We analyzed urine samples using a quadrupole time-of-flight (QTOF) mass spectrometer (Premier, Waters), in positive (ESI+) and negative (ESI−) electrospray ionization modes, using a 50 × 2.1 mm Acquity 1.7 µm C18 column (Waters Corp, Milford, MA). Urine samples were diluted with an equal volume of 50% aqueous acetonitrile containing debrisoquine (ESI+ internal standard) and 4-nitrobenzoic acid (ESI− internal standard). Samples were centrifuged at 14,000 × g for 20 minutes at 4°C to precipitate proteins. Five µl was chromatographed on a 50 × 2.1 mm Acquity BEH 1.7 µm C18 column (Waters) using an Acquity UPLC system (Waters). The gradient mobile phase consisted of 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). A typical 10-min sample run (at 0.5ml/min) consisted of 0.5 min of 100% solvent A followed by a linear gradient to 80% A at 4 min, to 5% A at 8 min. After a 0.5 min wash step, the column was equilibrated to initial conditions for 1.5 min. The eluent was introduced by electrospray ionization into the QTOF mass spectrometer (Premier, Waters) operating in positive (ESI+) or negative (ESI−) ionization mode. The capillary and sampling cone voltages were set to 3,000 and 30 V, respectively. Source and desolvation temperatures were set to 120 °C and 350 °C, respectively, and the cone and desolvation gas flows were set to 50.0 and 650.0 L/h, respectively. To maintain mass accuracy, sulfadimethoxine at a concentration of 300 pg/µl in 50% aqueous acetonitrile was used as a lock mass and injected at a rate of 50 µl/min. For MS scanning, data were acquired in centroid mode from 50 to 850 m/z and for tandem MS the collision energy was ramped from 5 to 35 V.
To avoid artifacts based on sample injection order, the order was randomized. Four different quality control sets were included with the runs to assess machine sensitivity and sample carry over. First, 169 “pooled” samples, containing aliquots from 108 randomly selected urine samples were processed randomly throughout the run. Second, a standard cocktail containing theophylline, caffeine, hippuric acid, 4-nitrobenzoic acid, and nortriptyline (designated as MetMix) was injected every 100 samples. Third, 32 blanks were randomly injected to assess sample carryover. Fourth, 48 samples with 4 high-purity nicotine metabolite standards, including cotinine, nicotine-N’-oxide, anabasine, and trans-3’-hydroxycotinine (Sigma-Aldrich), were spiked into urine. Fifth, 10% of the samples were randomly selected and processed in duplicate at the end of the run to evaluate chromatogram consistency. Finally, debrisoquine and 4-nitrobenzoic acid were spiked into samples for runs in ESI+ and ESI− modes, respectively. Raw chromatograms and extracted and normalized ion counts can be accessed in the MetaboLights database with study identifierMTBLS28 .
To avoid artifacts based on sample injection order, the order was randomized. Four different quality control sets were included with the runs to assess machine sensitivity and sample carry over. First, 169 “pooled” samples, containing aliquots from 108 randomly selected urine samples were processed randomly throughout the run. Second, a standard cocktail containing theophylline, caffeine, hippuric acid, 4-nitrobenzoic acid, and nortriptyline (designated as MetMix) was injected every 100 samples. Third, 32 blanks were randomly injected to assess sample carryover. Fourth, 48 samples with 4 high-purity nicotine metabolite standards, including cotinine, nicotine-N’-oxide, anabasine, and trans-3’-hydroxycotinine (Sigma-Aldrich), were spiked into urine. Fifth, 10% of the samples were randomly selected and processed in duplicate at the end of the run to evaluate chromatogram consistency. Finally, debrisoquine and 4-nitrobenzoic acid were spiked into samples for runs in ESI+ and ESI− modes, respectively. Raw chromatograms and extracted and normalized ion counts can be accessed in the MetaboLights database with study identifier
