Acquity uplc hss t3 1.8 μm

Gibberellins levels were determined using high-performance liquid chromatography–tandem mass spectrometry (AB SCIEX TripleTOF 5600+, Darmstadt, IN, USA). Approximately 0.5 g of fruits was ground in liquid nitrogen and extracted using solvent A (1 L 80% methanol containing 0.5 g citric acid and 0.2 g butylated hydroxytoluene). The sample was extracted with 4 ml of solvent A, and the extract was shaken at 4°C for 12 h. After centrifuging the sample at 10,000 ×g and 4°C for 15 min, the supernatant was collected. Next, 2 ml of solvent A was added to the sample, which was shaken again at 4°C for 1 h. The sample was centrifuged at 10,000 ×g at 4°C for 15 min, and the supernatant was collected. This step was repeated. All supernatant aliquots were mixed and dried under nitrogen gas, dissolved in 250 µl methanol and passed through a 0.22-µm filter membrane. The high-performance liquid chromatography analysis was performed using an ACQUITY UPLC HSS T3 (1.8 μm, Waters, USA) column (2.1 × 100 mm). The mobile phase solvent was the same as in Balcke et al. (2012) (link), and the injection volume was 2 µl. The mass spectrometry conditions were as follows: a spray voltage of 4,500 V, and air curtain, nebulizer, and auxiliary gas pressures of 15, 65, and 70 psi, respectively. The atomizing temperature was 400°C. Each sample consisted of three replicates from independent experiments.

Plant hormone (ABA, IAA, GA₁, GA₄, tZ, and tZR) levels were measured according to Kojima et al. (2009) (link) with minor modifications. Fresh powder samples (0.2 g) was homogenized in 5 mL pre-chilled acetonitrile for 12 h. The supernatant was collected by centrifugation at 12,000 x g for 15 min at 4°C. The pellet was resuspended in 5 mL pre-chilled acetonitrile for 2 h and then collected and combined with the supernatant after centrifugation as before. Next, 20 mg C18 powder and 0.5 g anhydrous magnesium sulfate powder was added to the combined supernatant, and the supernatant was collected after centrifugation as before. The supernatant was concentrated to near dryness using a centrifuge concentrator for 6–8 h and added with 0.2 mL methanol. The sample was filtered through a 0.22 μm organic nylon membrane and filled with a 2 mL sample bottle. The levels of plant hormones were measured using liquid chromatography-mass spectrometry (LC-MS/MS; QTRAP^® 6500, AB Sciex, USA) fitted with a chromatographic column (ACQUITY UPLC HSS T3, 1.8 μm, 2.1 × 75 mm, Waters). Hormones were separated at a flow rate of 0.3 mL min^-1 with the gradients of solvent A (0.1% formic acid) and solvent B (acetonitrile) set as follows: 0 min, 95% A + 5% B; 1 min, 95% A + 5% B; 8 min, 5% A + 95% B; 8.1 min, 95% A + 5% B; and 10 min, 95% A + 5% B.

Reagents of analytical grade were mainly used, with the exception of mobile phase reagents which were of high-performance liquid chromatography (HPLC) grade. Analytical standards comprised: MPA and NIV from Supelco (Bellefonte, PA, USA); PA from Santa Cruz Biotechnology (Dallas, TX, USA); AFB1, B2, G1, G2, and M1, BEA, CIT, DON, ENNA, and B, FB1 and B2, HT-2 and T-2 toxins, MON, OTA, PAT, TEA, TTX, and ZEA from Sigma-Aldrich (Steinheim, Germany). Ultrapure H₂O was supplied from Millipore System (Paris, France), C18 sorbent from Agilent Technologies (Santa Clara, CA, USA), acetonitrile (ACN) from Carlo Erba (Val de Reuil Cedex, France), anhydrous magnesium sulfate (MgSO₄) and sodium chloride (NaCl) from Honeywell (Seelze, Germany), and formic acid from Chem-Lab (Zedelgem, Belgium). ACQUITY UPLC^® HSS T3 1.8 μm (2.1 × 100 mm i.d.) was purchased from Waters (Milford, MA, USA), and HPLC vials and Syringeless Device Mini UniPrep filters (0.45 μm PVDF, polypropylene) from Whatman (Maidstone, UK).

SCFA concentrations were analyzed at LSI Medience Corporation (Tokyo, Japan), which is a contract laboratory. Briefly, approximately 150 mg of tissue or feces was transferred to a disruptor tube supplied by Yasui Kikai (Osaka, Japan) and shaken with an iron cone cooled in liquid nitrogen. The powders were suspended with 1 mL of water, and 50 μL of the suspension was transferred to a micro tube (when a specimen was serum, 50 μL of serum was transferred into a micro tube). After mixing with 200 μL of methanol using a shaker for 15 min, the samples were centrifuged at 20,000g for 1 min at 4°C. A volume of 200 μL of the supernatant was mixed with 2‐nitrophenylhidrazine and 1‐3‐dimethylaminopropyl‐carbodiimide solution and analyzed by liquid chromatography–tandem mass spectrometry (Nexera X2 LC‐30 AD, 8050; Shimadzu, Kyoto, Japan) equipped with a reverse‐phase LC column (ACQUITY UPLC HSS T3,1.8 μm, 2.1 × 50 mm; Waters, USA). The data were analyzed using the LabSolutions software (Shimadzu). The peak areas were normalized by internal standards, and the concentration of each SCFA was obtained using a standard curve.

For remdesivir and GS-441524 PK assessments at steady state, blood samples were collected on day 4, immediately after (C₀) and at 1 h (C₁) and 24 h (C₂₄) after IV remdesivir administration.^{18 (link)}Plasma samples were obtained by centrifugation (5000 rpm for 5 min at +4°C) of blood samples (tubes containing lithium heparin). Immediately after collection, 1 mL of plasma was isolated and stored at –20°C until measurement of remdesivir and GS-441524 concentrations. Plasma concentrations of remdesivir and its metabolite GS-441524 were determined by using validated UHPLC-MS/MS.^{19 (link)} Briefly, 50 μL of sample was mixed with 600 μL of acetonitrile:methanol (50:50, v:v) containing internal standard (quinaxoline). Three hundred microlitres of supernatant was diluted with 600 μL of HPLC-MS grade water and 10 μL was injected into the column Acquity UPLC HSS T3 1.8 μm (2.1 × 50 mm Waters). Remdesivir and GS-441524 were measured simultaneously with a limit of quantification of 5.86 and 1.96 ng/mL, respectively. Linearity was 5.86 to 3000 ng/mL for remdesivir and 1.96 to 1000 ng/mL for GS-441524, with R² >0.995 for both drugs. In order to decrease esterase plasma activity, all steps were performed on ice.