Agilent 1260 high performance liquid chromatography system

Tigecycline reference standard (99% purity, Lot L910S56 and LN70S134) was purchased from Beijing J&K Scientific Ltd., and internal standard (IS) tigecycline-d9 (95% purity, Lot 2-NYL-17-1-PFZ) was purchased from Toronto Research Chemical, Inc., HPLC-grade formic acid and methanol were purchased from Thermo Fisher Scientific. Ultrapure water was prepared using a Milli-Q water purification device (Millipore, Bedford, MA, United States). Drug-free plasmas were obtained from the Department of Blood Transfusion, PLA General Hospital. A total of 134 AB isolates were used for susceptibility tests. Quality control strain ATCC25922 was purchased from National Institutes for Food and Drug Control, China. Mueller–Hinton Agar and Mueller–Hinton Broth were purchased from Becton, Dickinson and Company. Chromatographic analysis was performed using the Agilent 1260 high-performance liquid chromatography system (Agilent Technologies Inc.), and mass spectral analysis was performed using the Agilent 6460A mass spectrometer (Agilent Technologies Inc.) with an Agilent MassHunter Workstation B.06.00 software for data processing.

HPLC condition: An Agilent 1260 high-performance liquid chromatography system (Agilent Technologies, Santa Clara, CA, USA), equipped with a quaternary pump system, an auto-sampler, a column oven, and a diode array detector, was employed in the current experiment. The sample was separated on an Agilent Poroshell 120 EC-C18 column (100 mm × 4.6 mm, 2.7 μm). The column temperature was set at 30 °C. The mobile phase was 0.1% aqueous formic acid (A) and acetonitrile (B), with a gradient elution procedure of 5% B (0 min), 5% B (3 min), 20% B (23 min), 24% B (28 min), 60% B (32 min), and 100% B (38 min). The flow rate was 0.6 mL/min. The detection wavelength was set at 290 nm, and the injection volume was 5.0 µL.
MS condition: The Agilent 6530 mass spectrometer system (Agilent Technologies, Santa Clara, CA, USA), with an electron spray ionization (ESI) source, was used in both positive and negative scanning modes from 50–1000 m/z. The electrospray ionization mass spectrometer conditions were as follows: the capillary voltage was set at 3500 V (+/−); the collision-induced dissociation voltage was set at 120 V; the drying gas (N₂) flow rate was 8 L/min; the drying gas temperature was set at 350 °C; the nebulizer pressure was 35 psi; and the sheath gas flow rate was 11 L/min.

Ruminal content samples were collected using an oral tube and a hand vacuum pump at 6 h after the morning feeding in 2 consecutive days in each sample collection week. To minimize saliva contamination, approximately 50 mL of ruminal fluid was discarded before sample collection. Ruminal pH was measured immediately after sampling. Rumen fluid was subsampled for analysis of volatile fatty acids (VFA, 5 mL with 1 mL of 25% metaphosphoric acid added), organic acids (5 mL), and microbiota (45 mL), and then stored at − 80 °C until analysis.
Ruminal VFA concentration was determined using gas chromatography (Agilent Technologies 7820A GC system, Palo Alto, CA, USA) as described by Li et al. [23 (link)]. Ruminal organic acid (fumarate, succinate, and lactate) concentration was determined using an Agilent 1260 high-performance liquid chromatography system as done in previous studies [24 (link), 25 (link)].