scenario by which isobaric interference may occur is the metabolism of a chlorine- or bromine-containing small molecule to a metabolite with the concomitant loss of 2 Da. This report describes the detection and characterization of two distinct dehydrogenation [M-2] metabolites during LC/MS/MS quantification of a chlorinated small molecule in rat plasma samples derived from a toxicokinetic study. The potential isotope-related impact of these metabolites on quantification of the parent compound was assessed. Several alternate precursor ion and product ion combinations were evaluated and shown to minimize the quantitative impact of the interfering metabolites without having to rely on their stringent chromatographic resolution from the parent compound. These results indicate that when quantifying chlorine- or bromine-containing small molecules from in vivo samples or in vitro metabolic incubations: (1) efforts to detect potential dehydrogenation metabolites should be undertaken and (2) if such metabolites are detected, the judicious choice of alternate multiple-reaction monitoring (MRM) transitions can limit their impact on quantification of the parent molecule without the need for robust chromatographic resolution.

Enzymatic Synthesis of Trideuterated Sialosides

“…Although X-Gal was beneficial during monitoring the conversion rates and for the isolation of the reaction products, it’s use as a substrate had also a downside: For the mass spectrometric analysis of the compound, the natural distribution of the containing chlorine and bromine isotopes of the 5-bromo-4-chloro-3-indoxyl portion somewhat complicate the overall mass spectra, as always two main peaks with the mass difference of 2 Da and a smaller mass peak with a mass difference of additional 2 Da were observed [26]. However, in case of the herein described sialylation method the expected sialosides will incorporate three deuteriums, and therefore the overall mass increase is 3 Da, which allows the discrimination from the 2 Da and 4 Da added by bromine and chlorine isotopes.…”

Bioanalytical Aspects of Clinical Mass Balance Studies in Oncology

“…Similarly, isobaric metabolites, with the same nominal mass but a different elemental composition (e.g., R-OSO 3 H instead of R-OPO 3 H 2 ) can cause interference. Even if the molecular mass differs by 1 Da (e.g., R-OH instead of R-NH 2 ) or 2 Da (e.g., R-OH instead of R-CH 3 ), interference can occur through a naturally occurring 13 C-, 15 N-, 37 Cl-or 81 Br-isotope of the analyte with the lower molecular mass [8,23,24]. In the latter two cases, whereby the elemental composition differs, high-resolution MS detection provides an alternative solution to distinguish between the analytes [25].…”

Interference by metabolites and the corresponding troubleshooting during the LC–MS/MS bioanalysis of G004, a bromine-containing hypoglycemic agent

“…The prevalent use of short chromatographic run times may increase the risk of suffering from the metabolite interference during bioanalysis of pharmacokinetic samples [3][4][5]. Particularly during the determination of bromine or chlorine containing compounds, such quantitative interference may exist when their metabolites could generate the ions called [M-2] species [6]. The m/z values of these [M-2] species are 2 Da less than the precursor ions of the parent compounds.…”

“…Chromatographic resolution was given up for the cost of longer run times and lower throughput. Several MRM transitions were evaluated to overcome the isotope-related metabolite interference in this report, which would enable more reliable parent compound quantification without the need for chromatographic separation of the parent drugs from their metabolites [6,21,22]. Furthermore, a standard addition method for the metabolite-related matrix effect evaluation was established by using the incurred plasma samples, which can be applied to the LC-MS/MS method validation for other drugs facing the problem of the metabolite interference.…”