Tsq quantum triple quadrupole mass spectrometer

Plasma XOR activity was measured using frozen samples that were maintained at −80 °C until the time of assay and was measured using the recently established assay using stable isotope-labelled [13C2,15N2] xanthine with liquid chromatography mass spectrometry (Nano Space SI-2 LC system, Shiseido, Tokyo, Japan) and a TSQ-Quantum triple quadrupole mass spectrometer (Thermo Fisher Scientific GmbH, Bremen, Germany) [17 (link),18 (link)]. The calibration curve of [13C2,15N2] UA showed linearity over the range of 4–4000 nM (r² > 0.995) with a lower limit of quantitation of 4 nM. The lower detection limit of XOR activity was 6.67 pmol/h/mL plasma, and intra- and inter-assay coefficients of variation of human plasma XOR activity were 6.5% and 9.1%, respectively [17 (link)].

Fentanyl, acetylfentanyl and their metabolites in the culture medium were tentatively quantified as reported previously [7 (link)]. Briefly, a 25 µL sample of the culture medium was treated with β-glucuronidase/aryl sulfatase as described above. Ten microliters of internal standard (IS) solution (50 ng of cis-3-methylfentanyl hydrochloride dissolved in 10 µL of water) was added to the reaction mixture, and then it was deproteinized with 0.25 mL of acetonitrile. After centrifugation (10,000 × g for 5 min), a portion of the supernatant was diluted five times with 0.1% formic acid. This sample was centrifuged at 10,000 × g for 5 min, and then the supernatant was analyzed by LC/MS. The conditions of analysis were as follows: apparatus, a NANOSPACE SI-2 LC system (Shiseido, Tokyo, Japan) connected to a TSQ Quantum triple quadrupole mass spectrometer (Thermo Fisher Scientific); column, mobile phase composition, flow rate, and MS interface were the same as for the identification of the metabolites; analysis mode, selected reaction monitoring (SRM).

C. albicans cells were harvested [82 (link)] and protein extracts prepared in 0.5 M citrate, pH 5.0) as GSNO was found to be unstable at pH 8.0. For GSNO assays, 5 μL of 1 mM NADH and 10 μL of 250 μM GSNO were added to 10 μL of protein extract, and samples were derivatized at 0, 2, 5, 10, 20 and 30 min. For GSSG assays, 5 μL of 1 mM NADPH and 10 μL of 250 μM GSSG were added to 10 μL of protein extract, and samples were derivatized at 0, 2, 5, 10, 20 and 30 min. The derivatization and analysis were performed as previously described [93 (link)] using a Thermo Surveyor LC system coupled to a TSQ Quantum, triple quadrupole mass spectrometer (Thermo Scientific, UK). The following SRM transitions were used for quantification; Glu-Glu (internal standard) m/z 277–241, GSNO m/z 337–307, GSNEM m/z 433–304 and GSSG m/z 613–355. Peak integration and quantification was performed using Xcalibur software (Version 2.0.7.SP2). GSH, GSSG and GSNO concentrations were then calculated relative to authentic standards. Data were normalised against total protein. Data represent the means and standard deviations from at least three independent experiments.
The redox potential was calculated using the GSH and GSSG concentrations calculated using the following equation based on the Nernst equation [20 (link)].
(30°C, pH 7.4)