Extraction and purification of indole-3-acetic acid (IAA) and abscisic acid (ABA) metabolites was done as described previously34 (link) with minor modifications. Frozen samples were homogenized using a MixerMill (Retsch GmbH, Haan, Germany) and extracted in 1 ml 50 mM sodium phosphate buffer (pH 7.0) containing 1% sodium diethyldithiocarbamate and stable isotope-labelled internal standards (5 pmol of [13C6]-IAA and [6H2]-ABA per sample added). The pH was adjusted to 2.7 with 1 M hydrochloric acid, and the samples were purified by solid phase extraction. The extracts were purified on Oasis HLB columns (30 mg, Waters Corp., Milford, USA), conditioned with 1 ml methanol, 1 ml water, and 0.5 ml sodium phosphate buffer (pH 2.7). After sample application, the column was washed with 2 ml 5% methanol and then eluted with 2 ml 80% methanol. Eluates were evaporated to dryness and dissolved in 30 ul of mobile phase prior to mass analysis using a 1290 Infinity Binary LC System coupled to the 6490 Triple Quad LC/MS System with Jet Stream and Dual Ion Funnel technologies (Agilent Technologies)35 (link).
6490 triple quad lc ms system
Manufactured by Agilent Technologies
Sourced in United States
The 6490 Triple Quad LC/MS System is a high-performance liquid chromatography-mass spectrometry (LC/MS) instrument designed for quantitative and qualitative analysis. It features a triple quadrupole configuration and delivers reliable, accurate, and sensitive results for a wide range of analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
1290 infinity binary lc system, by Agilent Technologies (4 mentions)
Dual ion funnel technologies, by Agilent Technologies (2 mentions)
1200 series system, by Agilent Technologies (2 mentions)
Zorbax extended c18 column, by Agilent Technologies (2 mentions)
Masshunter workstation software, by Agilent Technologies (2 mentions)
Dual ion funnel, by Agilent Technologies (2 mentions)
Oasis hlb column, by Waters Corporation (1 mentions)
Cary 60 uv vis spectrophotometer, by Agilent Technologies (1 mentions)
Jet stream, by Agilent Technologies (1 mentions)
6 protocols using 6490 triple quad lc ms system
1
Quantitative Analysis of Phytohormone Metabolites
2
Carotenoid Quantification by HPLC-MS
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Absorption UV–visible spectra were obtained at room temperature on a Cary 60 UV-Vis spectrophotometer (Agilent, Santa Clara, CA, USA) using the scan mode, with a 300–800 nm absorbance range. Acetone was used as blank and baseline correction.
The HPLC analysis of carotenoids in acetone was performed using a Zorbax extended -C18 column (Agilent, Santa Clara, CA, USA) (2.1–50 mm, 1.8 m) on an Agilent 1200 series system (Santa Clara, CA, USA). The optimization and validation of HPLC parameters were determined in previous experiments [16 (link)]. To determine the mass spectra of the different compounds, a 6490 Triple Quad LC/MS system (Agilent, Santa Clara, CA, USA) was used equipped with an electrospray ionization source (ESI) Jet stream operating in positive scan mode (m/z range of 300–900), with 0.1 a.m.u. (atomic mass unit). precision, and controlled by MassHunter Workstation Software (Agilent, B.05.00, Santa Clara, CA, USA). The following specific working conditions were used: capillary voltage 3000 V, gas flow rate 11 L min−1, gas temperature 290 °C, sheath gas flow rate 12 L min−1, sheath gas temperature 300 °C and nebulizer pressure 35 psi.
The HPLC analysis of carotenoids in acetone was performed using a Zorbax extended -C18 column (Agilent, Santa Clara, CA, USA) (2.1–50 mm, 1.8 m) on an Agilent 1200 series system (Santa Clara, CA, USA). The optimization and validation of HPLC parameters were determined in previous experiments [16 (link)]. To determine the mass spectra of the different compounds, a 6490 Triple Quad LC/MS system (Agilent, Santa Clara, CA, USA) was used equipped with an electrospray ionization source (ESI) Jet stream operating in positive scan mode (m/z range of 300–900), with 0.1 a.m.u. (atomic mass unit). precision, and controlled by MassHunter Workstation Software (Agilent, B.05.00, Santa Clara, CA, USA). The following specific working conditions were used: capillary voltage 3000 V, gas flow rate 11 L min−1, gas temperature 290 °C, sheath gas flow rate 12 L min−1, sheath gas temperature 300 °C and nebulizer pressure 35 psi.
