Blood was collected in a tube with EDTA and centrifuged (4°C, 10 minutes, 2000 g). Plasma was frozen and stored until assay. Total homocysteine was quantified with the LC‐MS Clinmass® ‘Homocysteine in plasma/serum’ kit (Recipe, Germany). Supernatants were analysed using a Shimadzu UFLC XR system consisting of two LC‐20ADXR binary pumps, a DGU20A5R vacuum degasser, a CT0‐20AC thermostated column oven and a SIL‐20ACXR cooled auto sampler (Shimadzu). The LC system was interfaced with an ABSciex 4500 triple quadrupole mass spectrometer (Les Ulis, France) operating with an electrospray ionization source (ESI) using nitrogen (purity: 99.99%). The intra‐ or inter‐assay CV was <5%.
4500 triple quadrupole mass spectrometer
Manufactured by AB Sciex
Sourced in France
The 4500 triple quadrupole mass spectrometer is a high-performance analytical instrument designed for quantitative and qualitative analysis. It features a triple quadrupole configuration, which allows for the isolation, fragmentation, and detection of specific ions within a sample. The 4500 provides accurate and sensitive measurements, making it a versatile tool for various applications in analytical chemistry and life sciences.
Automatically generated - may contain errors
Lab products found in correlation
Uflc xr system, by Shimadzu (3 mentions)
Acquity hss t3 c18 column, by Waters Corporation (2 mentions)
Dgu 20a5r vacuum degasser, by Shimadzu (1 mentions)
Lc 20 ad xr binary pumps, by Shimadzu (1 mentions)
Atlantis r t3 column, by Waters Corporation (1 mentions)
1260 lc system, by Agilent Technologies (1 mentions)
Au5400 chemistry analyzer, by Olympus (1 mentions)
Whatman 903 protein saver card, by GE Healthcare (1 mentions)
Adenosine, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
2 chloroadenosine, by Merck Group (1 mentions)
Whatman, by Cytiva (1 mentions)
Alkaline phosphatase, by Merck Group (1 mentions)
Multiquant software, by AB Sciex (1 mentions)
7 protocols using 4500 triple quadrupole mass spectrometer
1
Quantification of Plasma Homocysteine
2
Adenosine Quantification by LC-MS/MS
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After the extraction, adenosine concentration was measured by LC-MS/MS. Samples were analyzed using a Shimadzu UFLC XR system (Shimadzu, Marne la Vallee, France). The LC system was interfaced with an ABSciex 4,500 triple quadrupole mass spectrometer (Les Ulis, France), operating with an electrospray ionization source (ESI) using nitrogen (purity: 99.99%). Ten microliters of the extracted sample were injected onto a 2.1-mm- × -100-mm, 3-μm Atlantis R T3 column, Waters (Guyancourt, France). The starting mobile phase consisted of 3% methanol and 97% acidified water (0.1% formic acid), with a flow of 0.7 ml/min for 3.5 min. Then, the gradient of methanol was increased to 30% for 3 min. The column was re-equilibrated for 2 min to starting conditions.
3
Quantification of DEHTP Metabolites in Urine
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The on-line HPLC-MS/MS method used for quantification of specific urinary DEHTP metabolites has been described in detail by Lessmann et al. (2016a) . In short, to each urine sample aliquot, ammonium acetate buffer and internal standard solution were added. After enzymatic hydrolysis with β-glucuronidase from E. coli K12 (arylsulfatase free), the pH was adjusted with acetic acid and samples were frozen over night to precipitate proteins. After thawing, samples were centrifuged and the supernatant was injected into an Agilent Technologies LC 1260 system (Agilent 1260 autosampler, two Agilent 1260 binary pumps) coupled to an AB Sciex 4500 triple quadrupole mass spectrometer in negative ionization mode. On-line SPE column assembly, HPLC gradient and MS/MS conditions remain as described in Lessmann et al. (2016a) . The limit of quantification (LOQ) was 0.2 μg/L for mono(2-ethyl-5-carboxypentyl) terephthalate (5cx-MEPTP) and mono(2-ethyl-5-oxohexyl) terephthalate (5oxo-MEHTP), 0.3 μg/L for mono(2-ethyl-5-hydroxyhexyl) terephthalate (5OH-MEHTP), and 0.4 μg/L for mono [2-(carboxymethyl) hexyl] terephthalate (2cx-MMHTP). Urinary creatinine concentrations were determined according to a modified Ja ffé method (Jaffe, 1886) with an Olympus AU5400® Chemistry Analyzer.
