Sb c18 reversed phase column

The determination of BAs was conducted based on the work of Wang et al. [26 (link)]. H. alvei H4 strains (wild type, ΔluxI, ΔluxR and ΔluxIR) were cultivated in LB supplemented with 0.005% pyridoxal-5-phosphate and 0.1% precursor amino acid for 24 h. Then 1 mL of culture was mixed with 9 mL 10% trichloroacetic acid in a centrifuge tube. After standing for 2 h at 4 °C, the mixture was homogenized for 10 min (3000× g). A 200 μL volume of supernatant was derivatized using 80 μL 2 mol/L NaOH and 800 μL 10 mg/mL dansyl chloride dissolved in acetone. After water-bath heating at 45 °C for 40 min, 50 μL ammonium hydroxide and 550 μL acetonitrile were added into the dansyl derivatives, homogenized for 5 min (3000× g) and filtrated through a 0.22 μm filter. Finally, 10 μL aliquots were injected for HPLC analysis.
The concentrations of BAs were determined by an HPLC system (ZORBAX, Agilent, Tokyo, Japan). An SB-C₁₈ reversed-phase column (5 μm, 4.6 mm × 125 mm; Agilent, Tokyo, Japan) was used for chromatographic separation. The gradient elution program was operated with acetonitrile/water as the mobile phase.

Samples (1 g) of lyophilized liquid nitrogen were ground into a powder and extracted for 2 h at 4°C in 10 ml of 80% methanol. The extracts were centrifuged at 10,000 r/min for 5 min at 4°C. The supernatant was added to a Bond Elut column and eluted with 3 ml methanol. Then, the eluant was added into a Strata-X column and eluted with 3 ml methanol. Nitrogen flow was used to dry eluant and 200 μl methanol was added to redissolve. The sample was filtered through a 0.22 μm syringe for HPLC-MS/MS detection.
The extracted samples were quantified using a ZORBAX SB-C18 reversed-phase column (150 mm × 2.1 mm, 3.5 μm). Five microlitres of the sample were injected at a flow rate of 0.35 ml·min⁻¹ and a column temperature of 35°C.