Zorbax eclipse aaa

Ten milligrams the sample was dissolved in 1 mL of distilled water. The solution was 25× diluted, and 50 μL of the solution was dried up in an ampoule. Then 100 μL of 6 M HCl was added and the ampule was sealed under vacuum. Acidic hydrolysis was performed for 24 h at 110 °C. After that, the ampoule was opened and the solution was dried up in the Eppendorf 5301 vacuum concentrator (Eppendorf, Hamburg, Germany). Finally, 50 μL of 0.1 M HCl was added to the dried sediment.
The amino acid analysis was performed using Agilent 1200 series chromatographic system equipped with a fluorescent detector and ZORBAX Eclipse AAA (150 × 4.6 mm; 5μm) column (Agilent Technologies, Santa Clara, CA, USA). The mobile phases were 40 mM pH 7.8 phosphate buffer solution (Solution A) and 80% water solution of acetonitrile (Solution B). Borate buffer (pH = 10.2) and o-phtalaldehyde were used for amino acid derivatization. See [34 (link)] for details.

Determination of the amino acid content was carried out in accordance with the provisions of the European Pharmacopoeia 5.0 – 2.2.56. Amino acid analysis – Protein hydrolysis – Method 1 for hydrolysis and from the European Pharmacopoeia 5.0 – 2.2.56. Amino acid analysis – Methodologies of amino acid analysis: general principles – Method 5 and Method 7 for derivatization. HPLC identification and quantification were performed following Agilent ZORBAX Eclipse AAA – Technical Note (publication number 5980-1193).

The aerial and root parts of 3-day-old BR-F microcolonies were separated as described above, with additional washing of root cells with distilled H₂O, and total intracellular amino acids were extracted from cell suspensions in water by boiling for 5 min. The concentration was determined by HPLC with precolumn derivatization by OPA (o-phthaldialdehyde) [9 (link), 54 (link)] with a ZORBAX Eclipse AAA, 3.5 μm, 4.6 × 75 mm reverse phase column (Agilent), and fluorescence detection.

Free amino acids were analyzed following a previously described method^{29 (link)} with some modifications. First, 1.2 mL of 5% trichloroacetic acid was added to 100 mg of frozen pepper seed powder, and the mixture was sonicated at 22 °C for 30 min. After centrifugation at 12,000 × g at 4 °C for 20 min, 1 mL of the supernatant was collected and filtered through a 0.45 μm polyvinylidene fluoride membrane filter. After mixing 5 μL of 0.4 N borate buffer (pH 10.2) and 1 μL of sample, 1 μL of o-phthalaldehyde and 1 μL of fluorenylmethyloxycarbonyl were added for derivatization. Finally, 64 μL of distilled water was added, and the mixture was analyzed by HPLC. The column was equipped with a Zorbax Eclipse AAA (4.6 × 150 mm, Agilent, Santa Clara, CA, USA), and the flow rate was set to 2 mL·min-¹. Mobile phase A was set to 40 mM NaH₂PO₄ (pH 7.8), and B was set to acetonitrile:methanol:H₂O (45:45:10, v:v:v).

Glutamate levels were analyzed by RP-HPLC using an Agilent 1200 liquid chromatograph and fluorescence detector as previously described [14 (link)] with a few modifications. The experiments utilized 4.6 × 75 mm, 3.5 μm ZORBAX Eclipse AAA analytical columns (Agilent). A gradient elution program was optimized for glutamate measurement with a flow rate 0.75 ml/min. The intracellular glutamate levels in the whole brain lysates of mice and whole cell lysates were determined by Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit (Invitrogen) based on the manufacturer’s instruction. The brain tissue lysates and whole cell lysates were diluted to the same protein concentration before the assay.

Free amino acids were analyzed following a previously described method^{5 (link),46 (link)} with some modifications. First, frozen pepper seeds were completely ground into a fine powder using a mortar and pestle in liquid nitrogen. Then, 1.2 mL of 5% trichloroacetic acid was added to 100 mg of pepper seed powder and the mixture was sonicated at room temperature for 30 min. After reaching a concentration of 16,000×g at 4 °C, 1 mL of the supernatant was collected and filtered through a 0.45 μm polyvinylidene fluoride membrane filter. After mixing 5 μL of 0.4 N borate buffer (pH 10.2) and 1 μL of sample, 1 μL of o-phthalaldehyde, and 1 μL of fluorenylmethyloxycarbonyl were added for derivatization. Finally, 64 μL of distilled water was added and analyzed by HPLC. The column was equipped with a Zorbax eclipse AAA (4.6 × 150 mm, Agilent, Santa Clara, CA, USA) and the flow rate was set to 2 mL min⁻¹. The mobile phase A was set to 40 mM NaH₂PO₄ (pH 7.8), and B was set to acetonitrile:methanol:H₂O (45:45:10, v:v:v).

Amino acid quantification was performed using HPLC (Agilent 1100; Agilent Technologies) with a guard cartridge and a reverse-phase C₁₈ column (Zorbax Eclipse AAA; 3.5 μm, 150 by 4.6 mm; Agilent Technologies), according to the procedure specifically developed for this system (83 ) and subsequently adapted to aphid tissues (82 (link)). The derivatization process, at room temperature, was automated using the Agilent 1313A autosampler. Detection was performed by a fluorescence detector set at 340 and 450 nm of excitation and emission wavelengths, respectively (266/305 nm for proline). Under these conditions, oxidations can lead to several cysteine forms, which does not allow its accurate detection and quantification, so only 19 amino acids were quantified. For this quantification, norvaline was used as the internal standard, and the response factor of each amino acid was determined using a 250-μM standard mix of amino acids. The software used for the analysis was ChemStation for LC three-dimensional systems.