All samples were analyzed using an Agilent 6560 IM-QTOF (Santa Clara, CA). Each measurement was performed with the corresponding drift gas maintained at approximately 4 Torr and 25 °C. Other relevant instrument parameters can be found in the Supporting Information Table S1. Chromatographic separations were performed using Agilent 1290 Infinity II UHPLC (Santa Clara, CA) coupled to the 6560 IM-QTOF. Samples were injected (10 µL) onto an Agilent ZORBAX Extend-C18 column (2.1 × 50 mm, 1.8 μm) maintained at 30 °C. Mobile phase A was water (0.1% formic acid) and mobile phase B was methanol. The flow rate was maintained at 0.400 mL/min with gradient conditions shown in Table S2. Multiplexing (4-bit) was enabled for all LC-IM-MS measurements, which allowed for a maximum ion funnel trap fill time of 3900 μs; the release time was 150 μs.
Agilent 1290 infinity 2 uhplc
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 1290 Infinity II UHPLC is a high-performance liquid chromatography system designed for efficient and reliable separation and analysis of a wide range of samples. It features advanced technology and capabilities to deliver precise and reproducible results.
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Lab products found in correlation
6560 im qtof, by Agilent Technologies (1 mentions)
Zorbax extend c18 column, by Agilent Technologies (1 mentions)
6470 triple quadrupole mass spectrometer qqq ms, by Agilent Technologies (1 mentions)
Agilent 1100 hplc, by Agilent Technologies (1 mentions)
Xbridge beh phenyl column, by Waters Corporation (1 mentions)
6545 q tof, by Agilent Technologies (1 mentions)
Poroshell 120 phenyl hexyl column, by Agilent Technologies (1 mentions)
Deionized filtered water, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Agilent 6470 triple quadrupole mass spectrometer, by Agilent Technologies (1 mentions)
Dual agilent jet stream electrospray ionization ajs esi, by Agilent Technologies (1 mentions)
Masshunter workstation lc ms data acquisition software, by Agilent Technologies (1 mentions)
6545 q tof mass spectrometer, by Agilent Technologies (1 mentions)
Formic acid, by Thermo Fisher Scientific (1 mentions)
Hplc grade, by Thermo Fisher Scientific (1 mentions)
Agilent 6495a qqq ms, by Agilent Technologies (1 mentions)
11 protocols using agilent 1290 infinity 2 uhplc
1
Comprehensive Ion Mobility Mass Spectrometry
2
Plasma Sample Preparation and LC-MS/MS Analysis
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Solid-phase extraction sample processing was performed as described previously (27 (link)). Wells were conditioned with methanol and washed with aqueous 0.1% FA. Immediately after loading the plasma samples into the HLB μElution plate, the wells were washed with LC-MS grade water. Elution was done by adding a solution containing a high percentage of organic solvent (70% ACN/0.1% FA) to all wells. After SPE clean-up, the resulting peptide eluates were dried in a SpeedVac vacuum concentrator and stored at −80 °C until mass spectrometry (MS) analysis. QC samples and calibration curve standards were not subjected to SPE cleanup, but were dried and then frozen. Plasma samples, calibration curve standards, and QC samples were then resolubilized and analyzed on an Agilent 1290 Infinity II UHPLC coupled to an Agilent 6495B triple quadrupole (QqQ) MS.
3
Symbiodiniaceae Metabolomics in Anemones
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Twelve anemones of each of the eight Symbiodiniaceae treatments were assessed for survival at week 1, 2, 3, 4, 5, and 9. At week 9, 12 anemones per Symbiodiniaceae treatment were sampled for targeted metabolomics analysis (12 anemones × 8 treatments = 96 total). To avoid contamination from Artemia proteins and metabolites, anemones were starved for a week before sampling, when they were snap frozen in liquid nitrogen and stored at −80 °C. Two anemones of the same Symbiodiniaceae treatment were combined into a single sample to provide sufficient biomass for the analysis. Samples were homogenized and centrifuged to separate the host and Symbiodiniaceae fractions (Supplementary Methods 1.4). Each fraction was freeze dried to obtain its dry weight, then extracted and analyzed on the Agilent 1290 Infinity II UHPLC with an Agilent 6470 Triple Quadrupole LC-MS system (Supplementary Methods 1.5). The analysis followed the Agilent metabolomics dynamic MRM (dMRM) method where a curated database with retention times and optimized MS/MS acquisition parameters of ~200 central carbon metabolites are specified. Samples were run in two batches (batch one: B1, WT10, SS− (SS5), SS+ (SS8); batch two: SS− (SS3, SS9) and SS+ (SS1, SS7)).
