Sample volumes of 4 μl were injected onto a Waters Acquity UPLC® Bridged Ethyl Hybrid (BEH) C18 column (2.1 × 30 mm, 1.7 μm particle size, Waters, Milford, MA, USA), using 0.1% (v/v) formic acid in water (A) and methanol (B), with a run time of 2.2 min and a total flow rate of 0.6 ml/min. The temperature of the column oven was set at 40°C and the autoinjector rack at 4°C. Gradient elution started at 40% methanol and increased linearly to 70% in 1.5 min, followed by flushing with 100% methanol for 0.3 min and re‐equilibration at 40% until the end of the run. A Sciex QTRAP®5500 triple quadrupole mass spectrometer was used for detection. The optimized electrospray parameters were: 4000 V ion spray voltage; 15 psi curtain gas; “medium” collision gas; 60 psi ion source gas 1; 75 psi ion source gas 2; and 700°C temperature. Selected reaction monitoring in positive ion mode with unit resolution for both quadrupoles was performed at m/z 384.1 > 101.0, 85 V declustering potential and 15 V collision energy for EAI045, at m/z 302.1 > 134.0, 50 V declustering potential and 26 V collision energy for PIA, and at m/z 414.1 > 307.0, 96 V declustering potential and 31 V collision energy for PLX4720 (internal standard, IS). The dwell time was 50 ms, and the entrance potential was 10 V.
5500 qtrap triple quadrupole mass spectrometer
Manufactured by AB Sciex
Sourced in United States, Japan, Germany
The 5500 QTRAP triple quadrupole mass spectrometer is a high-performance analytical instrument designed for advanced quantitative and qualitative analysis. It features a triple quadrupole configuration with a linear ion trap, providing enhanced sensitivity, selectivity, and scan speed for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Prominence uflc hplc system, by Shimadzu (17 mentions)
Multiquant v2, by AB Sciex (14 mentions)
Amide hilic chromatography, by Waters Corporation (8 mentions)
Prominence uflc system, by Shimadzu (6 mentions)
Acquity uplc system, by Waters Corporation (4 mentions)
Analyst software version 1, by AB Sciex (3 mentions)
Acquity uplc, by Waters Corporation (3 mentions)
Amide hilic column, by Waters Corporation (3 mentions)
Exionlc ad, by AB Sciex (2 mentions)
Nexera uhplc system, by Shimadzu (2 mentions)
Xbridge amide column, by Waters Corporation (2 mentions)
Hilic plus rrhd column, by Agilent Technologies (2 mentions)
4000 qtrap triple quadrupole mass spectrometer, by AB Sciex (2 mentions)
Ammonium hydroxide, by Avantor (2 mentions)
Luna nh2 column, by Phenomenex (2 mentions)
Multiquant 2, by AB Sciex (2 mentions)
Inosine 15n4, by Cambridge Isotopes (2 mentions)
Thymine d4, by Cambridge Isotopes (2 mentions)
Glycocholate d4, by Cambridge Isotopes (2 mentions)
Tracefinder 3, by Thermo Fisher Scientific (2 mentions)
68 protocols using 5500 qtrap triple quadrupole mass spectrometer
1
UPLC-MS/MS Quantification of Anticancer Compounds
2
Metabolomic Analysis of Bone Marrow Adipose Tissue
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After overnight fasting (about 12 h), BMAT was isolated from the femurs based on the protocol of Tencerova and the co-authors with modifications24 . Briefly, the femurs were longitudinally bisected using a Dremel rotary tool with a 409 cutoff wheel, and the BMAT was then removed using a stainless-steel spatula. The BMAT was snap-frozen for further metabolomic analysis.
