DMS273 cells were seeded at 4 × 105 cells per tube in 100 μM MG and the inhibitor (0 or 10 μM) in DPBS (pH 7.4) (n = 4). After 2 h incubation at 37 °C, sample was centrifuged, and supernatant was collected. Extracellular d -lactate was measured by analysis of the supernatant with Q-dsAMC. After collection of supernatants, the cells were washed with DPBS (pH 7.4) and lysed by vortexing with MeOH/H2O (80/20, v/v). The lysate was centrifuged, and the supernatant was collected. Intracellular S-D-lactoylglutathione (SLG) was measured by analysis of the cell extract with LC-MS/MS. For detection of intracellular GSH, the cell extract (15 μL) was mixed with 15 μL 5 mM Dansyl-Cl in MeCN and 15 μL 100 mM borate buffer (pH 9.1) at r.t. for 30 min, then quenched by adding 15 μL MeCN containing 10% formic acid. The mixture was subjected to LC-MS/MS. LC-MS/MS analysis was performed on an Acquity UPLC H-Class system (Waters) equipped with an Acquity UPLC BEH C18 1.7 μm (2.1 × 50 mm) column (Waters) and an MS detector (Xevo TQD, Waters). Detection was performed in the positive mode. For SLG, the fragment of m/z = 366.2 > 170.1 was used (cone voltage: 25 V, collision voltage: 25 V). For GSH, the fragment m/z = 367.2 > 170.1 was used (cone voltage: 25 V, collision voltage: 25 V).
Acquity uplc beh c18 1.7 μm 2 1 50 mm column
Manufactured by Waters Corporation
Sourced in Ireland, United States, Canada
The Acquity UPLC BEH C18 1.7 μm (2.1 × 50 mm) column is a high-performance liquid chromatography (HPLC) column designed for use with ultra-high-performance liquid chromatography (UPLC) systems. The column features a stationary phase of 1.7 μm ethylene-bridged hybrid (BEH) particles with a C18 bonded phase. This column is suitable for the separation and analysis of a wide range of analytes.
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Lab products found in correlation
Acquity uplc h class system, by Waters Corporation (3 mentions)
Xevo tqd, by Waters Corporation (1 mentions)
Q exactive, by Thermo Fisher Scientific (1 mentions)
Quadrupole orbitrap, by Thermo Fisher Scientific (1 mentions)
Xevo tq s, by Waters Corporation (1 mentions)
Masslynx software, by Waters Corporation (1 mentions)
Pu 2080, by Jasco (1 mentions)
Isolera one purification system, by Biotage (1 mentions)
Jnm la400, by JEOL (1 mentions)
Acquity uplc, by Waters Corporation (1 mentions)
Acquity tuv, by Waters Corporation (1 mentions)
Xevo g2 q tof ms, by Waters Corporation (1 mentions)
9 protocols using acquity uplc beh c18 1.7 μm 2 1 50 mm column
1
Quantification of Intracellular Metabolites
2
Characterization of Cryptococcus gattii Extracellular Vesicles
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C. gattii EVs were vacuum dried and extracted with 1 ml of methanol during 1 h in an ultrasonic bath. The extracts were filtered (0.22 μm), dried under a gentle N2 flux and stored at −20°C. EV extracts were resuspended in 200 μl of MeOH and transferred into glass vials. Ultra high-performance liquid chromatography-mass spectrometry (UHPLC-MS) analyses were performed using a Thermo Scientific QExactive® hybrid Quadrupole-Orbitrap mass spectrometer with the following parameters: electrospray ionization in positive mode, capillary voltage at 3.5 kV; capillary temperature at 300°C; S-lens of 50 V and m/z range of 100.00–1500.00. Tandem Mass spectrometry (MS/MS) was performed using normalized collision energy (NCE) of 20, 30, and 40 eV; maximum 5 precursors per cycle were selected. Stationary phase was a Waters ACQUITY UPLC® BEH C18 1.7 μm (2.1 × 50 mm) column. Mobile phases were 0.1% (v/v) formic acid in water (A) and acetonitrile (B). Eluent profile (A:B) 0–10 min, gradient from 95:5 up to 2:98; held for 5 min; 15–16.2 min gradient up to 95:5; held for 3.8 min. Flow rate was 0.2 mL min−1. Injection volume was 3 μL. UHPLC-MS operation and spectra analyses were performed using Xcalibur software (version 3.0.63).