4
HPLC-MS/MS Analysis of Pharmaceutical Compounds
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A Waters XBridge BEH phenyl column, with the dimensions of 150 × 4.6 mm and particle size 2.5 µm, with a corresponding VanGuard phenyl guard column (Waters, Milford, MA, USA), was used as the stationary phase, thermostated at 35 °C. Ultrapure water (phase A) and MeOH (phase B), both containing 0.1% HCOOH, were used as the mobile phase in the gradient elution. The initial experiments were performed on an Agilent 1100 HPLC equipped with DAD and FLD (Agilent Technologies, Santa Clara, CA, USA). The flow rate was set to 0.8 mL/min, and 10 µL of the sample was injected. The applied mobile phase gradient is shown in Table 10 .
The method was then transferred to an Agilent 1290 Infinity II UHPLC coupled to a 6470 triple-quadrupole mass spectrometer (QQQ-MS) equipped with an Agilent Jet Stream electrospray (AJS-ESI) source (Agilent Technologies, Santa Clara, CA, USA) to achieve the necessary detection sensitivity. With the same stationary and mobile phases, the flow rate was adjusted to 0.6 mL/min using the gradient elution described inTable 11 . An adjusted gradient composition was necessary due to the smaller dwell volume of the UHPLC system. The column temperature was 35 °C, and 10 µL of the sample was injected into the system.
The method was then transferred to an Agilent 1290 Infinity II UHPLC coupled to a 6470 triple-quadrupole mass spectrometer (QQQ-MS) equipped with an Agilent Jet Stream electrospray (AJS-ESI) source (Agilent Technologies, Santa Clara, CA, USA) to achieve the necessary detection sensitivity. With the same stationary and mobile phases, the flow rate was adjusted to 0.6 mL/min using the gradient elution described in
5
HPLC-MS Quantification of Cannabidiol
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Quantification of CBD was achieved by HPLC coupled with mass spectroscopy (MS) using an Agilent 1290 Infinity II UHPLC (Agilent Technologies; Santa Clara, CA) equipped with an Agilent Poroshell 120 Phenyl Hexyl column (1.9 μm, 150 × 2.1 mm) coupled to an Agilent 6545 QTOF. Gradient elution was used with eluent A consisting of H2O and eluent B consisting of acetonitrile, both of which contained 20 mM formic acid. Gradient elution started at 10% eluent B, increasing to 100% over 10 min, and held at 100% for 2 min, all at a constant flow rate of 0.35 ml/min and a constant column temperature of 60°C. Ionization was achieved using positive electrospray ionization, and mass spectra were obtained with an m/z range of 100–1,600, acquired at a rate of 10 scans/s. All MS analyses were conducted with purine and HP‐0921 as internal standards for spectrum calibration. The monoisotopic ion mass of the CBD proton adduct was used for integration with a mass accuracy of 6 ppm and a calibration curve made with four levels (0.7, 0.35, 0.175, 0.088 mg/ml). Two replicates of CBD were used to determine the concentration of CBD in samples.
