Carotenoids and chlorophylls were extracted and measured as previously described [25 (link)]. The separation and detection were performed on a C30 column on an Agilent Technologies 1290 Infinity UHPLC and an Agilent Technologies 6230 TOF LC/MS. External calibration was performed using authentic reference compounds.
6230 tof lc ms
Manufactured by Agilent Technologies
Sourced in United States, Germany
The 6230 TOF LC/MS is a high-performance liquid chromatography-time of flight mass spectrometer (LC-TOF MS) system designed for accurate mass measurements. It provides precise mass determination of a wide range of compounds, enabling effective identification and quantification of unknown analytes.
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Lab products found in correlation
1290 infinity uhplc, by Agilent Technologies (2 mentions)
Masshunter profinder b 08, by Agilent Technologies (1 mentions)
Millex mp, by Merck Group (1 mentions)
Ultrasonic cleaner, by Cole-Parmer (1 mentions)
1290 infinity 2 uhplc dad, by Agilent Technologies (1 mentions)
Poroshell 120 ec c18 column, by Agilent Technologies (1 mentions)
Zorbax eclipse xdb, by Agilent Technologies (1 mentions)
1260 series, by Agilent Technologies (1 mentions)
1260 system, by Agilent Technologies (1 mentions)
Lc 20a hplc, by Shimadzu (1 mentions)
Fls980 spectrophotometer, by Edinburgh Instruments (1 mentions)
Integrating sphere detector, by Edinburgh Instruments (1 mentions)
Sp8 laser scanning confocal microscope, by Leica (1 mentions)
Zorbax extend c18, by Agilent Technologies (1 mentions)
1200 series autosampler, by Agilent Technologies (1 mentions)
1290 infinity 2 uhplc, by Agilent Technologies (1 mentions)
Total starch assay kit, by Megazyme (1 mentions)
Dmso d6, by Cambridge Isotopes (1 mentions)
Kinetex c18 column, by Agilent Technologies (1 mentions)
Luna c18, by Phenomenex (1 mentions)
18 protocols using 6230 tof lc ms
1
Carotenoid and Chlorophyll Extraction and Analysis
2
Quantitative Analysis of DiAcSpm in Urine
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Samples were analyzed using an Agilent Technologies 6230 TOF LC/MS. LC separation was achieved using a Cogent 4 Diamond Hydride column with an initial gradient of 85% LC/MS grade acetonitrile containing 0.2% formic acid, followed by a gradual increase in hydrophilicity to 95% LC/MS grade water containing 0.2% formic acid. Detected ions were indexed and characterized using their ion m/z and chromatographic retention time. Data were analyzed using Agilent Technologies Qualitative Analysis B.07, Agilent Technologies MassHunter Profinder B.08, and XCMS software. Compound identification was achieved using known m/z and retention time coupled to chemical standards of targeted compounds run with each set of urine samples. Identity of DiAcSpm was further confirmed using MS/MS fragmentation analyses of chemical standards and random patient urine samples. DiAcSpm chemical standards at 5 known concentrations (50 nM, 100 nM, 500 nM, 1 μM, and 5 μM) were included within each run to create standard curves for urinary DiAcSpm concentration calculation.
3
Extraction and Elution of CSA-131 from PICC Lines
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Extraction and elution assays were run in triplicate. To obtain total extraction of CSA-131, PICC line segments were submerged in a solution comprised of 80% isopropanol and 20% 1 N HCl and heated to 70 °C for 8 h. Segments were transferred to new vessels and incubated in additional extraction solution at room temperature. Subsequent extraction steps continued at room temperature until CSA-131 peaks were indetectable using mass spectrometry. Daily elution samples were acquired from PICC line segments by incubation in PBS at 37 °C. PBS was exchanged daily at the same time, and collected samples were run on the same day. Deuterated reagents had previously been used to synthesize CSA-131D25, which served as an internal standard for quantification via mass spectrometry on a 6230 TOF LC/MS (Agilent Technologies, Santa Clara, CA, USA).
