Blood was collected from WT and Slc7a2–/–mice at the time of euthanasia via cardiac puncture and serum was separated as described [8 (link), 10 (link)]. Serum were diluted 1:5 with acetonitrile and derivatized by sequential addition of 50 mM sodium carbonate and 2% benzoyl chloride (v/v in acetonitrile). An internal standard solution (13C6-benzoyl chloride derivatized arginine at a concentration of 8.7 pg/μl in 20% acetonitrile in water with 2% sulfuric acid) was also added to the samples. LC–MS analysis was performed using a Waters Acquity UPLC (Milford, MA, USA) coupled to a SCIEX 6500+ QTrap mass spectrometer (Framingham, MA, USA) operating in multiple reaction monitoring mode [48 (link)].
Acquity uplc
Manufactured by AB Sciex
Sourced in United States
The Acquity UPLC is a high-performance liquid chromatography (HPLC) system developed by AB Sciex. It is designed to provide efficient and rapid separation of complex samples, enabling improved resolution, sensitivity, and speed compared to traditional HPLC systems.
Automatically generated - may contain errors
Lab products found in correlation
Multiquant software, by AB Sciex (2 mentions)
Mass spectrometer api5500 q trap, by AB Sciex (2 mentions)
Sciex 6500 qtrap mass spectrometer, by AB Sciex (1 mentions)
Qtrap 5500 mass spectrometer, by AB Sciex (1 mentions)
Acquity uplc csh c18 column, by Waters Corporation (1 mentions)
Api 5000, by AB Sciex (1 mentions)
Analyst software version 1, by AB Sciex (1 mentions)
Acquity uplc, by Waters Corporation (1 mentions)
Sample manager, by Waters Corporation (1 mentions)
Xbridge c8, by Waters Corporation (1 mentions)
Qtrap 5500, by AB Sciex (1 mentions)
Analyst software 1, by AB Sciex (1 mentions)
Beh c18, by Waters Corporation (1 mentions)
Analyst 1, by AB Sciex (1 mentions)
Api 4000 triple quadrupole mass spectrometer, by AB Sciex (1 mentions)
Strata x, by Phenomenex (1 mentions)
Triple quad 5500 system, by AB Sciex (1 mentions)
Triple quad 6500, by AB Sciex (1 mentions)
5500 qqq ms, by AB Sciex (1 mentions)
12 protocols using acquity uplc
1
Serum Metabolite Profiling of WT and Slc7a2−/− Mice
2
Targeted Lipidomics Analysis of Plasma
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Lipids were extracted from 10 μL of plasma with 900 μL dichloromethane:isopropanol:methanol (25:10:65, v/v/v) containing the following internal standards at a concentration of 200 nM: glyceryl triheptadecanoate, 1,2-dinonadecanoin, cholesteryl heptadecanoate, 1,2-dilauroyl-sn-glycero-3-phosphocholine, 1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine, and N-heptadecanoyl-D-erythro-sphingosine. Lipid extracts were then dried under N2 and resuspended in isopropanol:acetonitrile:water (2:1:1, v/v/v). Targeted lipidomics analyses were performed on a UPLC-MS/MS using a Waters Acquity UPLC coupled to a Sciex QTrap 5500 mass spectrometer. Lipid classes were separated by reversed-phase chromatography on a Waters Acquity UPLC CSH C18 column, 1.7 micron, 2.1 x 50 mm. The column temperature was set to 55°C and a 10-min gradient was run with the following mobile phase solvents: (i) mobile phase A = 10 mM ammonium formate in water:acetonitrile:formic acid (40:60:0.1, v/v/v) and (ii) mobile phase B = 10 mM ammonium formate in acetonitrile:isopropanol:formic acid (10:90:0.1, v/v/v). Lipid species were then analyzed on a mass spectrometer using positive ion electrospray ionization in the multiple reaction monitoring mode. LC chromatogram peak integration was performed with Sciex MultiQuant software. All data reduction was performed with in-house software.