The extraction of metabolites was performed using the prechilled (−20 °C) extraction buffer containing MeOH-ACN-H2O (43:43:16, v/v/v) and stable isotope standards as described before25 ,26 (link). The metabolomic analysis was performed on an Agilent 1290 HPLC coupled with a SCIEX QTRAP 5500 triple quadrupole mass spectrometer (LC-MS/MS)26 (link). The metabolites were separated on a Luna NH2 HPLC column (250 mm × 2.0 mm, 5 μm, Phenomenex, USA). The MS was performed by multiple reaction monitoring (MRM) in both negative and positive mode with rapid polarity switching (50 ms). A total of 420 metabolites covering all the major metabolic pathways were targeted21 (link). Data were log 2 transformed before statistical analysis. Multivariate analysis (Partial least squares discriminant analysis, PLS-DA), pathway analysis, and ANOVA simultaneous component analysis were performed using Metaboanalyst 4.0 (www.metaboanalyst.ca ). The details were described in the Supplementary method .
The extraction of metabolites was performed using the prechilled (−20 °C) extraction buffer containing MeOH-ACN-H2O (43:43:16, v/v/v) and stable isotope standards as described before25 ,26 (link). The metabolomic analysis was performed on an Agilent 1290 HPLC coupled with a SCIEX QTRAP 5500 triple quadrupole mass spectrometer (LC-MS/MS)26 (link). The metabolites were separated on a Luna NH2 HPLC column (250 mm × 2.0 mm, 5 μm, Phenomenex, USA). The MS was performed by multiple reaction monitoring (MRM) in both negative and positive mode with rapid polarity switching (50 ms). A total of 420 metabolites covering all the major metabolic pathways were targeted21 (link). Data were log 2 transformed before statistical analysis. Multivariate analysis (Partial least squares discriminant analysis, PLS-DA), pathway analysis, and ANOVA simultaneous component analysis were performed using Metaboanalyst 4.0 (
3
Quantification of Eicosanoid Lipid Mediators
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The concentrations of PGE2, PGD2, PGF2α, 11β-PGF2α, 15-deoxy-PGJ2 and 8-iso-PGF2α were measured using a UFLC Nexera liquid chromatograph system (Shimadzu, Kyoto, Japan) coupled to a QTrap 5500 triple quadrupole mass spectrometer (Sciex, Framingham, MA, USA). Briefly, samples were spiked with the internal standard (IS) mixture and extracted using ethyl acetate (EtOAc) acidified with acetic acid. After vigorous shaking, samples were centrifuged, and the upper organic layer was evaporated to dryness under a nitrogen stream (37 °C). The dry residue was reconstituted in nitrogen-purged ethanol (EtOH) and injected onto an Acquity UHPLC BEH C18 (3.0 × 100 mm, 1.7 µm, Waters, Milford) analytical column. Analytes were separated under the gradient elution mode. The MS detection of studied eicosanoid and their deuterated internal standards was carried out in negative ion electrospray ionisation by applying the multiple reaction monitoring mode (MRM); this method and the instrument operation parameters were described previously [20 (link)].
The quantification of studied eicosanoid was performed based on the calibration curves plotted for each eicosanoid as the relationship between the peak area ratios of analyte/IS to the nominal concentration of the analyte. Eicosanoid levels in cell medium samples were calculated based on the regression equations estimated for each analyte.
The quantification of studied eicosanoid was performed based on the calibration curves plotted for each eicosanoid as the relationship between the peak area ratios of analyte/IS to the nominal concentration of the analyte. Eicosanoid levels in cell medium samples were calculated based on the regression equations estimated for each analyte.