4
Measuring Compound Solubility in PBS
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Initially, a small amount of solid compound (generally 0.5–1.5 mg) was measured into a 1.7-ml Eppendorf tube. PBS (pH 7.4) was added to give a maximum final concentration of 1 mg ml−1 of compound. The compound was vortexed for ∼30 s before being placed into a bath sonicator (Cole Parmer, ultrasonic cleaner) for 1 h. Longer incubation times (up to 24 h) were performed with select compounds and no difference in solubility was observed; thus, 1 h was used for all subsequent testing. The tubes were vortexed again for 30 s before being centrifuged at maximum speed (13,000 × g) for 10 min. The supernatant was then filtered through a 0.22-μm syringe filter (Millipore Millex MP). The filtrate was then analysed by liquid chromatography–mass spectrometry (λ=254 nm, electrospray ionization–time-of-flight in positive mode, Agilent Technologies 6230 TOF LC/MS). The filtrate was diluted 1:2 and 1:4 and all three samples (1 × , 0.5 × and 0.25 × ) were analysed in triplicate. Three independent replicates of each compound were performed. A calibration curve for each compound was generated from 1 to 40 μM by dissolving the compound in DMSO and making dilutions of the stock in DMSO. The calibration curve (measured by ultraviolet absorbance) was linear over this range. The concentration of the samples was calculated based on the calibration curves.
5
Carotenoid Extraction and Quantification
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For carotenoid analysis, 5 mg of homogenized samples were weighed out, followed by three times extraction with 500 µL tetrahydrofuran/methanol (1:1, v/v) as previously described (Harbart et al., 2022 (link)). The collected and combined supernatants were dried under a nitrogen stream and redissolved in 250 µL dichloromethan/2-propanol (1:5, v/v). After filtration through PTFE filters (0.2 µm) the extracts were analyzed by HPLC-DAD-ToF-MS using an Agilent Technologies 1290 Infinity UHPLC coupled with an Agilent Technologies 6230 ToF LC/MS. Briefly, the separation was performed in gradient mode on a C30 column (YMC Co. Ltd, Kyoto, Japan, YMC C30, 100 × 2.1 mm, 3 μm) with eluents containing A: methanol/water (96:4, v/v) and B: methanol/tert-butyl methyl ether/water (6:90:4, v/v/v) both added with ammonium acetate (20 mM) to enhance the ionization. Ionization was achieved using a multimode ion source in positive polarity. Carotenoids were (tentatively) identified based on their specific absorption and mass spectra, in comparison with the literature or authentic standards (Table S2
6
Synthesis of a Cyclic Peptide
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Ac-CDDECPDYVCGGSGK-NH2 was synthesized by SPPS on a Liberty Blue peptide synthesizer (CEM, USA) using standard Fmoc chemistry protocols, DIC/Oxyma-Pure coupling reagents and Rink amide MBHA resin support (0.2 mmol scale, 10 equiv. amino acid excess). Fmoc groups were removed with 20% piperidine. Peptide amino acids side chains were deprotected, and peptide was cleaved off the resin with a mixture of TFA/EDT/anisole/thioanisole/water (90:2.5:2.5:2.5:2.5) for 1.5 h at RT.
The cleaved peptide was lyophilized and purified by RP HPLC (Vydac 218TP101522 column) using methanol and water with 0.05% TFA as solvents. The purity was assessed by analytical RP-HPLC (Vydac 218TP54 column) and LC/MS (Agilent Technologies 6230 ToF LC/MS). The mass was confirmed by MALDI-ToF MS: Ac-CDDECPDYVCGGSGK-NH2 [M + H]+ 1588.6.
The cleaved peptide was lyophilized and purified by RP HPLC (Vydac 218TP101522 column) using methanol and water with 0.05% TFA as solvents. The purity was assessed by analytical RP-HPLC (Vydac 218TP54 column) and LC/MS (Agilent Technologies 6230 ToF LC/MS). The mass was confirmed by MALDI-ToF MS: Ac-CDDECPDYVCGGSGK-NH2 [M + H]+ 1588.6.
7
Phenolic Compound Extraction and Quantification
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The analyses were performed based on a modified method by Schmidt et al. (2010 (link)). Twenty milligrams of lyophilized sample material were extracted with 600 μL 60% methanol by ultra-sonication followed by 20 min shaking at 1,400 rpm. The sample was pelleted at 12,000 rpm at 20°C and re-extracted by 400 and 200 μL. The combined supernatant was evaporated and re-dissolved in 500 μL of 20% methanol. Prior analysis the samples were filtered through a SpinX (0.22 μM SpinX Costar Cellulose Acetate, 3 min 3,000 rpm). The phenolics were analyzed on an Agilent Technologies 6230 TOF LC/MS equipped with an ESI ion source in positive ionization mode. The gas temperature was set to 350°C at a flow rate of 10 L min−1, the vaporizer to 320°C and the nebulizer pressure was set to 35 psi. The voltage was set to 3,500 V and a fragmentor voltage of 350 V was applied. The separation was performed on a Prodigy ODS1 100 A (150 × 2.1 mm, 5 μm) column in gradient mode using 0.5% acetic acid and acetonitrile as mobile phases at a flow rate of 0.4 mL min−1. Stock solutions of the authentic standards were prepared individually and identification was achieved by co-chromatography with references substances. External standard calibration curves were used for quantification by dose–response curves.
