Prensado en caliente

Urine samples were collected from three groups of male Sprague-Dawley rats (10 rats/group) administered a single intraperitoneal dose of 2-bromophenol at either 100 mg/kg or 200 mg/kg in corn oil. A control group was treated with corn oil alone. Rats were housed in metabolic cages to enable the collection of urine and samples were taken at -16, 0, 8, 24, 48, 72, 96, 120, 144 and 168 hours after administration. The urine samples were stored at - 40 °C prior to analysis.

A 120 µL aliquot of urine was mixed with 120 µL water to dilute the salt concentration before protein removal with acetonitrile (1:3 v/v). These samples were vortex mixed and left at - 20 °C overnight before centrifugation for 5 min at 15,000 g at 4 °C. For analysis, 50 µL was removed and added to 250 µL water in 350 µL 96-well plates. A pooled quality control (QC) sample was prepared by combining 100 µL of each sample and diluting 1:4 with water. The 96-well plates were stored at -20 °C until analysis and the plates were centrifuged again for 5 min at 700 g before being placed into the autosampler at 4 °C. The plate from this study was analysed five times, as a replicate, to assess the robustness of the approach.

3.2.2 Chronic Acetaminophen Study

Urine samples were collected and stored from four groups of male Sprague-Dawley (10 rats/group) rats dosed orally with acetaminophen (APAP) as described in Chapter 2 (pages 98 to 99)

For the analyses described here a 20 µL aliquot of each urine sample was mixed with 60 µL methanol in order to precipitate proteins. The samples were vortex mixed and left at -20 °C overnight before centrifugation for 5 min at 15,000 g at 4 °C. For analysis, 20 µL was removed and added to 180 µL water in 250 µL 384-well plates. A pooled quality control

(QC) sample, used for system conditioning and assessment of analytical performance was prepared by combining 5 µL of each sample and diluting 1:9 with water. The well plates were centrifuged again for 5 min at 700 g before being placed into the autosampler at 4 °C.

3.2.3 Liquid Chromatography

Liquid chromatographic analysis was performed on an Acquity I-class UPLC system, equipped with a binary solvent manager, sample manager and column heater (Waters Corporation, Milford, MA, USA), interfaced with a Synapt G2-S HDMS mass spectrometer (Waters Corporation, Manchester, UK). The chromatographic separations were performed on a HSS T3 1.8 µm stationary phase of either 2.1 x 100 mm for the conventional analysis or 1 x 50 mm for the fast, microscale analysis. The chromatographic mobile phase was composed of 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B). The column temperature was maintained at 40 °C and linear gradient elution was performed at either 0.5 mL/min for the 2.1 x 100 mm column or 0.4 mL/min for the 1 x 50 mm column. For the conventional method the starting composition was 1 % B, held for 1.0 min before increasing to 15 % at 3.0 min, 50 % at 6.0 min, 95 % at 9.0 min for a 1.0 min wash and returning to 1 % B for a 2 min re-equilibration step (total cycle time 12 min). A 2 µL injection of sample was performed using the flow through needle. For the fast, microscale method the starting composition was 1 % B, held for 0.14 min before increasing to 15 % at 0.42 min, 50 % at 0.83 min, 95 % at 1.25 min for a 0.25 min wash and returning to 1 % B for a 1 min re-equilibration step (a total cycle time of 2.5 min). A 1 µL (2-bromophenol study) or 0.2 µL (acetaminophen study) injection of sample was performed using the flow through needle. In both cases the purge solvent was 95:5 (v/v) H2O/CH3CN and sample manager wash was

CH3OH. For each study, prior to the analysis of the samples themselves 50 consecutive

injections (2 µL volume) of the pooled samples were made at the start of the chromatographic run to ‘condition’ the column to ensure that the analytical system was fully equilibrated. The QC sample was injected at the beginning of the analytical run and every

For use of 1 mm I.D. columns, minor system modifications were necessary to reduce peak dispersion (Gray et al., 2015). For the microbore separations the standard outlet tubing i.d. (0.004”) was reduced to 0.0025” of minimum length and the ESI stainless steel capillary (125 µm i.d.) was replaced with a narrow bore variant (50 µm i.d.).

3.2.4 Mass Spectrometry

Mass spectrometry for metabolic profiling was performed on a Synapt G2-S HDMS accurate mass instrument with electrospray ionization (ESI) operated in positive (ESI+) ion mode and a Xevo G2 operated in the same way. For the Synapt G2-S the capillary voltage was 1.0 kV, cone voltage was 25 V, source temperature was set at 120 °C with a cone gas (nitrogen) flow rate of 50 L/h, a desolvation gas temperature of 600 °C and a nebulization gas (nitrogen) flow of 1000 L/h. The Synapt G2-S was operated in resolution (V optics) mode whereas the Xevo G2 was operated in sensitivity (V optics) mode and both detectors were set to acquire data over the m/z range 50-1200 with a scan time of 0.1 s for the conventional method or 0.05 s for the fast method . All mass spectral data were collected in centroid mode using the MSe _{data acquisition21 function to obtain fragmentation data simultaneously.}

In function one a low collision energy (4 eV) was used and in the second function a high collision energy (ramp 15-45 eV) was used for fragmentation. For mass accuracy, leucine enkephalin (MW = 555.62) was used as a lock mass at a concentration of 200 pg/µL (in 50:50 CH3CN/H2O, 0.1 % formic acid) infused at a flow rate of 20 µL/min via a lock spray interface. Lockmass scans were collected every 15 s and averaged over 3 scans to perform mass correction. The instrument was calibrated before analysis with 0.5 mM sodium formate solution. The data were collected using MassLynx V 4.1 software (Waters Corporation, Manchester, UK).

The raw data were processed by Progenesis QI data analysis software (Non-Linear Dynamics, Newcastle, UK) for peak picking, alignment and normalization, to produce peak intensities for retention time (RT) and m/z data pairs. Further statistical analysis was performed on the resulting normalized peak intensities using SIMCA P 13.0.2 (Umetrics, Umea, Sweden).

3.3 Results and Discussion