Acquity beh c8 column

Lipids (polar and nonpolar) were extracted from plasma (10 μl) using 190 μl of isopropanol containing 1‐dodecanoyl‐2‐tridecanoyl‐sn‐glycero‐3‐phosphocholine as an internal standard (Avanti Polar Lipids). After centrifugation (10 min, 9000 g, ambient temperature), supernatants (10 μl) were injected directly onto a 100 × 2.1 mm ACQUITY BEH C8 column (1.7 μm; Waters). The column was eluted at a flow rate of 450 μl/min isocratically for 1 minute at 80% mobile phase A (95:5:0.1 vol/vol/vol 10 mM ammonium acetate/methanol/acetic acid), followed by a linear gradient to 80% mobile‐phase B (99.9:0.1 vol/vol methanol/acetic acid) over 2 min, a linear gradient to 100% mobile phase B over 7 min, and then 3 min at 100% mobile phase B. The column temperature was kept at 30°C. MS analyses were carried out using electrospray ionization in the positive ion mode using full scan analysis over m/z 200–1100 at 70,000 resolution and 3 Hz data acquisition rate. Additional MS settings are as follows: ion spray voltage, 3.0 kV; capillary temperature, 300°C; probe heater temperature, 300°C; sheath gas, 50; auxiliary gas, 15; and S‐lens RF level 60.

LC–MS samples were prepared from stool ethanol extracts (10 μl) using 190 μl isopropanol containing 1-dodecanoyl-2-tridecanoyl-sn-glycero-3-phosphocholine as an internal standard (Avanti Polar Lipids; Alabaster, AL). After centrifugation (10 min, 9000 g, ambient temperature), supernatants (10 μl) were injected directly onto a 100 × 2.1-mm ACQUITY BEH C8 column (1.7 μm; Waters). The column was eluted at a flow rate of 450 μl/min isocratically for 1 min at 80% mobile phase A (95:5:0.1 v/v/vl 10 mM ammonium acetate/methanol/acetic acid), followed by a linear gradient to 80% mobile phase B (99.9:0.1 v/v methanol/acetic acid) over 2 min, a linear gradient to 100% mobile phase B over 7 min, and then 3 min at 100% mobile phase B. MS analyses were carried out using electrospray ionization in the positive ion mode using full scan analysis over m/z 200–1100 at 70,000 resolution and 3 Hz data acquisition rate. Additional MS settings are: ion spray voltage, 3.0 kV; capillary temperature, 300 °C; probe heater temperature, 300 °C; sheath gas, 50; auxiliary gas, 15; and S-lens RF level 60.

The bile acid profiling using LC-MS/MS platform was carried out by BIOTOOLS CO., LTD. Briefly, 20 mg of cecal contents were mixed with 1000 μL of an extraction buffer containing an internal standard mixture. After vortexing for 30 s, the samples were homogenized at 35 Hz for 4 min and then sonicated for 5 min in an ice-water bath. Subsequently, the samples were incubated for 1 h at −20 °C and centrifuged at 12000 rpm for 15 min at 4 °C. The resulting supernatant was used for bile acid analysis. Chromatographic separation was achieved using a Waters ACQUITY BEH C8 column (2.1 mm × 100 mm × 1.7 μm), and mass analysis was performed utilizing the Waters Xevo TQ-S system in positive-ion ESI mode.

Plasma lipids were profiled using a Shimadzu Nexera X2 U-HPLC (Shimadzu Corp.; Marlborough, MA). Lipids were extracted from plasma (10 μL) using 190 μL of isopropanol containing 1,2-didodecanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids; Alabaster, AL). After centrifugation, supernatants were injected directly onto a 100 × 2.1 mm, 1.7 μm ACQUITY BEH C8 column (Waters; Milford, MA). The column was eluted isocratically with 80% mobile phase A (95:5:0.1 v/v/vol 10mM ammonium acetate/methanol/formic acid) for 1 min followed by a linear gradient to 80% mobile-phase B (99.9:0.1 v/v methanol/formic acid) over 2 min, a linear gradient to 100% mobile phase B over 7 min, then 3 min at 100% mobile-phase B. MS analyses were carried out using electrospray ionization in the positive ion mode using full scan analysis over 200–1100 m/z at 70,000 resolution and 3 Hz data acquisition rate. Other MS settings were: sheath gas 50, in source CID 5 eV, sweep gas 5, spray voltage 3 kV, capillary temperature 300°C, S-lens RF 60, heater temperature 300°C, microscans 1, automatic gain control target 1e6, and maximum ion time 100 ms. Lipid identities were denoted by total acyl carbon number and total number of double bond number.

The degradation of EB and abamectin was monitored and quantified by UPLC-MS/MS. Samples (0.5 μL each) were separated by Acquity BEH C8 column (2.1 × 50 mm, 1.7 μm particle size, Waters, Milford, Massachusetts) using a Waters Acquity UPLC system (Waters ACQUITY UPLC I-Class) and eluted with a gradient of mobile phase consisting of A: 10 mM ammonium acetate [+0.1% (v/v) formic acid] and B: acetonitrile, with a constant flow rate of 0.3 mL min^-1. The gradient elution conditions were as follows: 0 min A: B 50: 50; 0.3 min A: B 50: 50; 2 min A: B 5:95; 2.5 min A: B 5: 95; 2.6 min A: B 0:100; 3 min A: B 0: 100; 3.1 min acetonitrile: Water 50: 50; 5 min acetonitrile: Water 50: 50. Samples separated by UPLC were directly analyzed using a tandem triple quadrupole mass-spectrometer (Waters Xevo TQ-S micro, Waters, Milford, Massachusetts) and run in positive ESI mode with multireaction monitoring (MRM) (S7 Table).
For identification of the EB and abamectin metabolites, samples were separated using a Shimadzu LC system (LC20ADXR with Agilent Poroshell 120 EC-C18 column 2.1 mm × 50 mm, 2.7 μm) and the same mobile phases and gradient as outlined above. The partitioned samples were then analyzed by high-resolution mass spectroscopy (Triple Tof 5600+, AB Sciex, USA) under +ESI mode with collision energy at 50V.

Lipidomic analysis was performed using an Ultimate 3000 ultrahigh performance liquid chromatography system coupled to a Thermo Q-Exactive Orbitrap mass spectrometer equipped with a heated electrospray ion source (Thermo Scientific, CA, USA). Lipid extracts were separated on a Waters ACQUITY BEH C8 column (2.1 × 100 mm, 1.7 μm) with the temperature maintained at 40 °C. The flow rate was 250 μL/min, and the mobile phases consisted of 60:40 water/acetonitrile (A), and 90:10 isopropanol/acetonitrile (B), both containing 10 mM ammonium formate and 0.1% formic acid. The samples were eluted with a linear gradient from 32 to 97% B over 25 min, maintained at 97% B for 4 min and re-equilibrated with 32% B for 6 min. The sample injection volume was 5 μL. The mass spectrometer was operated in positive ionization mode, and the full scan and fragment spectra were collected at a resolution of 70,000 and 17,500, respectively. Data analysis and lipid identification were performed using the software Lipidsearch 4.1.30 (Thermo Fisher, CA, USA). Mass labeled d13-PC (18:0) was used as an internal standard.