1290 infinity series uplc system

To clarify the changes produced by the metabolites of the intestinal flora of NASH mice after treatment with FTZ, we selected three representative samples from the HFD and FTZ (600 mg/kg) groups for a metabolomic study among the mice feces samples in the 16S rRNA gene sequencing by combining the results of previous experiments. The sample and extract solution (acetonitrile:methanol:water = 2:2:1) (1,000 μl) containing the internal standard (L-2-chlorophenylalanine, 2 μg/ml) were mixed, then homogenized, sonicated, and centrifuged. The supernatant was dried in a vacuum concentrator. The samples were reconstituted in 200 μl of 50% acetonitrile and centrifuged for 10 min, and the supernatant was ready for UPLC-MS/MS analysis. The UPLC and MS/MS instrument settings are shown in Supplementary Tables 2, 3. The UPLC separation was carried out using a 1290 Infinity series UPLC System (Agilent Technologies, CA, Palo Alto, USA).

A total of 25 mg of hepatic sample was weighted in an EP tube, and 1,000 μl extract solution (acetonitrile:methanol:water = 2:2:1) containing internal standard (L-2-Chlorophenylalanine, 2 μg/ml) was added. Then the samples were centrifuged for15 min (10,000 r/min, 4°C). 800 μl of the supernatant was taken to a fresh tube and dried in a vacuum concentrator at 37°C. Then, the dried samples were reconstituted in 200 μl of 50% acetonitrile by sonication on ice for 10 min. The constitution was then centrifuged for15 min (13,000 r/min, 4°C), and 75 μl of the supernatant was transferred to a fresh glass vial for LC/MS analysis. In addition, the quality control (QC) sample was prepared by mixing an equal aliquot of the supernatants from all samples.
The UPLC separation was carried out using a 1290 Infinity series UPLC System (Agilent Technologies), equipped with a UPLC BEH Amide column (2.1 * 100 mm, 1.7 μm, Waters). The mobile phase consisted of 25 mmol/L ammonium acetate and 25 ammonia hydroxide in water (A) and acetonitrile (B). The analysis was carried with elution gradient as follows: 0~0.5 min, 95% B; 0.5~7.0 min, 95%~65% B; 7.0~8.0 min, 65%~40% B; 8.0~9.0 min, 40% B; 9.0~9.1 min, 40%~95% B; 9.1~12.0 min, 95% B. The column temperature was 25°C. The auto-sampler temperature was 4°C, and the injection volume was 2 μl (pos) or 1 μl (neg), respectively.

Chromatographic separation was performed on a 1,290 Infinity series UPLC System (Agilent Technologies) with a UPLC BEH C₁₈ column (2.1 × 100 mm, 1.7 μm, Waters). The mobile phase consisted of 0.1% formic acid aqueous (mobile phase A), and 0.1% formic acid acetonitrile (mobile phase B), and the gradient elution program with a flow rate of 0.4 ml min⁻¹ was listed as follows: mobile phase B maintaining at 5% (0.00–0.50 min), from 5 to 30% (0.50–1.50 min), from 30 to 60% (1.50–4.00 min), from 60 to 99% (4.00–5.00 min) and maintaining at 95% (5.00–7.50 min).
Mass spectrometry information of small molecules was collected by the Q-TOF-MS spectrometer with an ESI source in negative or positive ion mode. The following MS conditions were used: the capillary voltage and cone voltage were 2.0 kV and 40 V, the desolvation gas temperature and source temperature were 440 and 115°C, and the flow rate of desolvation gas and cone gas was 80 L/h and 50 L, respectively, and the scan range was 50–1,000 m/z. The system control and data analysis were performed by Waters Progenesis QI v2.2 (Nonlinear Dynamics).