3
UPLC-PDA Method for Compound Characterization
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The Waters ACQUITY UPLC® H-Class system (Waters Corporation, Milford, MA, USA) comprised of quaternary pump, column oven, autosampler, and photodiode array detector was used for method development, forced degradation studies, and method validation. The Empower 3 software was used for controlling instrument operation and processing data. Chromatographic separation was achieved on an ACQUITY UPLC® BEH C18 1.7 μm, 2.1 × 50 mm column (Waters Chromatography Ireland Limited, Dublin, Ireland). The mobile phase consisting of 2 %v/v acetic acid in water (A) and acetonitrile (B) was delivered at a flow rate of 0.3 mL/min. The gradient elution program was optimized as follows: initial A-B of 55:45 at 0.0 min; linear-gradient A-B of 20:80 from 0.0−2.7 min; isocratic A-B of 20:80 from 2.7−4.5 min; linear-gradient A-B of 55:45 from 4.5−5.0 min; isocratic A-B of 55:45 from 5.0−7.0 min. The column oven temperature and detection wavelength were set at 33 °C and 400 nm, respectively. The injection volume was 2 μL.
4
Quantitative Bioanalysis of Nilotinib in Plasma and CSF
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Plasma and CSF samples (20 μl) were thawed initially on ice at room temperature and transferred to Eppendorf tubes containing 100 μl of water. Extraction solvent (500 μl) Acetonitrile/Methanol (50:50) containing the internal standard (5 ng/mL of Nilotinib_13C_2H3) was added to the sample. The mixture was vortexed and incubated for 20 min on ice to accelerate protein precipitation and dialysis through 25 μm membranes was performed to obtain unbound Nilotinib. After incubation, the samples were vortexed and centrifuged at 13,000 rpm for 20 min at 4°C. The supernatant containing unbound Nilotinib was freeze-dried using speed vacuum and reconstituted in 200 μL of Methanol: Water (50:50) and processed by mass spectrometry.
The samples were resolved on an Acquity UPLC BEH C18 1.7 μm, 2.1×50 mm column online with a triple quadrupole mass spectrometer (Xevo-TQ-S, Waters Corporation, USA) operating in the multiple reaction monitoring (MRM) mode. The instrument parameters were optimized to gain maximum specificity and sensitivity of ionization for the parent [m/z = 530.27 (Nilotinib), 438.25 and daughter ions [m/z = 289.01 (Nilotinib) using “IntelliStart” feature of MassLynx software (Waters Corporation, USA). The metabolite ratios were calculated by normalizing the peak area of endogenous metabolites within tissue samples normalized to the internal standard Nilotinib_13C_2H3.
The samples were resolved on an Acquity UPLC BEH C18 1.7 μm, 2.1×50 mm column online with a triple quadrupole mass spectrometer (Xevo-TQ-S, Waters Corporation, USA) operating in the multiple reaction monitoring (MRM) mode. The instrument parameters were optimized to gain maximum specificity and sensitivity of ionization for the parent [m/z = 530.27 (Nilotinib), 438.25 and daughter ions [m/z = 289.01 (Nilotinib) using “IntelliStart” feature of MassLynx software (Waters Corporation, USA). The metabolite ratios were calculated by normalizing the peak area of endogenous metabolites within tissue samples normalized to the internal standard Nilotinib_13C_2H3.
5
Plasma Stability of Investigational Compounds
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Plasma stability studies were conducted by WuXi AppTec Co. Ltd. ((Nanjing, China). The concentration of ST3ND-25 and ST3ND34 in plasma was determined using SCIEX Triple Quad 6500+ LC-system and Waters Acquity UPLC BEH C18 1.7 μm 2.1 × 50 mm Column. 2 μl of 100 μM compound stock solution in DMSO were added to 98 μl of thawed pooled plasma that was cleared by centrifugation for 5 min at 4000 rpm. The mixtures were incubated at 37°C and precipitated with 500 μl of acetonitrile containing internal standards, 200 ng/ml of tolbutamide and 200 ng/ml of labetalol. After 20 min of mixing, the 96-well plate with samples was centrifuged for 20 min at 4000 rpm and supernatants were analyzed by LC-MS/MS.