4
Metabolomic Analyses of CD and HFrD Mice
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Untargeted metabolome analyses and targeted analyses for short-chain fatty acids (SCFAs) were performed to assess the metabolic profile in feces and serum of 5 CD mice and 10/11 HFrD mice of each cohort, as well as in the cardia of 5 CD and 5 HFrD mice of each cohort. Untargeted metabolomics were performed using a Nexera UHPLC system (Shimadzu, Nakagyo-ki, Kyoto, Japan) coupled with a Q-TOF mass spectrometer (TripleTOF 6600, AB Sciex, Framingham, MA, USA). Separation of the samples was performed using a HILIC UPLC BEH Amide 2.1 × 100, 1.7 μm analytic column (Waters Corp., Milford, MA, USA), and a reversed-phase Kinetex XB-C18, 2.1 × 100, 1.7 μm analytic column (Phenomenex, Torrance, CA, USA), respectively. Targeted SCFA analysis was performed using a QTRAP 5500 triple quadrupole mass spectrometer (Sciex, Darmstadt, Germany) coupled with an ExionLC AD (Sciex, Darmstadt, Germany) ultrahigh performance liquid chromatography system. According to a method previously described [28 (link)], a multiple reaction monitoring (MRM) method was used for the detection and quantification of SCFA.
5
Quantitative Analysis of Bile Acids by LC-MS/MS
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Samples were analyzed using an LC-MS/MS system consisting of a QTRAP 5500 triple quadrupole mass spectrometer (Sciex, Darmstadt, Germany) coupled to an ExionLC AD (Sciex, Darmstadt, Germany) ultrahigh performance liquid chromatography system consisting of two LC pump systems ExionLC AD, an ExionLC degasser, an ExionLC AD autosampler, an ExionLC AC column oven, and an ExionLC controller. A multiple reaction monitoring (MRM) method was used for the detection and quantification of BA. For separation of these compounds, a 100 × 2.1 mm, 100 Å, 1.7 μm, Kinetex C18 column (Phenomenex, Aschaffenburg, Germany) and a SecurityGuard™ ULTRA Cartridges UHPLC C18 2.1 mm column (Phenomenex, Aschaffenburg, Germany) was used. The chromatography was performed with a column temperature of 40 °C and a constant flow rate of 0.4 mL/min using the mobile phase consisting of eluent (A) water and eluent (B) acetonitrile/water (95/5, v/v), both containing 5 mM ammonium acetate and 0.1% formic acid. The gradient elution is described in Table 7 . The injection volume for all samples was 1 μL, the column oven temperature was set to 40 °C, and the autosampler was kept at 10 °C. Data acquisition and instrumental control were performed with Analyst 1.7 software (Sciex, Darmstadt, Germany).
6
Quantitative LC-MS/MS Analysis Protocol
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LC-ESI-MS/MS was performed using a QTRAP5500 triple quadrupole mass spectrometer with a TurboV-ion source (Sciex, Darmstadt, Germany) coupled to an Agilent 1260 HPLC system (G 1322A Degasser, binary pump G1312B, oven G1316A, Agilent, Waldbronn, Germany) and a SIL-20A/HT autosampler (Shimadzu, Duisburg, Germany). Autosampler settings were set as follows: rinsing volume 200 μL (acetonitrile/water 50:50, v/v), needle stroke 52 mm, rinsing speed 35 μL/s, sampling speed 5.0 μl/s, purge time 1.0 min rinse dip time 0 s and rinse mode was set to “before and after aspiration.” Data acquisition and analysis was carried out with Analyst 1.6.3 software.
7
Entrectinib Separation and Quantification
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Chromatographic separation of entrectinib was achieved on Waters Alliance-e2695 , using Luna, 250×4.6 mm, 5 µm column and the mobile phase containing 0.1% formic acid and ACN in the ratio of 70:30% v/v. The flow rate was 1.0 ml/min. An injection volume was 10 µl and the column temperature was 30°C. The runtime was 10.0 min. The LC-MS/MS consists of SCIEX QTRAP 5500 triple quadrupole mass spectrometer equipped with electrospray ionization (ESI) with an automatic sample injector. The mass spectrometer was operated in positive ESI mode. The drying gas temperature was 120-150°C and the Dwell time was 1 s. Quantification was performed using multiple reaction monitoring (MRM) of the transitions.
