Agilent 1260 lc

Samples were analyzed for eight MC variants (dm-7-MC-RR, MC-RR, MC-YR, dm-7-MC-LR, MC-LR, MC-LY, MC-LW, and MC-LF). Calibration standards were obtained from the National Research Council (Ottawa, Canada) for dm-7-MC-LR, and from Enzo Life Sciences Inc. (Farmingdale, NY, United States) for the other variants. Measurements were performed on an Agilent 1260 LC and an Agilent 6460A QQQ (Agilent Technologies, Santa Clara, CA, United States). The compounds were separated on an Agilent Zorbax Eclipse XDB-C18 4.6 mm × 150 mm, 5 μm column using Millipore water with 0.1% formic acid (v/v, eluent A) and acetonitrile with 0.1% formic acid (v/v, eluent B). The elution program was set at 0–2 min 30% B, 6–12 min 90% B, with a linear increase of B between 2 and 6 min and a 5 min post run at 30% B. Sample injection volume was set at 10 μL, with a flow of 0.5 mL min^-1 at a column temperature of 40°C. The LC-MS/MS was operated in positive mode with an ESI source, nitrogen was used as a drying, sheath and collision gas. For each compound, two transitions were monitored in MRM mode: m/z 491.3 to m/z 135.1 and m/z 981.5 to m/z 135.2 (dm-7-MC-LR, ratio between product ions 17%), m/z 498.3 to m/z 135.1 and m/z 995.6 to m/z 135.1 (MC-LR, ratio between product ions 16%). This protocol is based on the protocol earlier described by Faassen and Lürling (2013) (link).

2.5 g of ketoprofen was dissolved in 20 ml of ethyl alcohol at room temperature, and then 150 mg of Sr/PTA-MOF was added in this solution. After stirring for 24 h, the nanoparticles were obtained by filtration and washed with distilled water, and finally dried at 150 °C for 24 h to further treat. The loading samples were labeled as Sr/PTA-MOFs-Ketoprofen.
Loaded ketoprofen amount in MOFs is measured as following: 5 mg of samples after ketoprofen loading were digested separately in 10 mL of NaOH (0.1 mol/L) overnight. Then 2 mL of supernatant was taken and then mixed with 2 mL of methanol. After filtering by 0.45 µm filter membrane, this solution was tested using Agilent 1260 LC high performance liquid chromatography. The mobile phase was V(acetonitrile): V(K₂HPO₄, pH = 2) = 1:1. And sunfire-C18 reverse-phase column (5 μm, 4.6 × 150 mm Waters) was employed. Besides, the flow rate was 1 mL · min⁻¹ and the column temperature was fixed at 25 °C. Ketoprofen in NaOH solutions (0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5 mg · mL⁻¹) were used as standards.

The components of the TBF fraction were determined as described elsewhere [23 (link)]. The TBFs were analyzed using a high-performance liquid chromatography (HPLC) system (Agilent 1260 LC, Agilent Technology Co., Ltd.) equipped with an Agilent TC-C18 column (4.6 mm × 250 mm, Agilent Technology Co., Ltd.). The sample was eluted using mobile phase A of water and mobile phase B of a water solution containing 95% (v/v) methanol. A linear gradient program was applied as follows: 20% (v/v) B for 0–5 min, 20–40% (v/v) B for 5–15 min, and 40% (v/v) B for 15–40 min. The detection wavelength was set at 260 nm. Pure rutin and quercetin were used as standards for HPLC analysis.

Insecticide penetration was measured as previously described, with slight modifications [32 (link), 41 (link)]. Flies were exposed to malathion at the LD₅₀ for 1 h before washing twice with 3 mL acetonitrile in a 50-mL centrifuge tube. The eluent was then concentrated and dried in a CoolSafe (LaboGene, Bjarkesvej, Denmark) and dissolved in 1 mL acetonitrile. The solution was centrifuged (12,000 g, 10 min, 4 °C) and the malathion content of the supernatant was determined by high performance liquid chromatography (HPLC, Agilent 1260 LC, Agilent Technologies, Palo Alto, CA, USA) as previously described [22 (link), 42 (link)]. Briefly, the 10 μL sample was eluted with reversed-phase analytical column (ZORBAX SB-C18, 4.6 × 100 mm, 3.5 μm, Agilent Technologies) under mobile phase (60% acetonitrile and 40% ultrapure water) with speed of 1.0 mL·min⁻¹. The malathion was analyzed with monitor at 27 °C and 230 nm. The penetration ratio of malathion was calculated as follows: $\mathrm{penetration}\mathrm{ratio}=\frac{\mathrm{total}\mathrm{applied}\mathrm{dose}-\mathrm{residual}\mathrm{malathion}}{\mathrm{total}\mathrm{applied}\mathrm{dose}}\times 100\%$ .

LC–MS/MS of pTyr peptides were carried out on an Agilent 1260 LC coupled to a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific). Peptides were separated using a 140-min gradient with 70% acetonitrile in 0.2 mol/l acetic acid at a flow rate of 0.2 ml/minute with an approximate split flow of 20 nl/minute. The mass spectrometer was operated in data-dependent acquisition with the following settings for MS1 scans: m/z range: 350–2,000; resolution: 60,000; AGC target: 3 × 10⁶; maximum injection time (maxIT): 50 ms. The top 15 abundant ions were isolated and fragmented by higher energy collision dissociation with the following settings: resolution: 60,000; AGC target: 1 × 10⁵; maxIT: 350 ms; isolation width: 0.4 m/z, collisional energy (CE): 33%, dynamic exclusion: 20 s. Crude peptide analysis was performed on a Q Exactive Plus mass spectrometer to correct for small variations in peptide loadings for each of the TMT channels. Approximately 30 ng of the supernatant from pTyr IP was loaded onto an in-house-packed precolumn (100 μm ID × 10 cm) packed with 10 mm C18 beads (YMC gel, ODS-A, AA12S11) and analyzed with a 70-min LC gradient. MS1 scans were performed at the following settings: m/z range: 350–2,000; resolution: 70,000; AGC target: 3 × 10⁶; maxIT: 50 ms. The top 10 abundant ions were isolated and fragmented with CE of 33% at a resolution of 35,000.