Identification of bound nitro musk-protein adducts in fish liver by gas chromatography–mass spectrometry: Biotransformation, dose–response and toxicokinetics of nitro musk metabolites protein adducts in trout liver as biomarkers of exposure

Ubiquitous occurrences of synthetic nitro musks are evident in the literature. The in vivo analysis of musk xylene (MX) and musk ketone (MK)-protein adducts in trout liver has been performed by gas chromatography–mass spectrometry using selected ion monitoring (GC–SIM-MS). Biotransformation, dose–response and toxicokinetics studies of 2-amino-MX (2-AMX), 2-amino-MK (2-AMK) and 4-amino-MX (4-AMX) metabolites, covalently bound to cysteine amino acids in proteins in fish liver, formed by enzymatic nitro-reduction of MX and MK, have been described. Trouts were exposed to single exposures of 0.010, 0.030, 0.10, and 0.30 mg/g MX and/or MK. Forty-two fish liver samples were collected from exposed- and control-fish subsequent to exposure intervals of 1 day, 3 days, and 7 days and were composited as per exposure schedules and times. Alkaline hydrolysis released bound metabolites from exposed liver composites that were extracted into n-hexane and then concentrated and analyzed by GC–SIM-MS. The presence of the metabolites in liver extracts was confirmed based on agreement of similar mass spectral properties and retention times with standards. In the dose–response study, the maximum adduct formation was 492.0 ng/g for 2-AMX, 505.5 ng/g for 2-AMK and 12588.5 ng/g for 4-AMX in liver at 0.03 mg/g MX and MK fish in 1 day after exposure. For toxicokinetics investigation, the highest amount of the target metabolites was found to be the same concentration as observed in the dose–response study for 1 day after exposure with 0.03 mg/g MX and MK fish and the half-lives of the metabolites were estimated to be 2–9 days based on assumption of first-order kinetics. Average recoveries exceeded 95% with a relative standard deviation (RSD) around 9%, and the limit of detection (LOD) ranged from 0.91 to 3.8 ng/g based on a signal to noise ratio of 10 (S/N = 10) could be achieved for the metabolites. No metabolites were detected in the controls and exposed non-hydrolyzed liver extracts. This is the first report on dose–response and toxicokinetics of nitro musk-cysteine-protein adducts in fish liver.

► Biotransformation, dose-response and toxicokinetics assessments of 2-AMX, 2-AMK and 4-AMX metabolites have been investigated by GC–SIM-MS.
► In dose-response study, maximum adduct formation 492.0, 505.5, 12588.5 ng/g for 2-AMX, 4-AMX, respectively, occurred at 0.03 mg/g dose in fish.
► In toxicokinetics examination, half-lives of the metabolites were 2–9 days based on assumption of first-order kinetics.
► Average recoveries for the metabolites exceeded 95% and LOD ranged from 0.91–3.8 ng/g.