Agilent 6230 tof ms

The activity of HSP hydroxylase (HspB) was assayed according to previous reports23 (link). The OD_620nm was determined using a UV-2550 spectrophotometer (Shimadzu, Kyoto, Japan). The concentrations of nicotine and HSP were measured by high-performance liquid chromatography (HPLC) (Agilent, Santa Clara, CA, USA) using an Eclipse XDB-C18 column (5 μm 4.6 × 150 mm, Keystone Scientific, Bellefonte, PA, USA). Briefly, the sample was analyzed with a mobile phase of 12% (v/v) methanol and 88% (v/v) 1 mM H₂SO₄ at a flow rate of 0.5 ml min⁻¹. Liquid chromatography-mass spectrometry (LC-MS) was performed on an Agilent 6230 TOF-MS equipped with electrospray ionization sources. DCW was calculated from the optical density (OD_{620 nm}) with a linear correlation factor (1 OD_620nm = 0.56 g DCW l⁻¹).

UHPLC-APCI-TOF-MS analysis of the acid hydrolysed lipids was conducted in order to obtain information about the tetraether lipids. About 0.4 to 0.8 mg of the acid hydrolysed lipid extract was dissolved in a mixture of hexane/isopropanol 99:1. Extracts were filtered by use of a 0.45 μm, 4 mm diameter PTFE filter. About 2 mg per mL core lipid containing extracts were used for analysis by ultra-high performance liquid chromatography linked to time-of-flight atmospheric pressure chemical ionisation mass spectrometry using a (UHPLC-APCI-TOFMS). Core lipid analysis was performed on an Agilent 1260 Infinity II UHPLC coupled to an Agilent 6230 TOF-MS. Separation was achieved on two UHPLC silica columns (BEH HILIC columns, 2.1 × 150 mm, 1.7 μm; Waters) in series maintained at 25°C. The injection volume was 10 µL. Lipids were eluted isocratically for 10 min with 10% B, followed by a linear gradient to 18% B in 15 min, then a linear gradient to 30% B in 25 min, then a linear gradient to 100% B in 30 min and finally 100% B for 20 min, where A is hexane and B is hexane: isopropanol (9:1). Flow rate was 0.2 mL/min and pressure 400 bar. Total run time was 120 min with a 20 min re-equilibration. Settings of the ion source (APCI) are as followed: gas temperature 200°C, vaporiser 400°C, drying gas 6 L/min, nebuliser 60 psig. The lipids were identified using a positive ion mode (600–1400 m/z).