Syncronis c18 hplc column

For reconfirming the presence of menaquinone in S. Typhimurium SL1344, ΔyqiC, and ΔyqiC′, the bacterial menaquinone was extracted using acetone, as described in the TLC procedure. The acetone extracts were centrifuged and filtered using Whatman No. 1 filter papers to remove cell debris. Subsequently, the filtered extracts were dried under decreasing pressure and treated with 1 mL of acetonitrile. The reconstituted mixtures were filtered using Sep-Pak C18 cartridges (Waters, Milford, MA, USA) and subjected to HPLC (Hitachi L-7400) on a Syncronis C18 HPLC column (Thermo Fisher Scientific, Waltham, MA, USA). Menaquinone was detected at 245 nm on a UV detector.

The dried sample extracts (1 mg/mL) were dissolved in aqueous methanol for UPLC-Q-TOF-MS analysis using an LTQ IT mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an electrospray interface, RS column compartment, and RS pump (Dionex Corporation, Sunnyvale, CA, USA). Chromatographic separation was performed on a Thermo Fisher Scientific Syncronis C18 HPLC column (100 × 2.1 mm internal diameter; 1.7 μm particle size) with an injection volume of 2 μL. The mobile phase comprised water (solvent A) with 0.1% formic acid (v/v) and acetonitrile (solvent B) with 0.1% formic acid (v/v) at a flow rate of 0.4 mL/min; the column temperature was 35 °C. The solvent gradient was 5% B for 1 min, increased to 100% B for 20 min, maintained for 2.5 min, decreased to 5% B for 1 min, and maintained at 10% B for the final 3 min. The total run time was 25 min. The mass spectra and photodiode array range in the negative mode were tuned for m/z 50–1200 and 200–600 nm, respectively. The collision voltage was 4 V, and the source voltage was ±1 kV.