8
Metabolomic Biomarkers of Diabetic Kidney Disease
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To identify the progression of DKD, we recruited an extra cohort of 106 patients at stage 2 and 3 for an average 3.9 years of follow-up study. Patients’ serums were collected since the starting point and the sCr, Bun and eGFR were measured every 3 months. The serum samples collected at the first time were subjected to targeted metabolomic analysis for the quantification of ADT, SAdo and pseudouridine by an ultra-high performance liquid chromatography (Shimadzu, Kyoto, Japan) coupled with the AB SCIEX Q-Trap 5500 triple quadrupole mass spectrometer (AB SCIEX, Toronto, Canada). During the follow-up period, patients who remained at their original stage were regarded as the “unprogressed” group; those who progressed to later stages accompanying with a 25% drop in eGFR [48 ] were “progressed” group. Random forest algorithm [20 (link)] were used to evaluate covariates that associated with DKD progression. The prognostic power of biomarkers during DKD stage progression was assessed via performing 100 iterations for each variate. Logistic regression analysis was used to evaluate the risk scores of interested metabolites by SPSS. The average AUC values were calculated and compared among all the variates. Detailed methods of targeted metabolite analysis with UPLC-QQQ-MS/MS and random forest analysis can be found in the Supplementary Materials and Methods (Supplementary Tables 14 –16 ).
9
Quantification of Cefazolin and Clindamycin Plasma Levels
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Liquid chromatography‐mass spectrometry (LC‐MS/MS) was used to measure the total plasma drug concentrations of cefazolin and clindamycin.
Drug concentrations for clindamycin were measured using LC‐MS/MS in a Thermo TSQ Quantiva triple‐stage quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) at the pharmacy laboratory of the University Medical Center Groningen, The Netherlands.
Drug concentrations for cefazolin were measured using LC‐MS/MS in an AB SCIEX QTRAP 5500 triple quadrupole mass spectrometer (AB SCIEX, Concord, Ontario, Canada) and Analyst software version 1.6.2 (AB SCIEX, Concord, Ontario, Canada) at the IBMP Institute for Biomedical and Pharmaceutical Research in Nürnberg‐Heroldsberg, Germany.
Limits of quantification are specified in Table4 . A certified research technician from the ISO‐certified laboratories performed the FDA validated drug analyses. In all analyses, quality control samples are included, as is obliged in FDA analyses and ISO and GCP certified laboratory.
Drug concentrations for clindamycin were measured using LC‐MS/MS in a Thermo TSQ Quantiva triple‐stage quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) at the pharmacy laboratory of the University Medical Center Groningen, The Netherlands.
Drug concentrations for cefazolin were measured using LC‐MS/MS in an AB SCIEX QTRAP 5500 triple quadrupole mass spectrometer (AB SCIEX, Concord, Ontario, Canada) and Analyst software version 1.6.2 (AB SCIEX, Concord, Ontario, Canada) at the IBMP Institute for Biomedical and Pharmaceutical Research in Nürnberg‐Heroldsberg, Germany.
Limits of quantification are specified in Table
10
Quantitative Analysis of Alisols Using UPLC-MS/MS
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The quantitative analysis and pharmacokinetic investigation were conducted using an AB SCIEX QTRAP 5500 triple quadrupole mass spectrometer (AB SCIEX, Foster, CA, United States). The separation of alisol A, alisol B, and alisol A 24-acetate was carried out using an ACQUITY HSS T3 column (2.1 × 100 mm, 2.5 μm) coupled to a I-Class UPLC system (Waters Corporation, Milford, MA, United States) equipped with a binary pump. The analytical column was set to 35°C. This experiment employed an injection volume of 10 μL and a flow rate of 0.2 ml/min. Gradient elution was carried out with 0.1% (v/v) formic acid in water (solvent B) and acetonitrile with 2 mM ammonium acetate (solvent A). The gradient elution was as follow: 0–6 min, 40–0% B; 6–8 min, 0–40% B; 8–12 min, 40% B. The following were the optimum MS parameters: The ESI ion source temperature was 500°C; the air curtain pressure was 30 psi; the collision activated dissociation (CAD) gas parameters were medium, and the ion spray voltage was 5500 V.
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