For LC-MS/MS analysis, chromatographic separation was carried out on the HPLC-MS system Q-TRAP 6500 LC-MS/MS System from AB Sciex equipped with Shimadzu LC-20A LC and AutoSampler system. The mixture was separated on a Synergi Fusion RP 4 m from Phenomenex (150 mm × 2.00 mm).
Tauro-conjugated and non-conjugated BAs were separated at a flow rate of 200 μL/min using a methanol–aqueous ammonium acetate (NH4OAc) gradient. Mobile phase A was water containing 5 mM ammonium acetate and 0.1% formic acid, mobile phase B was methanol, containing ammonium acetate at 5 mM and 0.1% formic acid. The gradient started at 65% B and increased to 85% B in 23 min, kept at 85% B for 5 min then decreased to 65% B in 1 min and kept at 65% B for 10 min. ESI was performed in negative ion mode and the ion source temperature was set at 280°C. The tune page parameters were automatically optimized injecting taurocholic acid at 1 μM as standard. The MS/MS detection was operated in MRM mode using a collision energy of 20 (arbitrary units) and the observed transitions are reported in Mencarelli et al., 2013 (link).
Tauro-conjugated and non-conjugated BAs were separated at a flow rate of 200 μL/min using a methanol–aqueous ammonium acetate (NH4OAc) gradient. Mobile phase A was water containing 5 mM ammonium acetate and 0.1% formic acid, mobile phase B was methanol, containing ammonium acetate at 5 mM and 0.1% formic acid. The gradient started at 65% B and increased to 85% B in 23 min, kept at 85% B for 5 min then decreased to 65% B in 1 min and kept at 65% B for 10 min. ESI was performed in negative ion mode and the ion source temperature was set at 280°C. The tune page parameters were automatically optimized injecting taurocholic acid at 1 μM as standard. The MS/MS detection was operated in MRM mode using a collision energy of 20 (arbitrary units) and the observed transitions are reported in Mencarelli et al., 2013 (link).
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