Extraction and measurement of BAs were carried out as described previously [23 (link)]. Briefly, after adding nine times the volume of methanol to the samples, approximately 7 mg of small intestinal samples were cut and homogenized in Precellys tubes with 1.4 mm ceramic beads. Supernatants were transferred to Eppendorf tubes and were centrifuged at 15,000× g at 4 °C for 10 min. After repeating the centrifugation step, supernatants were pipetted into HPLC vials and were analyzed by LC-MS in positive modus using an LCMS-8040 Liquid Chromatograph Mass Spectrometer (Shimadzu Corporation, Kyoto, Japan) with an Atlantis T3 3 μm column (2.1 × 150 mm, Waters). The column temperature was kept at 30 °C. Gradient started from 30% B (acetonitrile/methanol (3/1), v/v) and 70% A (water). After 5 min, B was increased to 100% within 20 min and kept constant for 20 min. Afterward, the mobile phase was set back to 30% B for 10 min. Both solvents contained 0.1% formic acid with 20 mM ammonium acetate.
Lcms 8040 liquid chromatograph mass spectrometer
Manufactured by Shimadzu
Sourced in Japan
The LCMS-8040 Liquid Chromatograph Mass Spectrometer is an analytical instrument designed to separate, identify, and quantify compounds in a sample. It combines the separation capabilities of liquid chromatography with the high-sensitivity detection of triple quadrupole mass spectrometry. The LCMS-8040 can be used for a variety of applications, including environmental analysis, food safety, and pharmaceutical research.
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Lab products found in correlation
Atlantis t3 3 μm column, by Waters Corporation (1 mentions)
Waters 2996 photodiode array detector, by Waters Corporation (1 mentions)
Sephadex lh 20, by Cytiva (1 mentions)
Silica gel, by Qingdao Marine Chemical (1 mentions)
J 815 spectropolarimeter, by Jasco (1 mentions)
Oasis max μelution plate, by Waters Corporation (1 mentions)
Nexera x2 lc system, by Shimadzu (1 mentions)
3 protocols using lcms 8040 liquid chromatograph mass spectrometer
1
Bile Acid Extraction and Measurement
2
Spectroscopic Characterization of Natural Products
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Optical rotations were performed using a JASCO P-1020 digital polarimeter (Jasco Corporation, Tokyo, Japan). ECD spectra were obtained with a JASCO J-815 spectropolarimeter (Jasco Corporation, Tokyo, Japan). UV spectra were obtained using a Shimadz UV-210A spectrometer (Shimadzu Corporation, Tokyo, Japan). NMR spectra were recorded with 400 and 600 MHz Bruker Avance NMR spectrometers (Bruker BioSpin AG, Fällanden, Switzerland). ESIMS was performed using a Shimadzu LCMS-8040 Liquid Chromatograph Mass Spectrometer (Shimadzu Corporation, Tokyo, Japan). HRESIMS spectra were obtained using a Thermo Scientific Ultimate 3000 UHPLC-Q Exactive spectrometer (ThermoFisher Scientific, Waltham, MA, USA). Semi-preparative HPLC was performed on a Waters 1525 system using a semi-preparative C18 (Waters SunFire C18 ODB Prep Column, 10 × 250 mm, 5 µm, 3 mL/min) column coupled with a Waters 2996 photodiode array detector (Waters Corporation, Milford, MA, USA). Thin-layer chromatography (TLC) was performed on plates precoated with silica gel GF254 (10–40 μm) (Qingdao Marine Chemical Factory, Qingdao, China). Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden) and silica gel (100–200 and 200–300 mesh, Qingdao Marine Chemical Factory) were used for column chromatography.
3
Quantifying Plasma CP-I and CMPF
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Plasma CP‐I and CMPF concentrations were measured simultaneously according to the methods reported by Suzuki et al.
17 (link) Briefly, Oasis MAX μElution Plate (Waters) was used to pretreat 250 μL of plasma by solid phase extraction. The extract was analyzed by ultra‐high‐performance liquid chromatography coupled to tandem mass spectrometry using the Nexera X2 LC system coupled to LCMS−8040 Liquid Chromatograph Mass Spectrometer (Shimadzu) equipped with electrospray ionization. The 15N4‐CP‐I and CMPF‐d5 were used as internal standards for CP‐I and CMPF, respectively. CP‐I was measured with (M + H)+ signal in positive ion mode, and CMPF was measured with (M – H)− signal in negative ion mode. The tandem mass spectrometry transitions monitored were mass‐to‐charge ratio (m/z) 655.4 → m/z 596.3 for CP‐I, m/z 659.3 → m/z 600.3 for 15N4‐CP‐I, m/z 239.0 → m/z 195.2 for CMPF, and m/z 244.2 → m/z 200.2 for CMPF‐d5. The assays were validated in accordance with the US Food and Drug Administration Guidance for Bioanalytical Method Validation.
20 The lower limit of quantification was 0.1 ng/mL for CP‐I and 50 ng/mL for CMPF. The within‐batch accuracy of the assay ranged from 92.1% to 110.2% for CP‐I, and from 99.1% to 109.3% for CMPF. The within‐batch precision was less than 7.6% for CP‐I and 3.4% for CMPF.
17 (link) Briefly, Oasis MAX μElution Plate (Waters) was used to pretreat 250 μL of plasma by solid phase extraction. The extract was analyzed by ultra‐high‐performance liquid chromatography coupled to tandem mass spectrometry using the Nexera X2 LC system coupled to LCMS−8040 Liquid Chromatograph Mass Spectrometer (Shimadzu) equipped with electrospray ionization. The 15N4‐CP‐I and CMPF‐d5 were used as internal standards for CP‐I and CMPF, respectively. CP‐I was measured with (M + H)+ signal in positive ion mode, and CMPF was measured with (M – H)− signal in negative ion mode. The tandem mass spectrometry transitions monitored were mass‐to‐charge ratio (m/z) 655.4 → m/z 596.3 for CP‐I, m/z 659.3 → m/z 600.3 for 15N4‐CP‐I, m/z 239.0 → m/z 195.2 for CMPF, and m/z 244.2 → m/z 200.2 for CMPF‐d5. The assays were validated in accordance with the US Food and Drug Administration Guidance for Bioanalytical Method Validation.
20 The lower limit of quantification was 0.1 ng/mL for CP‐I and 50 ng/mL for CMPF. The within‐batch accuracy of the assay ranged from 92.1% to 110.2% for CP‐I, and from 99.1% to 109.3% for CMPF. The within‐batch precision was less than 7.6% for CP‐I and 3.4% for CMPF.
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