Uhplc 1290 lc system

Plasma bile acids were quantified at the Sick Kids Analytical Facility for Molecules by liquid chromatography mass spectrometry (LC–MS) using the Biocrates Life Sciences Bile Acids Kit (Biocrates, Innsbruck, Austria) and analyzed with the Agilent UHPLC 1290 LC system coupled to an ABSciex QTRAP 5500 in negative ESI MRM mode. The samples were lyophilized, reconstituted with 75% ethanol and homogenized. Then, vortexed for 5 min followed by a 10 min centrifugation at 20,000 g, the supernatants were used for bile acid measurement following manufacturer’s instructions.

Serum C4 measurement was performed by LC-MS-MS. C4 was extracted using a salting-out method as previously described [24 (link), 25 (link)]. Serum samples were prepared alongside of authentic C4 standards. Briefly, 100 μL of serum was diluted with 200 μL of distilled water, to which 5 ng of d7-C4 (used as internal standard) and 500 μL of acetonitrile were added. Then, 100 mg of ammonium sulfate was added, tubes vortexed for 1 minute and centrifuged at 2,000 g at 4°C for 5 minutes. The supernatant acetonitrile phase was collected and dried under nitrogen at 35°C. The residues were reconstituted with 200 μL methanol, vortexed for 1 minute, incubated for 10 minutes and centrifuged at 20,000 g for 5 minutes. Clear supernatants were transferred to 250 μL inserts for LC-MS-MS analysis. The LC-MS-MS system consisted of an Agilent UHPLC 1290 LC system coupled to an ABSciex QTRAP 5500 in positive ESI MRM mode. Chromatographic separation was performed using a XB-C18 kinetex column (50 x 3.0 mm, 2.6 μm; Phenomenex) operated at a flow rate of 800 μL/minute and eluted isocratically with a mobile phase consisting of acetonitrile/water (98/2, v/v) with 0.1% trifluoroacetic acid. Quantitation of C4 was achieved by comparing the deuterium-to-protium ratio of the samples with a standard using the Analyst 1.6 software.

Serum bile acid concentrations of the discovery cohort were determined by high-performance liquid chromatography (HPLC) negative ion electrospray tandem mass spectrometry as described previously [23 (link)]. Those of the follow up cohort were measured by liquid chromatography–mass spectrometry (LC-MS) using the Biocrates^® Life Sciences Bile Acids Kit (Biocrates, Innsbruck, Austria) as per manufacturer’s instructions using an Agilent UHPLC 1290 LC system coupled to an ABSciex QTRAP 5500 in negative ESI MRM mode. The same kit was used to analyze fecal bile acids. Samples were lyophilized, reconstituted with 75% ethanol and homogenized then vortexed for 5 minutes and centrifuged at 20,000 g for 10 minutes. The resulting supernatant was used for bile acids measurement.