8
Quantifying Glucosinolates in Plant Tissues
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To measure glucosinolate compounds, floret and leave samples were grinded with liquid nitrogen and transferred to 2 mL microtubes, lyophilized and stored in a −80 °C freezer. Briefly, 10 mg of lyophilized and homogenized plant material were extracted in presence of 0.02 µmol of the internal standard 4-hydroxybenzyl GS with hot 70% methanol (LC-MS grade, Th. Geyer GmbH & Co. KG, Renningen, Germany) and samples were prepared as described before [36 (link),37 (link)]. The desulfo-GS were analyzed using a 1290 Infinity II UHPLC-DAD coupled with a 6230 ToF-LC/MS (Agilent Technologies, Waldbronn, Germany) with a Poroshell 120 EC-C18 column (Agilent Technologies, Waldbronn, Germany; 100 mm × 2.1 mm, 2.7 μm). UHPLC conditions were as follows: solvent A, MilliQ water; solvent B, 100% v/v acetonitrile. The 19 min run comprised 0.2% (v/v) B (2 min), 0.2% to 19.8% (v/v) B (10 min), a 2 min hold at 19.8% (v/v) B, 19.8% B to 50% (v/v) B (1 min), a 1 min hold at 50% (v/v) B, 50% to 0.2% (v/v) B (1 min), and finally a 2 min hold at 0.2% (v/v) B. The injection volume was 5 µL, and determination was conducted at a flow rate of 0.4 mL min−1 and 30 °C and a wavelength of 229 nm. The concentration of desulfo-GS was calculated by the peak area relative to the area of the internal standard.
9
Oligomer Identification by LC-TOF/MS
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Liquid chromatography-time-of-flight/mass spectrometry (LC-TOF/MS), in positive ionization mode, was used to qualitatively identify the released soluble oligomers. The analytes were separated using an HPLC (1260 series, Agilent Technologies, Palo Alto, CA) equipped with a reversed-phase C18 rapid resolution column (Zorbax Eclipse XDB, Agilent Technologies) of 50 mm by 2.1 mm and 1.8 µm particle diameter. Column temperature was 40°C. Mobile phase A consists of 20 mM ammonium formiate NH4COOH in ultrapure H2O and mobile phase B was ultrapure acetonitrile (see Supplementary Table S2 for details).
The flow rate was 0.5 ml min−1 and the injection volume was 20 µl. This HPLC system was connected to a time-of-flight mass spectrometer (6230 TOF LC/MS, Agilent Technologies) equipped with an electrospray interface under the following operating parameters: capillary 3500 V, nebulizer 40 psig, drying gas 8 L min−1, gas temperature 300°C, fragmentator 125 V, skimmer 65 V, and OCT 1 RF Vpp 750 V. The mass axis was calibrated using the mixture provided by the manufacturer over the m/z 50–3,200 range. A second orthogonal sprayer with a reference solution was used as a continuous calibration using the following reference masses: 121.050873 and 922.009798 m/z. Spectra were acquired over the 50–3,000 m/z range at a scan rate of two spectra per second.
The flow rate was 0.5 ml min−1 and the injection volume was 20 µl. This HPLC system was connected to a time-of-flight mass spectrometer (6230 TOF LC/MS, Agilent Technologies) equipped with an electrospray interface under the following operating parameters: capillary 3500 V, nebulizer 40 psig, drying gas 8 L min−1, gas temperature 300°C, fragmentator 125 V, skimmer 65 V, and OCT 1 RF Vpp 750 V. The mass axis was calibrated using the mixture provided by the manufacturer over the m/z 50–3,200 range. A second orthogonal sprayer with a reference solution was used as a continuous calibration using the following reference masses: 121.050873 and 922.009798 m/z. Spectra were acquired over the 50–3,000 m/z range at a scan rate of two spectra per second.
10
General Organic Chemistry Protocols
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