Because the concentration of TG in milk were high relative to other lipid species, a separate analysis (Supplementary File S3 ) for TG was performed whereby samples were diluted (1 in 100) with milliQ water before lipids were extracted from 10 μL with butanol:methanol, as described above. Analysis of milk triacylglycerols was performed on an Agilent 6490 QQQ mass spectrometer with an Agilent 1290 series UHPLC system. Concentrations of each triacylglycerol were calculated based on chromatographic peak area relative to the labelled triacylglycerol internal standard (23 (link)).
6490 qqq mass spectrometer
Manufactured by Agilent Technologies
Sourced in United States
The 6490 QQQ mass spectrometer is a triple quadrupole mass spectrometer designed for high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) applications. The instrument utilizes advanced collision cell technology to deliver high sensitivity and selectivity for quantitative and qualitative analysis of a wide range of analytes.
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Lab products found in correlation
Zorbax eclipse plus c18 column, by Agilent Technologies (2 mentions)
1290 series hplc system, by Agilent Technologies (2 mentions)
1290 series uhplc system, by Agilent Technologies (1 mentions)
Infinitylab poroshell 120 hilic z column, by Agilent Technologies (1 mentions)
Tryptophan 15n2, by Merck Group (1 mentions)
Sequant zic chilic, by Merck Group (1 mentions)
High performance liquid chromatography (hplc), by Agilent Technologies (1 mentions)
Synergi polar rp 80a column, by Phenomenex (1 mentions)
Masshunter software, by Agilent Technologies (1 mentions)
Agilent 1290 series hplc system, by Agilent Technologies (1 mentions)
10 protocols using 6490 qqq mass spectrometer
1
Milk triacylglycerol analysis by UHPLC-MS
2
Plasma Lipidomics Analysis by LC-MS/MS
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Analysis of plasma extracts was performed on an Agilent 6490 QQQ mass spectrometer with an Agilent 1290 series HPLC system and a ZORBAX eclipse plus C18 column (2.1×100 mm, 1.8μm, Agilent) with the thermostat set at 60°C [8 (link)]. Mass spectrometry analysis was performed in positive ion mode, with dynamic scheduled multiple reaction monitoring (MRM). Mass spectrometry settings and MRM transitions for each lipid class are shown in Supplementary Table 3 . Detailed description of the method is presented in Huynh et al. [8 (link)].
3
Quantitative Analysis of Nucleotides
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To determine nucleotides, cell extracts were analysed using an UHPLC system coupled to a 6490 QqQ mass spectrometer (Agilent Technologies). Cell extracts were injected (5 µl) and metabolites were separated using an InfinityLab Poroshell 120 HILIC-Z column (2.7 µm, 2.1 × 100 mm, Agilent). The mobile phases used for the metabolite separation were A: 50 mM ammonium acetate with 5 µM medrionic acid; and B: acetonitrile. The chromatographic gradient was isocratic for 0.5 min at 80% B, from 0.5 to 7.5 min decreased to 70% B and from 7.5 to 8.5 min decreased to 50%, and maintained for 30 sec. From 9.0 min to 9.2 min the percentage of B rose quickly to 80% and finally the column was equilibrated at 80% B until min 11. Flow rate was 0.7 mL/min. The QqQ mass spectrometer worked in MRM mode using the transitions in Supplementary Table S3 to determine nucleotides. The electrospray ionization source (ESI) worked in positive and negative mode. The quantification of nucleotides was based on peak areas; the indicated relative concentrations correspond to the peak area/cell number (Supplementary Dataset).