3
Quantification of Compound Stability
4
UPLC Analysis of Metabolite Samples
5
Quantification of Drugs by LC-MS/MS
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Quantification of drugs was performed by LC–MS/MS (Acquity UPLC coupled to a Sciex 6500+ or 5500QTRAP, respectively, and operating in multiple reaction monitoring (MRM). A total of 0.025 µM of Carbutamide was used as the internal standard, as mentioned before. Briefly, substances were separated with the BEH C18 or Xbridge C8 columns (30 × 2.1 mm, 1.7 µm; Waters Corp., Milford, MA, USA), using a two-step gradient elution with acetonitrile and water both acidified with 0.1% formic acid. The gradient conditions were 0–0.4 min 1–40% acetonitrile and 0.4–1 min 40–95% acetonitrile with a constant flow rate of 0.7 mL/min. The column temperature was set at 50 °C. Samples were kept at 10 °C. The mass spectrometer was operating in positive electrospray ionization (ESI) mode (5500 V, temperature 550 °C; curtain gas 40 psi, GS1 60 psi and GS2 70 psi). The transition methods are described in Table S1, Supplementary Materials , for each analyte and its internal standard. Data were processed using Sciex Analyst Software 1.7.2.
6
Plasma Quantitation of SB-435495 by LC-MS/MS
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All plasma analysis was performed at GlaxoSmithKline. Briefly, SB-435495 was extracted from plasma (25 μL aliquot) using a protein precipitation method. Following vortexing and centrifugation, an aliquot of the supernatant (100μL) was removed and diluted with 50μL of acetonitrile/10mM Ammonium Formate, pH3 (50/50, v/v) and mixed. Samples were quantitated for SB-435495 using a liquid chromatography mass spectrometry (UPLC-MS/MS) method. The LC-MS/MS system consisted of a Waters Acquity UPLC (Milford, MA) and an AB Sciex API 4000 triple quadrupole mass spectrometer (Framingham, MA) operating under Analyst 1.4.2 software (AB Sciex, Framingham, MA). The lower limit of quantification of SB-435495 was 1ng/ml and the upper limit of quantification was 1000ng/ml in the assay.
7
Eicosanoid Profiling in Hemolymph
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A mix of 26 deuterated internal standards was added to 10uL of hemolymph. Eicosanoids were extracted by solid phase extraction (SPE) using Phenomenex Strata-X polymeric reversed phase columns. Samples were brought to dryness and taken up in buffer A (water/acetonitrile/acetic acid 60/40/0.02, v/v/v). Samples were analyzed using a Waters Acquity UPLC interfaced with an AB Sciex 6500 QTrap instrument. Chromatographic separation was achieved by a step gradient starting with100% buffer A to 100% buffer B (acetonitrile/isopropanol 50/50, v/v) over 5 min. Standard curves were obtained in parallel using identical conditions. Data analysis was performed with Analyst and Mulitquant software packages (71 (link)). We monitored 159 MRMs.
8
UPLC-MS/MS Quantification of Compound
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The concentrations of compound in plasma
and feces were quantified using an UPLC method with tandem mass spectrometric
detection (LC–MS/MS). The LC–MS system consisted of
a Waters Acquity UPLC coupled to an AB Sciex mass spectrometer API
5500 Q-trap. The UPLC separation was carried out on a reversed phase
column (Acquity UPLC BEC C8, 1.7 μM, 2.1 mm × 50 mm) using
a mobile phase gradient with 0.1% formic acid and acetonitrile as
eluents. The detection of the drug was performed using multiple reaction
monitoring in the positive ionization mode. Plasma samples were spiked
with internal standard, and the analyte was extracted from the matrix
using tert-butyl methyl ether (tBME). The organic
phase was evaporated to dryness under a stream of nitrogen. The residue
was dissolved in acetonitrile/0.1% formic acid for LC–MS/MS
analysis. Feces samples were homogenized with 4 times their volume
ethanol/water (4:1). Aliquots of the aqueous ethanolic extracts were
further diluted with acetonitrile/0.1% formic acid, spiked with internal
standard, and directly injected into the system.
