The content of endogenous BAs in the liver was determined by Shanghai Biotree Biotech Co., Ltd. (Shanghai, China). Briefly, individual liver specimens were precisely weighed and extracted with extraction solution (acetonitrile-methanol-water, 2:2:1, 0.1% formic acid contained and mixed with isotopically labeled standard), and the resulting supernatants were subjected to UHPLC–MS/MS analysis after centrifugation. UHPLC separation was conducted on an Agilent 1290 Infinity series UHPLC System (Agilent Technologies, USA). A series of calibration standard solutions were previously detected.
Agilent 1290 infinity series uhplc system
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 1290 Infinity series UHPLC System is a high-performance liquid chromatography system designed for ultra-high-pressure liquid chromatography (UHPLC) applications. It provides improved separation efficiency, reduced analysis time, and enhanced resolution compared to traditional HPLC systems.
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5 protocols using agilent 1290 infinity series uhplc system
1
Quantifying Endogenous Liver BAs
2
Targeted Bile Acid Analysis in Liver
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Fifty milligrams liver samples were homogenized and sonicated on ice for 5 min. The liver samples were incubated for 1 h at − 20 ℃ and centrifuged at 4 ℃ (11,000 rpm, 15 min). 0.5 mL supernatant was subjected to a fresh glass vial for LC–MS/MS analyses [41 ]. Targeted bile acid analysis was conducted using an Agilent 1290 Infinity series UHPLC System (Agilent Technologies).
3
UHPLC Analysis of Compounds Using C18 Column
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All measurements were conducted on an Agilent 1290 Infinity Series UHPLC system (Agilent Technologies, Santa Clara, USA). Chromatographic reversed-phase separation with 2 μL injection volume was performed on a C18 Hypersil Gold column (150 mm × 2.1 mm; 1.9 μm particle size) with guard column (Thermo Fisher Scientific, Waltham, USA) at a flow rate of 0.3 mL/min and with a column temperature of 40 °C. The binary mobile phase was composed of water as mobile phase A and methanol as mobile phase B, both containing 0.1% formic acid and 5 mmol ammonium formate. The gradient conditions were as follows: 0-0.5 min A: 95%/B: 5%; 7.0 min A: 50%/B: 50%; 7.5 min A: 20%/B: 80%; 7.6 min A: 0%/B: 100%; 10.1-15 min A: 95%/B: 5%.
4
Quantification of Bile Acids in Serum
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The BA concentrations in serum were measured utilizing ultrahigh-performance liquid chromatography–tandem secondary mass spectrometry (UHPLC-MS/MS) and according to the protocol described in our previous study [18 (link)]. In brief, the serum was mixed with an extract solvent (acetonitrile/methanol, 1:1, containing 0.1% formic acid and an isotopically labeled internal standard mixture), vortexed for 30 s, sonicated for 10 min in an ice-water bath, incubated at −40 °C for 1 h, and centrifuged at 12,000 g and 4 °C for 15 min. Then, the prepared samples were detected using a Q-Exactive Focus mass spectrometer (Thermo Fisher Scientific), in combination with an Agilent 1290 Infinity series UHPLC System (Agilent Technologies, Santa Clara, CA, USA) equipped with a Waters ACQUITY UPLC BEH C18 column (150 × 2.1 mm, 1.7 μm, Waters). The typical ion source parameters were as follows: spray voltage = +3500/−3100 V; sheath gas (N2) flow rate = 40; aux gas (N2) flow rate = 15; sweep gas (N2) flow rate = 0; aux gas (N2) temperature = 350 °C; capillary temperature = 320 °C. All detected BAs were classified into primary BAs (PBAs), secondary BAs (SBAs), free BAs (FBAs), and conjugated BAs (CBAs) according to their synthetic sources and properties.
5
Ultra-High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry
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Analysis was performed on an Agilent
1290 Infinity Series UHPLC System (Agilent Technologies, Santa Clara,
CA, USA) with an online 1290 Infinity Series DAD detector (Agilent
Technologies) coupled to a Q-TOF 6540 high-resolution mass spectrometer
(Agilent Technologies). The instrumentation was controlled by Agilent
MassHunter B.05.01 software.
1290 Infinity Series UHPLC System (Agilent Technologies, Santa Clara,
CA, USA) with an online 1290 Infinity Series DAD detector (Agilent
Technologies) coupled to a Q-TOF 6540 high-resolution mass spectrometer
(Agilent Technologies). The instrumentation was controlled by Agilent
MassHunter B.05.01 software.
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