Acclaim rslc c18 column

An accurately weighed amount containing 130 mg of mulberry leaves and sesame extract was placed in a 5 mL volumetric flask with a stopper. Then, 4 mL of methanol was added to it, and the flask was sealed. The solution was subjected to ultrasound treatment at 25 °C (power 600 W, frequency 60 kHz) for 30 min and then placed at 25 °C. Next, methanol (4 mL) was added to the mark, and the solution was shaken well and filtered through a 0.22 μM membrane to obtain the desired filtrate. Accurately weighed (10 mg) chlorogenic acid, rutin, astragalin, sesamin, and sesamolin were mixed with 10 mg of α-linolenic acid and 10 mg of linoleic acid in a 10-mL volumetric flask. The mixture was dissolved in 1 mL of dimethyl sulfoxide, and then the final volume (10 mL) up to the mark was achieved by adding the 50% methanol aqueous solution. The reserve solution had a reference substance concentration of 1 mg/mL. Chromatographic conditions (Vanquish Flex Thermo Fisher Scientific, Waltham, MA, USA) were as follows: Acclaim RSLC C18 column, 100 mm × 2.1 mm, 2.2 μm; mobile phase, A phase: 0.1% formic acid aqueous solution and B phase: methanol; gradient elution, 0–1 min, 10% B and 90% A; 1–2 min, 10–20% B and 90–80% A; 2–8 min, 20–90% B and 80–10% A; 8–9 min, 90% B and 10% A; 9–10 min, 90% A and 10% B; column temperature, 40 °C; detection wavelengths, 205 nm, 287 nm, and 330 nm; flow rate, 0.5 mL/min.

Abscisic acid was determined in Physcomitrium patens samples using the LC-MS/MS system which consisted of Nexera X2 UPLC (Shimadzu) coupled QTRAP 6500+ mass spectrometer (Sciex). Chromatographic separations were carried out using the Acclaim RSLC C18 column (150×2.1 mm, 2.2μm, Thermo Scientific) employing acetonitrile/water+0.1% acetic acid linear gradient. The mass spectrometer was operated in negative ESI mode. Data was acquired in MRM mode using following transitions: 1) ABA 263.2->153.1 (−14 eV), 263.2->219.1 (−18 eV); 2) ABA -D6 (IS) 269.2->159.1 (−14 eV), 269.2->225.1 (−18 eV); declustering potential was −45 V. Freeze-dried moss samples were ground using the metal beads in homogenizer (Bioprep-24) to a fine powder. Accurately weighted (about 20mg) samples were spiked with isotopically labeled ABA -D6 (total added amount was 2 ng) and extracted with 1.5 ml acetonitrile/water (1:1) solution acidified with 0.1% formic acid. Extraction was assisted by sonication (Elma S 40 H, 15 min, two cycles) and solution was left overnight for completion of extraction. Liquid was filtered through 0.2 μm regenerated cellulose membrane filters, evaporated to dryness upon a stream of dry nitrogen and redissolved in 100 μl extraction solution.

Quantitative analysis of plant hormones was accomplished using LC-MS/MS system which consisted of a 1,200 series Rapid Resolution liquid chromatography system (vacuum micro degasser G1379B, binary pump G1312B, autosampler G1367C and thermal column compartment G1316B) coupled to 6,410 triple quadruple mass selective detector (Agilent Technologies, Santa Clara, CA, United States). Analytes were separated on an Acclaim C18 RSLC column (2.1 × 150 mm, particle size 2.2 μm, Dionex) upon HPLC conditions described in Supplementary Table 4.
Mass spectrometer was operated in negative ionization mode, ion source parameters were as follows: capillary voltage 3500V, drying gas (nitrogen) temperature and flow 350°C and 10 l/min, respectively, nebulizer pressure 35 psi, nitrogen (99.999%) was used as a collision gas. The LC-MS system was controlled and data were analyzed using MassHunter software (Agilent Technologies). Quantitative analysis of plant hormones was accomplished in multiple reaction monitoring (MRM) mode, isotopically labeled analogs were used as internal standards. MRM parameters are listed in Supplementary Table 5.