Identification of phase I and phase II metabolites of benfluron and dimefluron in rat urine using high‐performance liquid chromatography/tandem mass spectrometry

Abstract

Biotransformation products of two potential antineoplastic agents, benfluron and dimefluron, are characterized using our integrated approach based on the combination of high-performance liquid chromatography (HPLC) separation of phase I and phase II metabolites followed by photodiode-array UV detection and electrospray ionization tandem mass spectrometry (MS/MS). High mass accuracy measurement allows confirmation of an elemental composition and metabolic reactions according to exact mass defects. The combination of different HPLC/MS/MS scans, such as reconstructed ion current chromatograms, constant neutral loss chromatograms or exact mass filtration, helps the unambiguous detection of low abundance metabolites. The arene oxidation, N-oxidation, N-demethylation, O-demethylation, carbonyl reduction, glucuronidation and sulfation are typical mechanisms of the metabolite formation. The interpretation of their tandem mass spectra enables the distinction of demethylation position (N- vs. O-) as well as to differentiate N-oxidation from arene oxidation for both phase I and phase II metabolites. Two metabolic pathways are rather unusual for rat samples, i.e., glucosylation and double glucuronidation. The formation of metabolites that lead to a significant change in the chromophoric system of studied compounds, such as the reduction of carbonyl group in 7H-benzo[c]fluorene-7-one chromophore, is reflected in their UV spectra, which provides valuable complementary information to MS/MS data.

Analytical Methods for Quantification of Drug Metabolites in Biological Samples

“…Identification of phase I and phase II metabolites of two antineoplastic agents was demonstrated by use of Q-TOF [40]. In this study, 32 metabolites for dimefluron and 28 metabolites for benfluron were detected in the rat urine within 25 min chromatographic run.…”

“…Detection of metabolite was in this case possible only in negative ionization mode [26]. However, with the commonly and easily used UV detection, the metabolites have often the same chromophore as the parent drug (but not always [40], hence giving similar response. But the main limitation of this technique in pharmacokinetic studies lies in not sufficient sensitivity and also in lower selectivity as some compounds does not have UV absorption at a wavelength to distinct it from the background.…”

Metabolic pathways of anthelmintic drug monepantel in sheep and in its parasite (Haemonchus contortus)

“…[8] No information about the in vivo metabolism of MOP in sheep is available so far, although detailed knowledge of drug metabolism in target species is important for safe and effective therapy including the disclosure of drug-drug interaction. [12,13] The combination of ultra high-performance liquid chromatography (UHPLC) and quadrupole-time-of-flight analyzer (QqTOF) mass spectrometry is very powerful strategy [14] due to the use of selected tandem mass spectrometry (MS/MS) scans, [13,[15][16][17] accurate mass measurements [13,15] and software dedicated to the metabolite prediction or detection. [9] The resistance of nematodes to MOP has not been observed yet, but the gradual development of strains with decreased sensitivity to MOP is highly probable during the next few years.…”

“…Information about MOP biotransformation in target parasites is also not yet known, but the metabolism of anthelmintics in helminths could also contribute to the drug-resistance development. [15] In present study, UHPLC-MS/MS technique is used for the identification of MOP metabolites formed in sheep in vivo and in its parasite (H. contortus) ex vivo. The increased drug biotransformation represents one of possible mechanisms of the drug resistance development, therefore the knowledge of anthelmintics biotransformation in nematodes is important.…”

“…[12,13] The combination of ultra high-performance liquid chromatography (UHPLC) and quadrupole-time-of-flight analyzer (QqTOF) mass spectrometry is very powerful strategy [14] due to the use of selected tandem mass spectrometry (MS/MS) scans, [13,[15][16][17] accurate mass measurements [13,15] and software dedicated to the metabolite prediction or detection. [15] In present study, UHPLC-MS/MS technique is used for the identification of MOP metabolites formed in sheep in vivo and in its parasite (H. contortus) ex vivo. MOP metabolism in H. contortus sensitive inbred susceptible Edinburgh (ISE) strain and stable multi-resistant White River (WR) strain were compared with the aim of evaluating the relationship between MOP metabolism and potential MOP resistance in helminths.…”