Seed ABA was extracted following the methods described by Tombesi et al.57 (link). Analyses were performed on an Agilent 1260 HPLC (Agilent Ltd., California, USA) equipped with an Agilent C18 ZORBAX (5 mm × 150 mm × 4.6 mm) column at a flow rate of 0.013 ml s−1. The injection volume was 10 μL and the detection was made at 265 nm. The mobile phase of acetonitrile/methanol/water (8:40:52 v/v/v, 0.6% acetic acid) was previously filtered and degassed. ABA was identified by comparing the retention times with those of standard ABA, and the peak area quantified by an external standard method. Stock solutions of ABA standards was prepared by diluting a solution (0.1 mg mL−1 in acetonitrile) to obtain a range of concentrations from 0.1 to 10 μg mL−1.
C18 zorbax
Manufactured by Agilent Technologies
Sourced in United States
The C18 ZORBAX is a high-performance liquid chromatography (HPLC) column developed by Agilent Technologies. It is designed for the separation and analysis of a wide range of organic compounds. The column features a silica-based stationary phase with chemically bonded C18 ligands, providing efficient and reliable chromatographic performance.
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2 protocols using c18 zorbax
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Quantitative ABA Analysis via HPLC
2
Amino Acid Quantification by LC-MS/MS
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Amino-acid mixture standard was purchased from Sigma-Aldrich (São Paulo, Brazil) and D3-testosterone (ISTD) from LGC Standards (London, England). Amino acid quantification was carried out using a TSQ Quantiva from Thermo Scientific (San Jose, USA) with a Dionex Ultimate 3000 HPLC system (Germering, Germany). Chromatographic separation was achieved using a reversed phase column (C18 Zorbax, 50 × 3 mm, 1,7 μm, Agilent, Santa Clara, USA). The analyte was eluted from the column using a gradient with the eluent changing from 5% to 100% methanol in water within 3 min. The column was washed for 1.2 min in 100% methanol and equilibrated for 3 min at the initial eluent composition. All solvents contained 0.1% formic acid. The flow rate, column temperature, and injection volume were 300 μL/min, 40°C and 5 μL, respectively.
Amino-acids were monitored by selected reaction monitoring (SRM) in the positive ion mode. The transitions selected for amino-acid quantification and ISTD are listed in Table S2. The curve was constructed using a mix of amino-acids in triplicate at 1; 2,5; 5; 10 and 20 nmol/mL. All samples were spiked with D3-Testosterone (ISTD) at 5 ng/mL. The area ratios of the total extracted ion of the product ions and the product ion of the IS were plotted versus the concentration.
Amino-acids were monitored by selected reaction monitoring (SRM) in the positive ion mode. The transitions selected for amino-acid quantification and ISTD are listed in Table S2. The curve was constructed using a mix of amino-acids in triplicate at 1; 2,5; 5; 10 and 20 nmol/mL. All samples were spiked with D3-Testosterone (ISTD) at 5 ng/mL. The area ratios of the total extracted ion of the product ions and the product ion of the IS were plotted versus the concentration.
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