6
Analytical Characterization of Organic Compounds
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Nuclear magnetic resonance (NMR) spectra were recorded on a JEOL JNM-LA400 at 400 MHz for 1H NMR and at 100 MHz for 13C NMR. Mass spectra (MS) were measured on a JEOL JMS-T100LP AccuTOF LC-plus 4G (electrospray ionization). Preparative high-performance liquid chromatography (HPLC) was performed on an Inertsil ODS-3 (10.0 × 250 mm) column (GL Sciences Inc.) using an HPLC system composed of a pump (PU-2080, JASCO) and a detector (MD-2015). Preparative medium pressure liquid chromatography (MPLC) was performed on an Isolera One purification system (Biotage) equipped with a Biotage SNAP Ultra C18 column (for reverse phase separation) or on an MPLC system composed of a pump and a detector (EPCLC-AI-580S, Yamazen) and equipped with a silica gel column (silica gel 40 μm or Amino 40 μm, Yamazen) (for normal phase separation). Liquid chromatography–mass spectrometry analysis was performed on an Acquity UPLC H-Class System (Waters) equipped with an Acquity UPLC BEH C18 1.7 μm (2.1 × 50 mm) column (Waters) and an MS detector (QDa, Waters).
7
Quantification of HGA and MCPG Metabolites
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The quantification of HGA and MCPG plus their metabolites in serum and urine was carried out by UPLC-MS/MS as described in detail earlier (9 (link)–12 (link)). For all analyses, 25 μL of material were extracted with 300 μL of methanol containing the internal standards. Analysis was performed after butylation. For chromatographic separation 5 μL of the final extracts were injected onto an Acquity UPLC BEH C18 1.7 μm, 2.1 ×50 mm column (Waters). Gradient chromatography was performed using acetonitrile/water modified by 0.1% formic acid and 0.01% trifluoroacetic acid. Quantifications were done on a Xevo TQMS UPLC-MS/MS system (Waters, Eschborn, Germany), calculation of concentrations was conducted with single-point calibration. Concentrations of C4 to C10 acyl conjugates were also determined by this method in order to differentiate branched and unbranched C4 and C5 metabolites. The isomers of 2-MBC appeared in two separate peaks, the values of both isomers were added for quantification.
8
UPLC-MS/MS Analysis of Compounds
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The ultra-performance liquid chromatography (UPLC) system was from Waters (ACQUITY UPLC I-Class system) coupled to Xevo™ TQ-S micro tandem mass spectrometry with positive electrospray ionization (+ESI) source. The UPLC chromatographic separation utilized ACQUITY UPLC ™ BEH C18 1.7 μm, 2.1 × 50 mm column from Waters (Mississauga, ON Canada). ACQUITY UPLC ™ in-line filter unit with 0.2 µm stainless steel filter from Waters (Mississauga, ON Canada).
9
UPLC-QTof-MS for Accurate Mass Analysis
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Mass spectrometry analysis. The liquid chromatography equipment coupled to mass spectrometry (LC/MS) system was equipped with an UPLC chromatograph (ACQUITY UPLC from Waters), equipped with a UV/vis detector (Acquity TUV), coupled to a quadrupole timeof-flight mass spectrometer (Xevo G2-QTof-MS from Waters) (UPLC-QTof-MS), equipped with an electrospray ionization source (ESI). UPLC analyses were performed using an ACQUITY UPLC BEH C18 (1.7 μm; 2.1 × 50 mm) column (Waters), and isocratic elution with 25 mM formic acid (pH = 3.2) and ACN (97:3) at a flow rate of 0.5 mL min -1 . The mass spectrometer was operated in positive and negative ion modes. For high mass accuracy, the Q-Tof was calibrated using 0.1% phosphoric acid in 50:50 MeOH/H 2 O (vol/ vol). The instrument drift was compensated by applying a lock mass correction. Therefore the samples were injected in the chromatograph, the components were separated and then the mass spectra were recorded for the each peak of the corresponding chromatograms. In addition, mass chromatograms, i.e. representations of mass spectrometry data as chromatograms (the x-axis representing time and the y-axis signal intensity), were recorded using different scan ranges.
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