3
HPLC Analysis of Carotenoids in Acetone
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The HPLC analysis of carotenoids in acetone was performed using a Zorbax extended -C18 column (Agilent, Santa Clara, CA, USA) (2.1 × 50 mm, 1.8 µm) on an Agilent 1200 series system (Santa Clara, CA, USA). To determine the mass spectra of the different compounds, a 6490 Triple Quad LC/MS system (Agilent, Santa Clara, CA, USA) was used equipped with an electrospray ionization source (ESI) jet stream operating in positive scan mode (m/z range of 300–900) with 0.1 a.m.u (atomic mass unit) precision and controlled by MassHunter Workstation Software (Agilent, B.05.00, Santa Clara, CA, USA). The following specific working conditions were used: capillary voltage 3000 V, gas flow rate 11 L min−1, gas temperature 290 °C, sheath gas flow rate 12 L min−1, sheath gas temperature 300 °C, and nebulizer pressure 35 psi. The percentage represented by each carotenoid was calculated by dividing the sum of the areas of a carotenoid by the sum of the areas of all identified carotenoids.
4
Mass Spectrometry Analysis of ABA
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Leaf samples were extracted, purified, and analyzed according to method described in Šimura et al.[48 ] Mass spectrometry analysis of D6‐ABA and ABA was performed by an UHPLC‐ESI‐MS/MS system comprising of a 1290 Infinity Binary LC System coupled to a 6490 Triple Quad LC/MS System with Jet Stream and Dual Ion Funnel technologies (Agilent Technologies, Santa Clara, CA, USA). The quantification was carried out in Agilent MassHunter Workstation Software Quantitative (Agilent Technologies, Santa Clara, CA, USA).
5
Extraction and Quantification of Auxin Compounds
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Extraction and purification of the targeted compounds (IAA, oxIAA, IAA-Asp, IAA-Glu, IAAglc, oxIAA-glc) were performed according to (Novak et al., 2012) . Briefly, 10 mg of frozen material per sample was homogenized using a bead mill (27 Hz, 10 min, 4°C; MixerMill, Retsch GmbH, Haan, Germany) and extracted in 1 ml of 50 mM sodium phosphate buffer containing 0.1% sodium diethyldithiocarbamate and a mixture of 13 C6 isotopically labelled internal standards (Olchemim, Olomouc, Czech Republic). After centrifugation (20 000 g, 15 min, 4°C), the supernatant was transferred into new Eppendorf tubes. The pH was then adjusted to 2.5 with 1 M HCl and samples were immediately applied to preconditioned solid-phase extraction columns (Oasis HLB, 30 mg of 1 ml; Waters Inc., Milford, MA, USA). After sample application, each column was rinsed with 2 ml 5% methanol. Compounds of interest were subsequently eluted with 2 ml of 80% methanol. UHPLC-MS/MS analysis was performed according to the method described in (Pencik et al., 2018) , using an LC-MS/MS system consisting of a 1290 Infinity Binary LC System coupled to a 6490 Triple Quad LC/MS System with Jet Stream and Dual Ion Funnel technologies (Agilent Technologies, Santa Clara, CA, USA). The quantification was carried out in Agilent MassHunter Workstation Quantitative Analysis software (Agilent Technologies, Santa Clara, CA, USA).
6
Cytokinin Quantification by LC-MS/MS
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Cytokinins and their related metabolites were extracted from seedlings with modified Bieleski buffer and consequently purified by using MCX separation cartridges (Waters) [52] . Samples were analyzed in 4 biological replicates. Stable isotope-labeled standards (Olchemim) for all measured compounds were added to samples before extraction. Mass spectrometry analysis and quantification was performed by LC-MS/MS, which consisted of the 1290 Infinity Binary LC System coupled to 6490 Triple Quad LC/MS System with Jet Stream and Dual Ion Funnel technologies (Agilent Technologies) [53] . Concentrations were then calculated using a standard isotope dilution method. All solvents used were of analytical or higher grade (Sigma Aldrich GmbH).
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