Catherine Duckett

Metabolism of 3-chloro-4-fluoroaniline in rat using [14C]-radiolabelling, 19F-NMR spectroscopy, HPLC-MS/MS, HPLC-ICPMS and HPLC-NMR

The metabolic fate of 3-chloro-4-fluoroaniline was investigated in rat following intraperitoneal (i.p.) administration at 5 and 50 mg kg−1 using a combination of HPLC-MS, HPLC-MS/MS, 19F-NMR spectroscopy, HPLC-NMR spectroscopy and high-pressure liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS) with 35Cl and 34S detection. The metabolism of 3-chloro-4-fluoroaniline at both doses was rapid and extensive, to a large number of metabolites, with little unchanged compound excreted via the urine. Dosing at 5 mg kg−1 with [14C]-labelled compound enabled the comparison of standard radioassay analysis methods with 19F-NMR spectroscopy. 19F-NMR resonances were only readily detectable in the 0–12 h post-dose samples. Dosing at 50 mg kg−1 allowed the facile and specific detection and quantification of metabolites by 19F-NMR spectroscopy. Metabolite profiling was also possible at this dose level using HPLC-ICPMS with 35Cl-specific detection. The principal metabolites of 3-chloro-4-fluoroaniline were identified as 2-amino-4-chloro-5-fluorophenyl sulfate and 2-acetamido-4-chloro-5-fluorophenyl glucuronide. N-acetylation and hydroxylation followed by O-sulfation were the major metabolic transformations observed.

Metabolism of 2-fluoro-4-iodoaniline in earthworm Eisenia veneta using 19F-NMR spectroscopy, HPLC-MS, and HPLC-ICPMS (127I)

A combination of 19F-NMR spectroscopy, HPLC-MS/MS, HPLC-MS with constant neutral loss scanning of 127, and HPLC-ICPMS with iodine detection has enabled the profiling, quantification, and limited characterization of the metabolites produced in the earthworm Eisenia veneta, following exposure to 2-fluoro-4-iodoaniline. Mass spectrometric analysis of the worm tissue and coelomic fluid afforded the identification of two Phase II metabolites, N-glutamyl and N-glucoside conjugates, indicating the importance of these pathways in the detoxification of xenobiotics for earthworms. Several further metabolites were observed and quantified by 19F-NMR spectroscopy and HPLC-127I-ICPMS, although these were of low abundance and their structures were not unequivocally identified. The parent compound and the glutamyl conjugate were found to be the major xenobiotic components of both the coelomic fluid and the worm tissue, representing approximately 23 and approximately 35%, respectively, of the dose that was recovered from the earthworm tissue extract.

Profiling biological samples using ultra performance liquid chromatography–inductively coupled plasma–mass spectrometry (UPLC-ICP-MS) for the determination of phosphorus and sulfur-containing metabolites

In this preliminary study UPLC-ICP-MS has been utilized to profile a range of different bio-fluids and tissue extracts for sulfur and phosphorus-containing metabolites. Particular attention has been given to the livers, plasma and urine from lean and obese Zucker rats, with a view to differentiating between them based solely on their respective sulfur or phosphorus profiles and/or their total sulfur and phosphorus content. In addition, bile and tumour extracts have been analysed to observe the nature of their profiles. To the best of our knowledge this is the first time ICP-MS has been used in a non-targeted metabonomic study. Results have shown lower limits of quantification for sulfur and phosphorus methods of 0.25 and 0.15 ng on column with CVs of 14.7% and 10.9% respectively. Total phosphorus analysis of the Zucker rat aqueous liver extracts, plasma and urine has shown the pattern of phosphorus concentrations to be statistically significantly different in the lean and obese Zucker rats. Chromatographic separation of the Zucker rat organic liver extracts and plasma allowed further differentiation between the lean and obese rats using their phosphorus profiles alone. In conclusion, this preliminary study has shown the potential of UPLC-ICP-MS to quantitatively discriminate between different species biofluids, fluids and tissues based solely on their phosphorus or sulfur concentrations and/or metabolomes.

Development and validation of a NANOGold™ immunoassay for the detection of vascular endothelial growth factor (VEGF) in human serum using inductively coupled plasma mass spectrometry

This work aimed to develop and validate a NANOGold based assay, quantified using inductively coupled plasma mass spectrometry (ICP-MS), for the detection of human vascular endothelial growth factor (hVEGF) in serum. The initial assay range based on calibration standards was 62.5-2000 pg/mL with a detection limit of approximately 30 pg/mL. After validation using spiked validation controls, a quantification range between 175 and 1928 pg/mL was obtained. The inter-assay precision was between 2.3 and 18.9% with accuracy between -8.8 and -3.1%. Additional performance parameters, including dilutional linearity, matrix specificity and time-factored drift, were within +/-20%, as defined by the validation acceptance criteria for the validation of macromolecule immunoassays used within our clinical environment. Serum samples from healthy donors were analysed to determine the endogenous levels of VEGF present; these ranged from 164 to 580 pg/mL with a mean of 273 pg/mL. The intra- and inter-assay precision obtained from the healthy donor samples were 1.3-10.7% and 4.2-17.5%, respectively. This demonstration of a validated immunoassay opens further possibilities, utilising the simultaneous detection capabilities of ICP-MS for the detection of multiple analytes in a single validated immunoassay, for routine use within a clinical environment.