6
Characterization of Novel Cyclic Peptides
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Isopedopeptin B and D were obtained from previous work (Nord et al. 2020 (link)), whereas pedopeptin A and B, were obtained as described in the supplementary material file. The peptides L-Ala—L-Phe—L-Val/L-Ser/L-Thr—L-Ala were purchased from Genescript (Piscataway, NJ, USA), and the peptide L-Ala—L-Phe—L-OHVal—L-Ala was from Bachem (Bubendorf, BL, Switzerland). L-OHVal was from Acros Organics BVBA (Geel, Belgium), L-2,3-diaminopropanoic acid from Alfa Aesar (Kandel, Germany) and L-2,4-diaminobutanoic acid were from Sigma Aldrich (MO, USA). L- and DL-FDLA were synthesised as previously described (Marfey 1984 (link)), but with Leu-NH2 (Sigma Aldrich) instead of Ala-NH2. Concentrated DCl in D2O was from Cambridge Isotope Laboratories Inc (Tewksbury, MA, USA) and H218O from Promochem Standard Supplies AB (Kungsbacka, Sweden). UHPLC-MS was performed on a maXis Impact Q-TOF MS (Bruker Daltonic GmbH, Bremen, Germany) connected to an Agilent 1290 Infinity II UHPLC (Agilent, Palo Alto, CA, USA). HPLC gradient grade MeCN (Sigma-Aldrich, St. Louis, MO, USA), deionized filtered water (Millipore, Billerica, MA, USA) and formic acid (Sigma Aldrich) were used for preparation of UHPLC solvents.
7
Quantitative Analysis of Phenolic Compounds
8
UHPLC-MS/MS Analysis of Compounds
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The instrument used for chemical analysis was an Agilent 1290 Infinity II UHPLC (Agilent Technology, Palo Alto, CA, USA) hyphenated with 6470 triple quadrupole mass spectrometry with electrospray ionization source (ESI) (Santa Clara, CA, USA). Agilent MassHunter Optimizer (version B.07.00) was used for standard compound-related parameters optimization, and MassHunter Workstation software Data Acquisition (version B.08.00) and Quantitative Analysis (version B.07.01) were used for data processing. The columns used for compound separation were Prontosil C18 AQ (2.1 × 150 mm, 3 μm) (for method (a)) and Prontosil C18 AQ (2.1 × 100 mm, 3 μm) (for method (b) and method (c)) (Bischoff, Leonberg, Germany).
9
Metabolomic Profiling by LC-MS
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The compound library was analyzed by LC-MS using an Agilent 6545 Q-TOF mass spectrometer with Agilent 1290 Infinity II UHPLC (Stanford ChemH Metabolomics Knowledge Center). Chromatography was done on a ZorbaxRapid Resolution High Definition Column, 1.8 μm (Agilent) column with HPLC-grade (Thermo-Fisher) water with 0.1% Formic acid as solvent A and HPLC-grade acetonitrile with 0.1% Formic acid (Thermo-Fisher) as solvent B. A volume of 10 μL of sample in DMSO were injected between 250 nM and 1 μM, and run at a constant rate of 0.4 mL per minute at 40°C. Separation was performed with the following gradient: 0–18 min, 3–50% B; 18–27 min, 50–97% B; 27–30 min, 97% B; followed by a 5 minute equilibration at 3% B. LC Eluent was sent to the MS starting at 0 min. The MS was in Dual Agilent Jet Stream electrospray ionization (AJS ESI) in positive mode, source gas temperature at 300°C, gas flow rate of 11 l/min, and nebulizer pressure of 35 psi. Data was collected using the MassHunter Workstation LC/MS Data Acquisition software (Agilent). Data files were converted into mzML format using MSConvert (Proteowizard).
Analysis was performed using Matlab, with code available athttps://github.com/btownshend .
Analysis was performed using Matlab, with code available at
10
Oligosaccharide Linkage Analysis by UHPLC-MS
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Linkage analysis was adapted from Galermo et al. with the following modifications55 (link),56 (link). Briefly, fractionated oligosaccharides and a pool of oligosaccharide standards were reacted with saturated NaOH and iodomethane in DMSO. Residual NaOH and DMSO were removed by extraction with DCM and water. The DCM layer was dried to completion under vacuum centrifugation. Samples were hydrolyzed and derivatized in the same manner as the monosaccharide analysis. Samples did not undergo chloroform extraction and were reconstituted in 70% (v/v) methanol/water. Fractions were analyzed on an Agilent 1290 infinity II UHPLC coupled to an Agilent 6495A QqQ MS ran in MRM mode. A pool of oligosaccharide standards was used to assign the glycosidic linkages present.
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