4
Targeted Lipidomic Analysis of ADNI Plasma
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The Alzheimer Disease Metabolomics Consortium (ADMC) deposited the generated baseline targeted lipidomic data for ADNI plasma samples to the Laboratory of Neuroimaging (LONI). ADMC uses metabolomics and lipidomics platforms to cover a broad area of biochemistry and to enable interrogation of the AD metabolome with the goal of building a metabolomics database for ADNI. Analysis of plasma extracts was performed on an Agilent 6490 QQQ mass spectrometer with an Agilent 1290 series high‐performance liquid chromatography (HPLC) system and a ZORBAX eclipse plus C18 column (2.1 × 100 mm 1.8 μm, Agilent). Mass spectrometry analysis was performed in the positive ion mode with dynamic scheduled multiple reaction monitoring (MRM). Mass spectrometry results were integrated using Agilent software (MassHunter B9.00). The ADMC lab eventually examined a total of 579 plasma lipid molecules.
5
Targeted Metabolomic Analysis of Plasma Samples
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Plasma samples were obtained at baseline (n = 325), stored at −80°C and analyzed at 15–18 years post-enrollment depending on the enrollment date. We quantified free plasma levels of downstream metabolites of the KP, tryptophan, kynurenine, 3-hydroxykynurenine, kynurenic acid, anthranilic acid, 3-hydroxyanthranilic acid and quinolinic acid, in addition to indole metabolites indoxyl sulfate and indole-3-acetate (Fig. 1 ), using a targeted liquid chromatography-mass spectrometry (LC-MS) platform. Tryptophan 15N2, kynurenic acid D5, anthranilic acid 13C6, indoxyl sulfate 13C6, indoxyl acetate D5 and all authentic standards were purchased from Sigma Aldrich (St. Louis, MO, USA). A total of 50 µL of plasma was extracted with 200 µL of chilled acetonitrile spiked with internal standards, vortexed and centrifuged. The supernatant was transferred to glass vials that were vacufuged. The dried samples were reconstituted in 50 µL of water with 0.1% formic acid. The samples were then analyzed with a 6490 QQQ mass spectrometer with a 1290 Liquid Chromatography machine attached (Agilent, Santa Clara, CA, USA) [14 (link)].
6
Quantitative Lipid Analysis by LC-MS/MS
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LC-MS/MS was performed according to previously published methods, with slight modification for tissue samples98 (link). Sample extracts were analysed using either (i) an AB Sciex Qtrap 4000 mass spectrometer coupled to an Agilent 1200 HPLC system for CL assessment, as described preciously99 (link) or (ii) an Agilent 6490 QQQ mass spectrometer coupled with an Agilent 1290 series HPLC system for assessment of all other lipid species98 (link). Lipids run on the Agilent 6490 were measured using scheduled multiple reaction monitoring with the following conditions: isolation widths for Q1 and Q3 were set to “unit” resolution (0.7 amu), gas temperature 150 °C, nebuliser 20 psi, sheath gas temperature 200 °C, gas flow rate 17 L/min, capillary voltage 3500 V and sheath gas flow 10 L/min. The list of multiple reaction monitoring (MRM) used and the chromatographic conditions were described previously98 (link). Chromatographic peaks were integrated using Mass Hunter (B.09.00, Agilent) and assigned to lipid species using MRMs and retention time, with quantification derived from the ratio of each peak with its corresponding internal standard.
7
Comprehensive Lipidomic Profiling of Alzheimer's Cohorts
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Extensive details on the lipidomic profiling of the BHS, ADNI, and AIBL cohorts have been published previously.16 , 26 , 55 Lipid extractions were performed on plasma (AIBL) and serum (ADNI, BHS) samples as described previously.57 Lipidomic profiling (569 lipid species from 32 classes) was carried out using scheduled multiple reaction monitoring on an Agilent 6490 QqQ mass spectrometer.57
8
Quantification of NAD+ and NADH in Kidney Cortex
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Kidney cortex tissues were pulverized and sonicated in 80:20 acetonitrile:water (pH 9.0 with ammonium hydroxide) containing 13C6-nicotinamide for internal standard. The homogenate was incubated on ice for 5 min and centrifuged for 10 min at 17,000g at 4 °C. LC-MS analysis was performed on an Agilent system consisting of a 1290 UPLC module coupled with a 6490 QqQ mass spectrometer (Agilent Technologies). Metabolites were separated on SeQuant ZIC-cHILIC (3 μm, 100 × 2.1 mm; Merck) with the following gradient: 0 to 2.5 min at 95% B, 2.5 to 8.5 min at 25% B, 8.5 to 8.6 min at 95% B, 8.6 to 12.6 min at 95% B. Solvent A was 50 mM ammonium acetate pH 8.0 in water, and solvent B was 100% acetonitrile. Column temperature was set at 30 °C, and flow rate was 0.3 ml/min. 13C6-nicotinamide (129.1 → 85.1 m/z), NAD+ (664.1 →136 m/z), and NADH (666.1 → 136 m/z) were monitored in MRM mode. Data were collected in positive mode.