and feces were quantified using an UPLC method with tandem mass spectrometric
detection (LC–MS/MS). The LC–MS system consisted of
a Waters Acquity UPLC coupled to an AB Sciex mass spectrometer API
5500 Q-trap. The UPLC separation was carried out on a reversed phase
column (Acquity UPLC BEC C8, 1.7 μM, 2.1 mm × 50 mm) using
a mobile phase gradient with 0.1% formic acid and acetonitrile as
eluents. The detection of the drug was performed using multiple reaction
monitoring in the positive ionization mode. Plasma samples were spiked
with internal standard, and the analyte was extracted from the matrix
using tert-butyl methyl ether (tBME). The organic
phase was evaporated to dryness under a stream of nitrogen. The residue
was dissolved in acetonitrile/0.1% formic acid for LC–MS/MS
analysis. Feces samples were homogenized with 4 times their volume
ethanol/water (4:1). Aliquots of the aqueous ethanolic extracts were
further diluted with acetonitrile/0.1% formic acid, spiked with internal
standard, and directly injected into the system.
9
Enzymatic Activity Determination of XimA
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For determination of enzymatic activity, we used 100 µl of the reaction mixture containing 50 mM Tris-HCl buffer (pH 7.5), 5 mM MgSO4, 5 mM ATP, 10 µg 3, 10 mM L-threonine and 1 mg XimA. After incubation at 30°C for 12 h, the reaction was quenched by adding 1 ml methanol. The protein was removed by centrifugation at 13,000 g for 10 min, and the supernatant was then evaporated at 50°C. The remaining residue was freeze-dried for 24 h and then dissolved in 100 µl methanol. Enzymatic products were analyzed by UPLC-Q-TOF-MS as described above. The control assay was carried out under the same conditions with heat-inactivated enzyme.
Reactions to determine the Km of XimA toward xiamenmycin B contained 50 mM Tris-HCl buffer (pH 7.5), 5 mM MgSO4, 5 mM ATP, 10 mM L-threonine, 40 µM XimA and various concentrations of xiamenmycin B ranging from 0.2 µM to 345 µM. The reaction products were detected by Ultra Performance Liquid Chromatography and a Triple Quadrupole Mass Spectrometer (Waters ACQUITY UPLC, AB SCIEX SelexION Triple Quad 5500 System).
Reactions to determine the Km of XimA toward xiamenmycin B contained 50 mM Tris-HCl buffer (pH 7.5), 5 mM MgSO4, 5 mM ATP, 10 mM L-threonine, 40 µM XimA and various concentrations of xiamenmycin B ranging from 0.2 µM to 345 µM. The reaction products were detected by Ultra Performance Liquid Chromatography and a Triple Quadrupole Mass Spectrometer (Waters ACQUITY UPLC, AB SCIEX SelexION Triple Quad 5500 System).
10
Plasma Metabolomic Analysis by GC-MS and LC-MS/MS
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Sample preparation, and GC-MS and LC-MS/MS analysis were performed as our previous studies25 (link),26 (link). Briefly, plasma samples were extracted and analyzed on GC-MS and LC-MS/MS. For GC/MS, samples were analyzed on an Agilent 7890 A/5975 C Inert Triple Axis Detector (Agilent, USA). The original spectral data from GC−MS were converted to NetCDF format and then processed by XCMS software for peak finding, integration and alignment. LC-MS/MS analysis was employed to quantify low abundance neurotransmitters, using a Waters ACQUITY UPLC and AB Sciex Triple Quad6500 mass spectrometry system. Data collection and analysis for LC-MS/MS were performed using Analyst software (AB Sciex, v. 1.5.2) on the default parameters for automatic identification and integration of the MRM transition. The additional details information of targeted metabolomic analysis was shown in Supplemental Materials .
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