Uhplc 1290 infinity 2 series

LC/MS/MS analysis was performed using an Agilent UHPLC 1290 Infinity II Series coupled to an Agilent QQQ/MS 6490 Series (Agilent Technologies, Santa Clara, CA, USA). Chromatographic separation was performed using an ACQUITY UPLC BEH C18 column (1.7 μm; 2.1 × 100 mm; Waters, Milford, MA, USA). The acetic acid in water was produced as acetonitrile (95:5, v/v) (solvent A) and acetonitrile:water (95:5, v/v) (solvent B). The flow rate was 0.3 mL/min, the injection volume was 50 µL, and the column temperature was set to 25 °C. The triple quadrupole was operated in ESI+ mode. The transitions used for each compound were m/z 777.7 > 731.7 and 777.7 > 604.9 for thyroxine and 783.7 > 737.7 for thyroxine-13C6.
The method validation parameters for free T4 (reproducibility, repeatability, accuracy, linear range, LOQ, and LOD) were evaluated. Overall, intraday (n = 5) and interday (n = 3) precisions were less than 7.2% and 12.6%, respectively, and accuracy was between 82.8% and 110.5%. The linear range was between the LOQ (3.86 pmol/L) and 25.74 pmol/L, and LOD was 1.29 pmol/L.

The main hesperidin metabolites were analyzed in the urine of participants. Urine samples were collected for 24 h before V2 and V5 visits from each participant, before and after the supplementation, and were frozen in liquid nitrogen after collection and thawed for its analysis. For analysis, 50 µL of urine was mixed with 100 µL of water with 1% formic acid containing the internal standard. Then, the mixture was injected into LC-MS/MS (UHPLC 1290 Infinity II Series coupled to a QqQ/MS 6490 Series Agilent Technologies, Sta. Clara, CA, USA). Metabolites were quantified by external standard calibration using rac-Hesperetin-d3 as the internal standard.

Set of lipid standards (Avanti Polar lipids): LPC(18:0); PC(32:0); SM(36:1); DG(36:0); TG(52:3); ChoE(16:0); MAG(18:0).
Set of labeled lipid internal standards (SPLASH, Avanti Polar lipids): LPC(18:1-d7); PC(33:1-d7); SM(36:2-d9); DG(33:1-d7); TG(48:1-d7); ChoE(18: 1-d7); MAG(18:1-d7).
Instrumentation: UHPLC 1290 Infinity II Series coupled to a qTOF/MS 6550 Series, both Agilent Technologies (Agilent Technologies). Analytical column: Kinetex 2.6 µm EVO-C18, 100 Å, 100 × 2.1 mm (Phenomenex).
Sample preparation: For the extraction of more hydrophobic lipids, a liquid–liquid extraction with chloroform: methanol (2:1) based on the Folch procedure was performed by adding ten volumes of chloroform: methanol (2:1) containing internal standard mixture (Lipidomic SPLASH) to plasma. Then, the samples were mixed and incubated at −20 °C for 30 min. Afterwards, water with NACl (0.8%) was added and the mixture was centrifuged at 15,000 rpm. Lower phase was recovered, evaporated to dryness and reconstituted with methanol:methyl-tert-butyl ether (9:1), and analyzed using UHPLC-qTOF (model 6550 of Agilent Technologies, Santa Clara, CA, USA) in positive electrospray ionization mode.