Oasis max column

Plasma aflatoxin B1-lysine (AFB₁-lys) was measured at the Department of Environmental Health and Engineering of the Johns Hopkins Bloomberg School of Public Health, using a minor variation of the method reported by McCoy and colleagues [15 , 20 (link)]. Briefly, plasma (150 μL) was spiked with an internal standard (0.5 ng AFB₁-d4-lysine in 100 μL), combined with Pronase (EMD Millipore, Billerica MA, USA) protease solution (3.25 mg in 0.5 mL phosphate-buffered saline), and incubated for 18 hours at 37°C. Solid-phase extraction–processed samples (Oasis MAX columns; Waters, Milford, MA, USA) were analyzed with ultra-high pressure liquid chromatography (UHPLC)-isotope dilution mass spectrometry on a ThermoFisher Scientific (San Jose, CA, USA) system composed of a Vanquish UHPLC and a TSQ Quantis triple quadrupole mass spectrometer in positive electrospray ionization mode [21 (link), 22 (link)].

Auxin (indole-3-acetic acid, IAA) and stress-related hormones such as jasmonates (jasmonic acid, JA and jasmonoyl-isoleucine, JA-Ile), SA and ABA were purified as described previously with minor modifications [62 (link)]. Briefly, lyophilized samples (5 mg DW) were homogenized with a MixerMill (Retsch GmbH, Haan, Germany), extracted in 1 mL 50 mM sodium phosphate buffer (pH 7.0) containing 1% sodium diethyldithiocarbamate and stable isotope-labelled internal standards (5 pmol each of [¹³C₆]IAA, [²H₄]SA, [²H₆]JA, [²H₂]JA-Ile and [²H₆]ABA; OlChemIm) and then alkalized by adding 1 mL of 5% NH₄OH/H₂O (v/v). The resulting solution was applied to a mixed-mode anion exchange columns (Oasis^® MAX column, 1 cc/30 mg, Waters, Milford, MA, USA) conditioned with 100% MeOH, equilibrated with 5% NH₄OH/H₂O (v/v), and washed with H₂O. The column was then washed with 2 mL 5% NH₄OH followed by 2 mL 100% MeOH, and the acidic phytohormones were eluted using 2 mL 2% HCOOH in 100% MeOH (v/v). The samples were evaporated to dryness under a stream of nitrogen and stored in a freezer at −20 °C until analysis.

The stress-related phytohormones, JAs (jasmonic acid, JA, and jasmonoyl-L-isoleucine, JA-Ile) and ABA were determined as previously described (Floková et al., 2014 (link)) with minor modifications. Briefly, lyophilized samples (5 mg dry weight, DW) were homogenized with a MM 301 vibration mill (Retsch GmbH, Haan, Germany), extracted in 1 mL 50 mM sodium phosphate buffer (pH 7.0) containing 1% sodium diethyldithiocarbamate and stable isotope-labeled internal standards (10 pmol [²H₆]JA, 10 pmol [²H₆]ABA and 0.1 pmol [²H₂]JA-Ile; OlchemIm, Olomouc, Czechia) then alkalized by adding 1 mL of 5% NH₄OH/H₂O (v/v). The resulting solution was purified by passage through a mixed-mode anion exchange column (Oasis^® MAX column, 1 cc/30 mg, Waters, Milford, MA, United States) conditioned with 100% MeOH and equilibrated with H₂O and 5% NH₄OH (1 ml of each solution). After sample loading, the column was washed with 2 mL 5% NH₄OH followed by 2 mL 100% MeOH, then the acidic phytohormones were eluted using 2 mL 2% HCOOH in 100% MeOH (v/v). The samples were evaporated to dryness under a stream of nitrogen and stored in a freezer at -20°C until analysis.