The assay protocol of XOR activity in humans was reported previously26 (link)–28 (link). In brief, 100 μL of plasma samples (purified by Sephadex G25 resin) were mixed with a Tris buffer (pH 8.5) containing [13C2,15N2] xanthine as a substrate, NAD+, and [13C3,15N3] UA as an internal standard. The mixtures were incubated at 37 °C for 90 min, mixed with 500 µL of methanol, and centrifuged at 2000× g for 15 min at 4 °C. The supernatants were transferred to new tubes and dried using a centrifugal evaporator. The residues were reconstituted with 150 μL of distilled water, filtered through an ultrafiltration membrane, and measured using LC/TQMS. LC/TQMS comprised a Nano Space SI-2 LC system (Shiseido Co., Ltd., Tokyo, Japan) and a TSQ Triple Quadrupole LC–MS system (ThermoFisher Scientific GmbH, Bremen, Germany) equipped with an ESI interface. Calibration standard samples of [13C2,15N2] UA were also measured, and the amounts of production were quantitated from the calibration curve. XOR activities were expressed in pmol/mL/h26 (link)–28 (link).
Nano space si 2 lc system
Manufactured by Shiseido
Sourced in Japan
The Nano Space SI-2 LC system is a laboratory instrument designed for liquid chromatography analysis. It is a high-performance liquid chromatography (HPLC) system that enables the separation, identification, and quantification of various chemical compounds in a sample. The core function of the Nano Space SI-2 LC system is to perform precise and efficient liquid chromatography analysis.
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5 protocols using nano space si 2 lc system
1
Quantitative Assay of XOR Activity
2
Quantification of TMAO, Phenyl Sulfate, and Indoxyl Sulfate
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Plasma concentrations of TMAO, phenyl sulfate (PS), and IS were measured using liquid chromatography–tandem mass spectrometry (LC‐MS/MS), as described previously (Kanemitsu et al., 2017 (link)). Briefly, chromatographic separation was performed on a Nanospace SI‐2 LC system (Shiseido) using a Scherzo SS‐C18 analytical column (50 × 2.0 mm i.d., 3.0 µm, Imtakt). A guard column (2 × 5 mm, 3 μm) was used to protect the analytical column containing the same material as the analytical column, which was fitted between the analytical column and the autosampler. The column effluent was monitored using a TSQ Ultra triple quadrupole mass spectrometer (Thermo Fisher Scientific) equipped with a heated electrospray ionization source system. Samples were analyzed in the single reaction monitoring mode using the ion transitions: m/z 76.05→58.10 for TMAO, m/z 172.99→93.30 for PS, and m/z 212.03→131.95 for IS. Deuterated internal standards were used for all analytes.
3
Plasma Xanthine Oxidase Activity Assay
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Plasma XOR activity was determined using freshly thawed samples that had been stored at -80°C until the time of the assay with our recently established assay using stable isotope-labeled ( 13 C 2 , 15 N 2 ) xanthine with LC/MS (Nano Space SI-2 LC system, Shiseido, Tokyo, Japan, and TQMS, Thermo Fisher Scientific GmbH, Bremen, Germany), as previously described [14, 15] . In brief, 100-µL aliquots of plasma were purified on a Sephadex G25 column and then mixed with Tris buffer (pH 8.5) containing ( 13 C 2 , 15 N 2 ) xanthine as the substrate and NAD + , with ( 13 C 3 , 15 N 3 ) uric acid used as an internal standard. The mixtures were incubated at 37°C for 90 min, then methanol (500 μL) was added and centrifugation was performed at 2,000 g for 15 min at 4°C. Next, the supernatants were transferred to new tubes and dried using a centrifugal evaporator. The residues were reconstituted in 150 μL of distilled water, filtered through an ultrafiltration membrane, and measured using LC/TQMS. Calibration standard samples were measured for ( 13 C 2 , 15 N 2 ) uric acid, and the amounts of ( 13 C 2 , 15 N 2 ) uric acid produced were calculated based on the calibration curve. XOR activity is expressed as pmol/mL/h of ( 13 C 2 , 15 N 2 ) uric acid produced. The intra-and inter-assay coefficients of variation were 6.5 and 9.1%, respectively [15] .
4
Quantification of Fentanyl and Metabolites
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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).
5
Plasma Xanthine Oxidoreductase Activity Assay
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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)].
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