4
Adenosine Quantification in Blood Samples
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Sample collection has been previously described (24) . Briefly, after washing out the lumen of the radial catheter with a solution of 1 mL of papaverine and 1 mL of dipyramidole, blood was withdrawn together with a cold stop solution in vacutainer tubes to prevent both adenosine uptake by red blood cells and deamination into inosine. Samples were immediately centrifuged (4°C, 1500 × g).
APL was performed on admissionbefore the administration of any inotrope or vasopressorand after 24 h. APL was performed as previously described by liquid chromatography-tandem mass spectrometry after extraction (25) using a Shimadzu UFLC XR system (Shimadzu, Marne la Vallée, France). The system was interfaced with an ABSciex 4500 triple quadrupole mass spectrometer (Les Ulis, France) operating with an electrospray ionization source using nitrogen.
Liquid chromatography-mass spectrometry-grade methanol and water were purchased from VWR (Fontenay-sous-Bois, France). Formic acid, adenosine, and 2-chloroadenosine were obtained from Sigma Chemical (Saint-Quentin Fallavier, France). Whatman 903 protein saver cards™ for sample collection and preparation were acquired from GE Healthcare (Cardiff, UK).
APL was performed on admissionbefore the administration of any inotrope or vasopressorand after 24 h. APL was performed as previously described by liquid chromatography-tandem mass spectrometry after extraction (25) using a Shimadzu UFLC XR system (Shimadzu, Marne la Vallée, France). The system was interfaced with an ABSciex 4500 triple quadrupole mass spectrometer (Les Ulis, France) operating with an electrospray ionization source using nitrogen.
Liquid chromatography-mass spectrometry-grade methanol and water were purchased from VWR (Fontenay-sous-Bois, France). Formic acid, adenosine, and 2-chloroadenosine were obtained from Sigma Chemical (Saint-Quentin Fallavier, France). Whatman 903 protein saver cards™ for sample collection and preparation were acquired from GE Healthcare (Cardiff, UK).
5
Quantitative Mitochondrial Nucleoside Analysis
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Mitochondrially-enriched RNA was digested to component nucleosides with an enzyme cocktail of benzonase, phosphodiesterase and alkaline phosphatase (Merck), all used as according to the manufacturer's instructions. Nucleosides were separated by reverse phase liquid chromatography - eluent A was 0.1% (v/v) formic acid in water, and eluent B was 0.1% (v/v) formic acid in acetonitrile, and a non-linear gradient of 2–15% B resolved nucleosides across a Waters Acquity HSS T3 C18 column. The eluent was sprayed into a Sciex 4500 triple quadrupole mass spectrometer and characterized by tandem mass spectrometry using a multiple reaction monitoring approach. Injection amounts were normalized by internal calibration with isotopically-labelled uridine and quantification performed using external calibration of a range of nucleoside standards.
6
Vistusertib Quantification by LC-MS/MS
7
Quantitative Nucleoside Profiling by LC-MS
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Isolated RNA was digested to component nucleosides with an enzyme cocktail of Benzonase, phosphodiesterase, and alkaline phosphatase (Merck), all used according to the manufacturer’s instructions. Nucleosides were separated by reverse-phase liquid chromatography; eluent A was 0.1% v/v formic acid in water, and eluent B was 0.1% v/v formic acid in acetonitrile, and a non-linear gradient of 2%–15% B resolved nucleosides across an Acquity HSS T3 C18 column (Waters). The eluent was sprayed into a 4500 triple quadrupole mass spectrometer (Sciex) and characterized using tandem mass spectrometry with a multiple reaction monitoring approach. Injection amounts were normalized by internal calibration with isotopically labelled uridine and quantification extrapolated from external calibration of a range of nucleoside standards using MultiQuant software (Sciex).
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