Velos ltq system

One gram of frozen leaf material per sample was ground with liquid nitrogen. For extraction, 5 ml 80% (v:v) methanol was added. The samples were treated with a Miccra D-9 homogenizer (MICCRA GmbH, Heitersheim, Germany) for 1 min and then placed into a Sonorex RK510 ultrasonic bath (BANDELIN electronic GmbH & Co. KG, Berlin, Germany) for 15 s. Subsequently, 2 ml of the suspension were centrifuged at 10,000×g for 5 min. 350 μl of the supernatant were added to 700 ml ultrapure water, mixed, and centrifuged again at 12,000 rpm for 5 min. The supernatant was then filtered with a Chromafil disposable filter O-20/15 MS (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany). Analytical UHPLC–MS analysis was performed on a Velos LTQ System (Thermo Fisher Scientific, Waltham, United States). Separation was achieved on a Synergi Polar 4 μm 3 × 150 mm column. The mobile phase consisted of 5% acetonitrile and 0.05% formic acid (solvent A), and acetonitrile LCMS grade (solvent B). The gradient used was: 95% A/5% B, 0–1 min; 10% A/90% B, 11–13 min; 95% A/5% B, 13.1 min; 95% A/5% B, 16 min. The flow rate was 0.5 ml min⁻¹, and the injection volume was 1 μl. The SA standard was purchased from Merck KGaA (Darmstadt, Germany). The standard was prepared in concentrations of 0.1, 0.5, 1.0 mg L⁻¹ with water/acetonitrile (70:30 v:v). Phytohormone content in μg g⁻¹ was calculated using the formula:

For determination of the antifungal compound benzoate 50 μl aliquots of sorption filter extract were mixed with 50 μl of H₂O followed by UHPLC-MS analysis on a Velos LTQSystem (Thermo Fisher Scientific, Waltham, MA, United States). Identification and quantitative analysis were conducted on an ACCLAIM^TMC30 column, 150 mm × 3 mm (ThermoScientific, Waltham, MA, United States) with gradient elution (solvent A): H₂O: acetonitrile (95:5) and (solvent B): acetonitrile and an external standard.

A total of 20 μl aliquots from 0.6 ml sorption filter extract were mixed with 15 μl of derivatization agent (ACCQFLUOR REAG, Waters, Milford, MA, United States) and 65 μl of borate buffer, incubated 10 min at 55°C, followed by addition of 400 μl acetonitrile: H₂O (1:4) with modifications of the method of Cohen and Michaud (1993) (link). Determination of amino acids (glutamic acid, asparagine, serine, glutamine, glycine, threonine, histidine, alanine, proline, cysteine, thyrosine, methionine, isoleucine, leucine, and phenylalanine) was performed by HPLC-MS, using a Velos LTQSystem (Thermo Fisher Scientific, Waltham, MA, United States) equipped with a ACCUTAG^TM column, 4 μm particle size, 150 mm × 3.9 mm (Waters, Milford, MA, United States) and additional comparison with external standards. Gradient elution was performed with (A) ammonium formate: methanol: H₂O (40:9:60) and (B) acetonitrile.