9
LC-MS/MS Method for Plasma Gem-Thr and Gemcitabine
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To determine concentrations of Gem-Thr and gemcitabine in rat plasma, plasma samples were deproteinized by adding two volumes of acetonitrile containing internal standard (phenacetin). After mixing, mixtures were centrifuged at 14,000× g for 10 min.
Aliquots of supernatants (2-µL) were injected into the LC-MS/MS system, which consisted of an Agilent HPLC and an Agilent 6490 QQQ mass spectrometer equipped with an ESI+ Agilent Jet Stream ion source (Agilent Technologies, Santa Clara, CA, USA). The separation of each drug and IS from endogenous plasma substances was achieved on a Synergi Polar-RP 80A column (150 × 2.0 mm, 4 μm; Phenomenex, Torrance, CA, USA). The mobile phase consisted of 0.1% formic acid and acetonitrile (20:80, v/v) at a flow rate of 0.2 mL/min. The column and autosampler tray were maintained at 25 and 4 °C, respectively. Gemcitabine, Gem-Thr, and the IS were quantified by multiple-reaction monitoring (MRM) in positive electrospray ionization mode. Respective precursor-to-product ion transitions were as follows: Gemcitabine, 264.1→112.1, Gem-Thr, 387.1→343.2, and IS (phenacetin), 180.2→162.2. Data acquisition was performed using Mass Hunter software (ver. A.02.00; Agilent Technologies).
Aliquots of supernatants (2-µL) were injected into the LC-MS/MS system, which consisted of an Agilent HPLC and an Agilent 6490 QQQ mass spectrometer equipped with an ESI+ Agilent Jet Stream ion source (Agilent Technologies, Santa Clara, CA, USA). The separation of each drug and IS from endogenous plasma substances was achieved on a Synergi Polar-RP 80A column (150 × 2.0 mm, 4 μm; Phenomenex, Torrance, CA, USA). The mobile phase consisted of 0.1% formic acid and acetonitrile (20:80, v/v) at a flow rate of 0.2 mL/min. The column and autosampler tray were maintained at 25 and 4 °C, respectively. Gemcitabine, Gem-Thr, and the IS were quantified by multiple-reaction monitoring (MRM) in positive electrospray ionization mode. Respective precursor-to-product ion transitions were as follows: Gemcitabine, 264.1→112.1, Gem-Thr, 387.1→343.2, and IS (phenacetin), 180.2→162.2. Data acquisition was performed using Mass Hunter software (ver. A.02.00; Agilent Technologies).
10
Comprehensive Lipid Profiling of Plasma and Serum
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Lipids were extracted from 10 L plasma (AIBL) or serum (ADNI), with the addition of an internal standard mix (Supplementary Table 1), using the single phase butanol/methanol extraction method as described previously 21 . Analysis of plasma extracts was performed on an Agilent 6490 QQQ mass spectrometer with an Agilent 1290 series HPLC system. Mass spectrometry settings and transitions for each lipid class are shown in Supplementary Table 1. Additional experiments using pooled samples were utilised under varying conditions to get acyl composition data. Detailed description of the method and characterisation of lipid composition and identification of isobaric and isomeric species have been described previously 22 . The additional experiments to characterise the lipid species include acid hydrolysis, fragmentation in the presence of lithium ions in positive ionisation mode and fragmentation in negative ionisation mode 22 .
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