Phosphorus and sulfur metabonomic profiling of tissue and plasma obtained from tumour‐bearing mice using ultra‐performance liquid chromatography/inductively coupled plasma mass spectrometry

RATIONALE Metabonomic studies use complex biological samples (blood plasma/serum, tissues, etc.) that when analysed with high-performance liquid chromatography/mass spectrometry (HPLC/MS) or nuclear magnetic resonance (NMR) generate profiles that may contain many thousands of features. These profiles can be difficult to interpret with the majority of the features contributing little to the study. As such there is an argument for the development of techniques that can simplify the problem by targeting particular classes of compounds. METHODS In this study ultra-performance liquid chromatography/inductively coupled plasma mass spectrometry (UPLC/ICP-MS) was used to profile tumour tissue and plasma samples for phosphorus- and sulfur-containing metabolites. These samples were xenograft tumours (derived from breast, lung and colon cell lines) and plasma obtained from nude mice. Plasma was also obtained from non-tumour-bearing mice as a control. Due to isobaric interferences this method took advantage of the dynamic reaction cell within the ICP-MS system to react the phosphorus and sulfur ions with oxygen. The PO+ and SO+ ions were then monitored free of interferences. The total phosphorus and sulfur within each sample was also recorded using flow injection ICP-MS. A robust quality control system based on pooled sample replicate analysis was used throughout the study. RESULTS Determination of the total phosphorus and sulfur content of each sample was sufficient in itself for statistical differentiation between the majority of the cell lines analysed. Subsequent reversed-phase chromatographic profiling of the organic tumour and plasma extracts revealed the presence of a number of well-retained phosphorus-containing compounds that showed tumour-specific profiles. Reversed-phase profiling was not suitable for the sulfur-containing compounds which eluted with the solvent front. CONCLUSIONS This study has shown the potential use of UPLC/ICP-MS to differentiate between tumour cell lines, using both plasma and tumour tissue samples, based solely on metabolites that contain phosphorus or sulfur. Whilst further work is required to identify these compounds this methodology shows the ability of the described methods to provide targets for future biomarker discovery studies. Copyright

The metabolism of 4-bromoaniline in the bile-cannulated rat: application of ICPMS (79/81Br), HPLC-ICPMS & HPLC-oaTOFMS

Abstract 1. An excretion balance study was performed following i.p. administration of 4-bromoaniline (50 mg kg−1) to bile-cannulated rats, using bromine-detected (79/81Br) ICPMS for quantification. Approximately 90% of the dose was recovered in urine (68.9 ± 3.6%) and bile (21.4 ± 1.4%) by 48 h post-administration. 2. HPLC-ICPMS (79/81Br) was used to selectively detect and profile the major urinary and biliary-excreted metabolites and determined that the 0–12 h urine contained at least 21 brominated metabolites with 19 bromine-containing peaks observed in the 6–12 h bile samples. 3. The urinary and biliary metabolites were subsequently profiled using HPLC-oaTOFMS. By exploiting the distinctive bromine isotope pattern ca. 60 brominated metabolites were detected in the urine in negative electrospray ionisation (ESI) mode while bile contained ca. 21. 4. While a large number of bromine-containing metabolites were detected, the profiles were dominated by a few major components with the bulk of the 4-bromoaniline-related material in urine accounted for by 4-bromoanaline O-sulfate (∼75% of the total by ICPMS, 84% by TOFMS). In bile a hydroxylated N-acetyl compound was the major metabolite detected, forming some ∼65% of the 4-bromoaniline-related material by ICPMS (37% by TOFMS).

Metabolism of [14C]-5 -chloro -1,3 -benzodioxol-4 -amine in male Wistar-derived rats following intraperitoneal administration

[14C]-5-chloro-1,3-benzodioxol-4-amine was administered intraperitoneally (i.p.) to bile duct-cannulated rats (Alpk:ApfSD, Wistar derived) at 25 mg kg−1 to determine the rates and routes of excretion of the compound and to investigate its metabolic fate. A total of 89.1% of the dose was excreted in the 48 h following administration, the majority being recovered in the urine during the first 12 h. The main metabolite in both urine and bile, detected by high-performance liquid chromatography (HPLC) with radioprofiling and mass spectrometry, was identified as a demethylenated monosulfate conjugate. Unchanged parent compound formed a major component of the radiolabel excreted in urine and, in addition to unchanged parent and demethylenated sulphate conjugate, a large number of minor metabolites were detected in urine and bile. The overall metabolic fate of 5-chloro-1,3-benzodioxol-4-amine in the rat was complex, with some similarities to previously studied methylenedioxyphenyl compounds.

Quantitative Investigation of Terbinafine Hydrochloride Absorption into a Living Skin Equivalent Model by MALDI-MSI

The combination of microspotting of analytical and internal standards, matrix sublimation, and recently developed software for quantitative mass spectrometry imaging has been used to develop a high-resolution method for the determination of terbinafine hydrochloride in the epidermal region of a full thickness living skin equivalent model. A quantitative assessment of the effect of the addition of the penetration enhancer (dimethyl isosorbide (DMI)) to the delivery vehicle has also been performed, and data have been compared to those obtained from LC-MS/MS measurements of homogenates of isolated epidermal tissue. At 10% DMI, the levels of signal detected for the drug in the epidermis were 0.20 ± 0.072 mg/g tissue for QMSI and 0.28 ± 0.040 mg/g tissue for LC-MS/MS at 50% DMI 0.69 ± 0.23 mg/g tissue for QMSI and 0.66 ± 0.057 mg/g tissue for LC-MS/MS. Comparison of means and standard deviations indicates no significant difference between the values obtained by the two methods.